Вы находитесь на странице: 1из 73

Acid base concept

Arrhenius's concept: An acid is a substance which An acid dissociation constant, Ka, (also known as acidity
dissociates in aqueous solution, releasing the hydrogen constant, or acid-ionization constant) is a quantitative measure
ion H+ (a proton): of the strength of an acid in solution. It is the equilibrium
HA A− + H+ constant for a chemical reaction known as dissociation in the
The equilibrium constant for this dissociation reaction is context of acid-base reactions. The equilibrium can be written
known as a dissociation constant. The liberated proton symbolically as:
combines with a water molecule to give a hydronium (or HA A− + H+
oxonium) ion H3O+, and so Arrhenius later proposed that
the dissociation should be written as an acid–base where HA is a generic acid which dissociates by splitting into
reaction: A−, known as the conjugate base of the acid, and the hydrogen
HA + H2O A− + H3O+ ion or proton, H+, which, in the case of aqueous solutions,
Brønsted and Lowry generalized this further to a proton exists as a solvated hydronium ion.
exchange reaction. The dissociation constant is usually written as a quotient of the
acid + base conjugate base + conjugate acid. equilibrium concentrations, denoted by [HA], [A−] and [H+]:
The acid loses a proton, leaving a conjugate base; the
proton is transferred to the base, creating a conjugate [H+] [A−]
acid. For aqueous solutions of an acid HA, the base is Ka= [HA]
water; the conjugate base is A− and the conjugate acid is
the hydronium ion. The Brønsted–Lowry definition
applies to other solvents, such as dimethyl sulfoxide: the Due to the many orders of magnitude spanned by Ka values, a
solvent S acts as a base, accepting a proton and forming logarithmic measure of the acid dissociation constant is more
the conjugate acid SH+. commonly used in practice. pKa, which is equal to −log10 Ka,
The designation of an acid or base as "conjugate" may also be referred to as an acid dissociation constant:
depends on the context. The conjugate acid BH+ of a base
B dissociates according to pKa= -log10 Ka
The larger the value of pKa, the smaller the extent of
BH+ + OH − B + H2O dissociation. A weak acid has a pKa value in the approximate
which is the reverse of the equilibrium range −2 to 12 in water. Acids with a pKa value of less than
about −2 are said to be strong acids; a strong acid is almost
H2O (acid) + B (base) OH− (conjugate base) + BH+ completely dissociated in aqueous solution, to the extent that
(conjugate acid). the concentration of the undissociated acid becomes
undetectable. pKa values for strong acids can, however, be
The hydroxide ion OH−, a well known base, is here estimated by theoretical means or by extrapolating from
acting as the conjugate base of the acid water. Acids and measurements in non-aqueous solvents in which the
bases are thus regarded simply as donors and acceptors of dissociation constant is smaller, such as acetonitrile and
protons respectively. dimethylsulfoxide.
Water is amphiprotic: it can react as an acid or a base. In particular, the pH of a solution can be predicted when the
Another example of an amphiprotic molecule is the analytical concentration and pKa values of all acids and bases
bicarbonate ion HCO3− which is the conjugate base of the are known; conversely, it is possible to calculate the
carbonic acid molecule H2CO3 in the equilibrium equilibrium concentration of the acids and bases in solution
when the pH is known.
H2CO3 + H2O HCO3− + H3O+ pH: pH is a measure of the acidity or basicity of a solution. It
but also the conjugate acid of the carbonate ion CO32− in is defined as the cologarithm of the activity of dissolved
(the reverse of) the equilibrium hydrogen ions (H+). Hydrogen ion activity coefficients cannot
be measured experimentally, so they are based on theoretical
HCO3− + OH− CO32− + H2O. calculations. The pH scale is not an absolute scale; it is
Carbonic acid equilibria are important for acid-base relative to a set of standard solutions whose pH is established
homeostasis in the human body. by international agreement.
Henderson–Hasselbalch equation
pH is defined as minus the decimal logarithm of the
hydrogen ion activity in an aqueous solution. By virtue of
its logarithmic nature, pH is a dimensionless quantity. pH = pKa – log [AH]/[A-]
pH= - log aH = log10 1/aH
where aH is the (dimensionless) activity of hydrogen ions.
The reason for this definition is that aH is a property of a
single ion which can only be measured experimentally by
means of an ion-selective electrode which responds,
according to the Nernst equation, to hydrogen ion activity.
pH is commonly measured by means of a combined glass
electrode, which measures the potential difference, or
electromotive force, E, between an electrode sensitive to
the hydrogen ion activity and a reference electrode, such
as a calomel electrode or a silver chloride electrode. The
combined glass electrode ideally follows the Nernst
equation:
Eo - E
E = Eo + RT/nF loge (aH); pH = 2.303 RT/F
At half-neutralization [AH]/[A−] = 1; since log(1) =0 , the pH
at half-neutralization is numerically equal to pKa. Conversely,
where E is a measured potential , Eo is the standard
when pH = pKa, the concentration of AH is equal to the
electrode potential, that is, the electrode potential for the
concentration of A−.
standard state in which the activity is one. R is the gas
constant T is the temperature in Kelvin, F is the Faraday The buffer region extends over the approximate range pKa ± 2,
constant and n is the number of electrons transferred, one though buffering is weak outside the range pKa ± 1. At pKa ±
in this instance. The electrode potential, E, is proportional 1, [AH]/[A−] = 10 or 1/10.
to the logarithm of the hydrogen ion activity.
If the pH is known, the ratio [AH]:[A−] may be calculated.
This definition, by itself, is wholly impractical because the
This ratio is independent of the analytical concentration of the
hydrogen ion activity is the product of the concentration
acid.
and an activity coefficient. The single-ion activity
coefficient of the hydrogen ion is a quantity which cannot Buffer solution of a desired pH can be prepared as a mixture of
be measured experimentally. To get round this difficulty a weak acid and its conjugate base. In practice the mixture can
the electrode is calibrated in terms of solutions of known be created by dissolving the acid in water, and adding the
activity. requisite amount of strong acid or base. The pKa of the acid
must be less than two units different from the target pH.
pH in living systems
Polyprotic acids are acids that can lose more than one proton.
Compartment pH The constant for dissociation of the first proton may be denoted
as Ka1 and the constants for dissociation of successive protons
Gastric acid 0.7 as Ka2, etc. Phosphoric acid, H3PO4, is an example of a
Lysosomes 4.5 polyprotic acid as it can lose three protons.

Granules of chromaffin cells 5.5 equilibrium pKa value


Urine 6.0
H3PO4 = H2PO4− + H+ pKa1 = 2.15
Neutral H2O at 37 °C 6.81
Cytosol 7.2 H2PO4− =HPO42− + H+ pKa2 = 7.20
Cerebrospinal fluid (CSF) 7.3
HPO42− = PO43− + H+ pKa3 = 12.37
Blood 7.34 –
7.45
Mitochondrial matrix 7.5
Pancreas secretions 8.1
Buffer solutions also play a key role in analytical chemistry.
Applications and significance of pKa They are used whenever there is a need to fix the pH of a
A knowledge of pKa values is important for the solution at a particular value. Compared with an aqueous
quantitative treatment of systems involving acid–base solution, the pH of a buffer solution is relatively insensitive to
equilibria in solution. Many applications exist in the addition of a small amount of strong acid or strong base.
biochemistry; for example, the pKa values of proteins and The buffer capacity of a simple buffer solution is largest when
amino acid side chains are of major importance for the pH = pKa. In acid-base extraction, the efficiency of extraction
activity of enzymes and the stability of proteins.Protein of a compound into an organic phase, such as an ether, can be
pKa values cannot always be measured directly, but may optimised by adjusting the pH of the aqueous phase using an
be calculated using theoretical methods. Buffer solutions appropriate buffer. At the optimum pH, the concentration of
are used extensively to provide solutions at or near the the electrically neutral species is maximised; such a species is
physiological pH for the study of biochemical reactions, more soluble in organic solvents having a low dielectric
the design of these solutions depends on a knowledge of constant than it is in water. This technique is used for the
the pKa values of their components. Important buffer purification of weak acids and bases.
solutions include MOPS, which provides a solution with
Useful buffer mixtures
pH 7.2, and tricine which is used in gel electrophoresis.
Buffering is an essential part of acid base physiology
Components pH range
including acid-base homeostasis, and is key to
understanding disorders such as acid-base imbalance. The
HCl, Sodium citrate 1-5
isoelectric point of a given molecule is a function of its pK
values, so different molecules have different isoelectric Citric acid, Sodium citrate 2.5 - 5.6
points. This permits a technique called isoelectric
focussing, which is used for separation of proteins by 2-D Acetic acid, Sodium acetate 3.7 - 5.6
gel polyacrylamide gel electrophoresis.
Na2HPO4, NaH2PO4 6-9

Borax, Sodium hydroxide 9.2 - 11


Common buffer compounds used in biology
pKa
Common Buffer Mol.
at Full Compound Name
Name Range Weight
25°C
3{[tris(hydroxymethyl)methyl]amino}propanes
TAPS 8.43 7.7–9.1 243.3
ulfonic acid
Bicine 8.35 7.6–9.0 163.2 N,N-bis(2-hydroxyethyl)glycine
Tris 8.06 7.5–9.0 121.14 tris(hydroxymethyl)methylamine
Tricine 8.05 7.4–8.8 179.2 N-tris(hydroxymethyl)methylglycine
4-2-hydroxyethyl-1-piperazineethanesulfonic
HEPES 7.48 6.8–8.2 238.3
acid
2{[tris(hydroxymethyl)methyl]amino}ethanesul
TES 7.40 6.8–8.2 229.20
fonic acid
MOPS 7.20 6.5–7.9 209.3 3-(N-morpholino)propanesulfonic acid
PIPES 6.76 6.1–7.5 302.4 piperazine-N,N′-bis(2-ethanesulfonic acid)
Cacodylat
6.27 5.0–7.4 138.0 dimethylarsinic acid
e
SSC 7.0 6.5-7.5 189.1 saline sodium citrate
MES 6.15 5.5–6.7 195.2 2-(N-morpholino)ethanesulfonic acid
There are different systems of units such as MKS,
PHYSICAL VARIABLES CGS, SI, and FPS units. Units of one system can be
converted into units of another system. SI units is the
Scientists measure all kinds of stuff in the lab. While
officially accepted system and is widely in use. There
many observations are qualitative—what color, what
are two clusterings of metric units in science and
state, etc.—many observations are quantitative.
engineering. One cluster, based on the centimeter, the
Measuring the mass of a reactant, or the volume of a
gram, and the second, is called the CGS system. The
liquid, performing a titration, and other more
other, based on the meter, kilogram, and second, is
sophisticated measurements require careful
called the MKS system. Similarly, FPS system is the
determination of value, which must be recorded
old British system that uses foot, pound, and second as
along with the proper unit. Both the magnitude of
the basic units.
the number and the unit are essential for
communicating information to other chemists,
wishing to repeat an experiment. The magnitudes of SI Unit
these measurable observations are called the All systems of weights and measurements, metric and
physical variables. non-metric, are linked through a network of inter-
Variables are of two types, national agreements supporting the International
Substantial variables: These variables have a unit. System of Units. The International System is called
They are measured against a precise physical the SI, using the first two initials of its French name
standard. These standards are called units. E.g. mass, Le Système International d’Unités. The SI is maintai-
length, volume, time, viscosity, heat, ned by a small agency in Paris, the inter-national
temperature, etc. Bureau of Weights and Measures (BIPM, for Bureau
Natural variables: These are variables known as International des Poids et Mesures), and it is updated
dimensionless numbers or groups. They do not every few years by an international conference, the
require any units to express their measurement. General Conference on Weights and Measures
Examples for unitless variables are refractive index, (CGPM, for Conférence Générale des Poids et
specific gravity, specific viscosity, etc. Mesures), attended by representatives of all the
There are some physical phenomena that do not industrial countries and international scientific and
have any units, such as the Reynolds number. The engineering organizations.
Reynolds number (Re) is a dimensionless TABLE: Base Measurements and Base Units - SI
measurement in fluid mechanics used to
characterize the nature of flow of fluid through
tunnels and pipes.

DIMENSIONS AND UNITS


The physical variables that we use in physics and
chemistry can be classified into two categories—
fundamental quantities and derived quantities.
There are some physical variables which form the
basis of all measurements and quantities and are
known as fundamental quantities or dimensions or
base quantities. The units to express them are known Few units
as base units. The units to express them are also Ampere [A]
derived from base units. These units are called The ampere is the basic unit of electrical current. It is the
derived units and their dimensions are the current that produces a specified force between two
combination of fundamental quantities. parallel wires, which are one meter apart in a vacuum. It
Area = length × length is named after the French physicist Andre Ampere
Volume = length × length × length (1775-1836).
Velocity = distance/time kelvin [K]
The kelvin is the basic unit of temperature. It is
UNITS 1/273.16th of the thermodynamic temperature of the
Physical variables are measured against certain triple point of water. It is named after the Scottish
standards known as units. Base units are those used mathematician and physicist William Thomson 1st Lord
to express the dimensions or the fundamental Kelvin (1824-1907).
quantities, and the derived units are those derived
from the fundamental or base units.
Mole [mol]
Watt [W]
The mole is the basic unit of substance. It is the
The watt is used to measure power or the rate of doing
amount of substance that contains as many elementary
work. One watt is a power of 1 joule per second. It is
units as there are atoms in 0.012 kg of carbon-12.
named after the Scottish engineer James Watt (1736-
Candela [cd]
1819).
The candela is the basic unit of luminous intensity. It
is the intensity of a source of light of a specified
MEASUREMENT CONVENTIONS
frequency, which gives a specified amount of power
The magnitude of these variables should be expressed
in a given direction.
with correct conventions for further analysis and
Farad [F]
understanding. It indicates the conditions of measure-
The farad is the SI unit of the capacitance of an
ment and analysis and is essential mainly for comparing
electrical system, that is, its capacity to store
the magnitudes. For example, the magnitudes of the
electricity. It is a rather large unit as defined and is
substantial variables like density and pressure are
more often used as a microfarad. It is named after the
always expressed with respect to temperature. Density
English chemist and physicist Michael Faraday (1791-
is defined as mass per volume at a fixed temperature,
1867).
whereas the specific gravity is the ratio of density of a
Hertz [Hz]
material with that of water. Therefore, it is a dimension-
The hertz is the SI unit of the frequency of a periodic
less variable. Density of water at 4°C is exactly 1.000 g.
phenomenon. One hertz indicates that 1 cycle of the
cm–3, therefore we can say immediately that the
phenomenon occurs every second. For most work
density of ethanol is 0.789 g.cm–3.
much higher frequencies are needed such as the
kilohertz [kHz] and megahertz [MHz]. It is named
ERRORS IN DATA AND CALCULATIONS
after the German physicist Heinrich Rudolph Hertz
Measurements are prone to errors. Therefore, all
(1857-94).
techniques for data analysis must consider this error in
Joule [J]
measurements. Experimental errors while taking
The joule is the SI unit of work or energy. One joule is
measurements are sometimes unavoidable and may
the amount of work done when an applied force of 1
depend on accuracy. For example, consider the
newton moves through a distance of 1 meter in the
measurement of length. Measure the length of a table as
direction of the force. It is named after the English
5 meters. Here, we are actually comparing the length of
physicist James Prescott Joule (1818-89).
the table with that of a standard that is 1 meter long. In
Newton [N]
this comparison, there is always some uncertainty
The newton is the SI unit of force. One newton is the
regarding its accuracy. It depends on the accuracy of
force required to give a mass of 1 kilogram an
the scale that you have used for measuring the length. If
acceleration of 1 meter per second per second. It is
the length measured is between 5 and 6 and the scale
named after the English mathematician and physicist
that was used did not have any subdivisions of meters
Sir Isaac Newton (1642-1727).
marked on that, the measurement is not accurate. To get
Ohm [Ω]
a more accurate measurement of the length, use a scale
The ohm is the SI unit of resistance of an electrical
where the meter is subdivided into centimeters and the
conductor. Its symbol is the capital Greek letter
length of the table can be measured to the accuracy of
‘omega’. It is named after the German physicist Georg
centimeters, say 5 meters and 3 centimeters.
Simon Ohm (1789-1854).
Experimentally-determined quantities always have
Pascal [Pa]
errors to varying degrees. The reliability of the
The pascal is the SI unit of pressure. One pascal is the
conclusions drawn from this data must take
pressure generated by a force of 1 newton acting on an
experimental errors into considerations for calculations.
area of 1 square meter. It is a rather small unit as
Minimization of errors by adopting accurate
defined and is more often used as a kilopascal [kPa]. It
measurement scales, estimation of the errors and
is named after the French mathematician, physicist,
principles of error propagation in calculations are very
and philosopher Blaise Pascal (1623-62).
important in all sciences to prevent deceptive and
Volt [V]
confusing interpretation of facts.
The volt is the SI unit of electric potential. One volt is
the difference of potential between two points of an
ABSOLUTE AND RELATIVE UNCERTAINTY
electrical conductor when a current of 1 ampere
Experimental and measurement errors always create
flowing between those points dissipates a power of 1
uncertainty in the final data. This problem can be
watt. It is named after the Italian physicist Count
solved by introducing the rules of significant figures.
Alessandro Giuseppe Anastasio Volta (1745-1827).
In this method, we specify the range of error by
which each of the given values can be varied. Each
of the readings will be uncertain within this range of
error. This error value is known as absolute error.
The same error can be represented in terms of
percentage, and then it is called relative error.
For example, when representing the temperature of a
solution it will be 37 ± 3°C. Here, ± 3°C represents
the actual temperature range by which the reading
is uncertain or can be varied and this is known as the
absolute error. When the same error is represented as
a percentage it is known as relative error. 37 ± 3°C
can be represented as 37 ± 1.25%. Here, the error,
1.25 % is called relative error.

TYPES OF ERRORS
Experimental errors can be broadly classified into
two categories:
a. Systemic errors and
b. Random errors
When an error affects all measurements in the same
way it is called a systemic error. In most cases, the
cause of this error is known and introducing a
correction factor can minimize the error. For
example, a watch showing an error of + five minutes
(five minutes fast). In this case we can reduce five
minutes from the time shown by the clock to get the
correct time. A balance that shows an error of – 0.5
gm can be adjusted for that error effectively if the
fact is known. If an error occurs due to unknown
reasons it is called a random error or an accidental
error. This type of error can be detected by repeating
the experiments under the same conditions. If
different experimental values or results when
repeating the experiments without changing the
experimental conditions are found,
then there are random errors. These errors can be
quantified and minimized by applying methods of
statistical analysis.
The results or data of an experiment should be
reliable and reproducible. The term precision refers
to the reliability and reproducibility of results. It
also indicates the magnitude by which the data is
free from random errors. We also use the term
accuracy to refer to the quality of the data. When
there is a minimum of both systemic and random
errors or when it is almost zero and the results are
reproducible, then we refer to the data as accurate.
Liquid chromatography of biomolecules
Proteins, peptides, DNA, RNA, lipids, and organic
cofactors have various characteristics such as electric
charge, molecular weight, hydrophobicity, and surface
relief. Purification is usually achieved by using methods
that separate the biomolecules according to their
differences in these physical characteristics, such as ion
exchange, gel filtration, and affinity chromatography.
Ion-Exchange Chromatography of Proteins
In ion exchange chromatography, the stationary solid
phase commonly consists of a resin with covalently
attached anions or cations. Solute ions of the opposite
charge in the liquid, mobile phase are attracted to the
ions by electrostatic forces. Adsorbed sample
components are then eluted by application of a salt
gradient which will gradually desorb the sample
molecules in order of increasing electrostatic
interaction with the ions of the column (Figs. 1– 2).
Because of its excellent resolving power, ion
exchange chromatography is probably the most
important type of chromatographic methods in many
protein preparations.
The choice of ion exchange resin for the purification
of a protein largely depends on the isoelectric point,
pI, of the protein. At a pH value above the pI of a
Fig. 1 Example of ion exchange chromatography. (a)–
protein, it will have a negative net charge and adsorb
(c) Loading the column: mobile anions (or cations) are
to an anion exchanger. Below the pI, the protein will
held near cations (or anions) that are covalently attached
adsorb to a cation exchanger. For example, if the pI is
to the resin (stationary phase). (d)–(f) Elution of the
4 then in most cases it is advisable to choose a resin
column with a salt gradient: the salt ions weaken the
which binds to the protein at a pH > 4. Since at pH > 4
electrostatic interactions between sample ions and ions of
this protein is negatively charged, the resin has to be
the resin; sample molecules with different electrostatic
an anion ion exchanger, e.g., DEAE. One could also
properties are eluted at different salt concentrations,
use a pH < 4 and a cation exchanger, but many
typically between 0–2 M. (g) Interaction of sample
proteins are not stable or aggregate under these
molecules with ions attached to the resin: at a suitable
conditions. If, in contrast, the protein we want to
pH and low salt concentration, most of the three types of
purify has a pI = 10, it is positively charged at usually
biomolecules to be separated in this example reversibly
suitable conditions for protein ion exchange
bind to the ions of the stationary phase.
chromatography, i.e., at a pH around 7. Thus, in
general for this protein type we have to choose a
cation ion exchange resin, e.g., CM, which is
negatively charged at neutral pH.
The capacity of the resin strongly depends on the pH
and the pI of the proteins to be separated (Fig. 4;
Table), but also on the quality of the resin, the applied
pressure, and the number of runs of the column
(Fig.5). To improve the life of the resin, it should be Fig. 2 Two ion exchangers: diethyl-amino-ethyl (DEAE)
stored in a clean condition in the appropriate solvent and carboxy methyl (CM). The positive charge of DEAE
and not be used outside the specified pH range and attracts negatively charged biomolecules. CM is suitable
pressure limit. for purification of positively charged biomolecules
Fig. 3 Example for the salt concentration during
adsorption of a sample to an ion exchange column,
subsequent elution of the sample, and cleaning of the
column. Example of a purification protocol: First the
solution of biomolecules and impurities in buffer
contained in a syringe is loaded onto the column. The
biomolecules and some of the impurities bind to the
ions attached to the resin. Loading is completed and
non-binding molecules are partly rinsed through the
column with some further buffer. The next step is to
apply a salt gradient with a programmable pump which
mixes buffer with extra salt containing buffer. The
steep salt gradient at the beginning elutes most of the
weakly binding impurities. At a certain salt
concentration, the biomolecules to be purified elute
from the column. Elution is monitored with an
absorption detector at 280 nm wavelength and the
sample fraction collected. After each run the column is
cleaned with 1–2 M KCl. This removes most of the
strongly binding sample impurities

The experimental set-up (Fig. 6) often just consists of a


bottle with buffer, a bottle with buffer with salt, a
programmable FPLC or HPLC pump, the column, a
detector and recorder of absorption at 280 nm, or
occasionally at 220 nm, and a sample collector. If the
right conditions for protein preparation are unknown, a
Fig. 4 Charge properties of anion and cation pre-run is performed with a small fraction of the sample.
Attention should be paid not to overload the column in
exchangers. DEAE has a significant capacity at preparative runs since this can shift peak positions and
low and medium pH; CM is highly capacious at lead to substantial sample losses. In many cases of
high and medium pH modern high expression of recombinant proteins, it is
possible to obtain a protein with 99% purity with a single
ion exchange chromatographic step.
Fig. 6 Typical setup for chromatographic purification of proteins with ion exchange FPLC. The pump
mixes the salt gradient for sample elution after the sample was loaded, e.g., with a syringe.

Fig. 5 Change of the capacity of ion exchange


columns due to usage. High performance columns
operated at the appropriate pressure and pH can
last many 1000 runs
Gel Filtration Chromatography

Gel filtration chromatography (sometimes referred to as size


exclusion chromatography) separates biomolecules based on
differences in their molecular size. The process employs a gel
media suspended in an aqueous buffer solution which is
commonly packed into a chromatographic column. These
columns can vary in size from very small (for example, spin
columns of <1 mL bed volume for analytical separations) to very
large (for preparative scale applications). The gel media consists
of spherical porous particles of carefully controlled pore size
through which biomolecules diffuse to different extents based on
differences in their molecular sizes. Small molecules diffuse
freely into the pores and their movement through the column is
retarded, whereas large molecules are unable to enter the pores
and are therefore eluted earlier. Hence, molecules are separated
in order of decreasing molecular weight, with the largest
molecules eluting from the column first.
Applications for gel filtration chromatography typically fall into one
of two categories, either: (i) fractionation, or (ii) group separation.
In fractionation applications, molecules are separated according
to small differences in size, as would be required in a purification
or characterization protocol. A group separation separates all
high-molecular-weight molecules from all low-molecular weight
molecules effectively yielding two groups, as would be required
when exchanging buffer components or salts, or removing low-
molecular-weight contaminants from valuable higher molecular-
weight samples (proteins, oligonucleotides, plasmids, or Fig. 1. A. Gel filtration chromatography of a series of protein
polysaccharides). molecular mass standards on a pre-packed Superdex 200.
The main advantage for gel filtration chromatography over these Standards were 1. thyroglobulin (Mr 669,000), 2. ferritin (Mr
techniques is the ability to separate analytical to preparative 440,000), 3. human IgG (Mr 150,000), 4. human transferrin (Mr
amounts of material under native, non-denaturing conditions with 81,000), 5. ovalbumin (Mr 43,000), 6. myoglobin (Mr 17,600), 7.
samples recovered in a form suitable for direct downstream vitamin B12 (Mr 1,355). Conditions were: 50 mMsodium
processing (such as other chromatographic steps, or analysis in phosphate, 150 mM NaCl, pH 7.0, flow rate = 0.25 mL/min (19
biological assay systems). The instrumentation required for gel mL/cm2/h). (B) Diagrammatic representation of the measurement
filtration chromatography is also relatively inexpensive and readily of the void volume (V0), elution volume (Ve) and total volume (Vt)
obtainable. The main disadvantages of gel-filtration chromato- for a gel filtration column. V0 is the elution volume of molecules
graphy are a relative lack of resolution, an inability to analyze too large to enter the pores of the gel media, whereas Vt is the
more than one sample at any one time (unless multiple columns total volume of the column determined with a small molecule. Ve
are utilized), and samples are recovered in a more dilute form represents the elution volume of a molecule of intermediate
than the starting material (frequently ≥ 2–3-fold dilution). molecular mass. Determination of these parameters is best done
with standard (continued) proteins (see panel A) under optimal
conditions for flow rate and sample size. From these
measurements the coefficient Kav can be derived, Kav = (Ve–
V0)/(Vt–V0). (C) The selectivity curve for a particular gel filtration
media is a plot of Kav vs log molecular weight, and the data
shown here has been derived from panel A. Selectivity curves as
provided by the manufacturer are used to choose the gel filtration
media that best suits the application, whereas in the laboratory
they are useful for estimation of the molecular mass of an
unknown protein.
Stationary Phase Separation based on size and shape

The stationary phase consists of semi-permeable, porous Proteins are separated roughly according to their molecular
beads with a well-defined range of pore sizes. The semi- weight because this is the major contribution to molecular size.
permeable porous beads are crosslinked polymers. However, the shape will affect its apparent size in solution.
Degree of crosslinking is controlled carefully to yield different Hence, gel filtration is NOT recommended for separating proteins
pore sizes. The stationary phase is said to have a with only a small difference in molecular weight.
fractionation range (due to the different pore sizes), meaning
Determination of molecular weight
that molecules within that molecular weight range can be
separated. Porous material must swell up and imbibe/absorb Consider the separation of a mixture of i. glutamate dehydrogenase
the liquid phase. The created solvent-filled ‘sponge’ allows (MW 290,000),lactate dehydrogenase (MW 140,000), serum albumin
diffusion of molecules. Therefore, stationary phase may be (MW 67,000), ovalbumin (MW 43,000), and cytochrome c (MW
hydrophilic to imbibe aqueous media, or lipophilic to imbibe 12,400) on a gel filtration column: fractionation range 15,000 -
non-polar organic solvents. 150,000).
When the protein mixture is applied to the column, glutamate
Mobile Phase
dehydrogenase would elute first because it is above the upper
The mobile phase contains a mixture of solutes. Small solutes fractionation limit. Therefore it is totally excluded from the inside of
will diffuse in and out of the pores (obeying Fick’s law). Their the porous stationary phase and would elute with the void volume
path through the column is longer and The elution time will be (V0). Proteins larger than the exclusion range of the resin are unable
longer to enter the pores and pass quickly through the column in the spaces
Chromatogram between the resin. This is known as the void volume of the column.
Cytochrome c is below the lower fractionation limit and would be
completely included, eluting last. The other proteins would be
partially included and elute in order of decreasing molecular weight.

• The separations can be described by this equation


Vr = Vo + KVi
• Vr is the retention volume of the protein, V0, void volume, is
the volume of mobile phase between the beads of the stationary
The extent of retention depends on the size of the included phase inside the column, Vi , included volume, is the volume
of mobile phase inside the porous beads and K is the partition
molecules relative to the pores. Smallest molecules will enter all
coefficient (the extent to which the protein can penetrate the
pores. Intermediate molecules, due to velocity of mobile phase, pores in the stationary phase, with values ranging between 0
will not be able to diffuse into the pores that they may fit, thus will and 1). In the mixture of proteins given, the
be retained less effectively. Initial peak contains the totally • partition coefficient, K=0 for glutamate dehydrogenase (totally
EXCLUDED solutes. Final peak contains the totally included excluded), K = 1 for cytochrome c (totally included), 0 > K > 1
solutes. for the other proteins, which are within the fractionation range
Advantages of Gel Filtration for the column.

1. Can handle biomolecules that are sensitive to changes in


pH, concentration of metal ions or harsh environmental
conditions.
2. Separations can be performed in the presence of essential
ions, detergents, urea, guanidine hydrochloride, at high or
low ionic strength, at 37 °C or in the cold room according to
the requirements of the experiment.
Applications
Affinity chromatography
Desalting
Desalting is necessary for purification of biochemicals. Gel with Affinity chromatography is a method enabling purification
low exclusion limit MW 1000-2000 is used. Short column and of biomolecules and other macromolecules with respect to
individual structure or function. It utilizes the highly
high flow rate can be used because of the vast difference in specific binding of the macromolecule to a second
size of solutes and contaminants. Macromolecules will be molecule which is attached to the stationary phase. The
eluted with little dilution and salts retained on the column. principle of operation is as follows: (a) the sample is
injected into the column; (b) buffer is rinsed through the
Concentration of Dilute Solutions
column, so that sample molecules with no affinity to the
Solution is mixed with a small quantity of dry gel that will absorb stationary phase are eluted from the column, but sample
10 to 20 times its weight in water. Some salts and small molecules with a high affinity for the stationary phase are
retained in the column; (c) the retained sample molecules
molecules are taken up also. Final macromolecules in a
are eluted from the column by buffer with a high salt
solution of almost unchanged pH and ionic strength but concentration or a different pH or a different solvent
significantly decreased volume. composition (Fig. below). The preparation of the protein
can be performed by using a number of protein tags. The
Molecular Weight Determination
tags should not cause artificial interactions and should not
Size is approximately proportional to molecular weight, M. alter the conformation of the tagged protein. Very common
are poly-histidine tags that are attached to the protein by
Elution time, VE, can be expressed by: genetic engineering (Fig. below). The tag typically consists
VE = a + b log M of 8–12 histidine residues. It binds to nickel compounds at
the surface of the chromatography beads. Fig. illustrates a
a and b are constants and dependent on the mobile and somewhat different variant of affinity chromatography in
stationary phases. which misfolded proteins are continuously refolded by
chaperones and eluted with buffer.
Protein purification
Proteins are separated according to the difference in their
molecular masses as described previously.
Procedure
1. Column must first be equilibrated with desired buffer.
2. pass several column volumes of buffer through column.
3. important step because the equilibration buffer is the buffer in
which the protein sample will elute.
4. Next, sample is loaded onto the column and allowed to enter
the resin.
5. Then more equilibration buffer is passed through the column
to separate sample and elute it from column.
6. Fractions are collected as the sample elutes from the column.
7. Larger proteins elute in the early fractions and smaller
proteins elute in subsequent fractions.
Types of Column
exclusion range for some common gel filtration chromatography Fig. Purification of antibodies with affinity chromatography:
media. The antigen is chemically bound to the beads of the column
Sephadex G-50 1-30 kDa and the mixture of antibodies is rinsed through the column.
Antibodies with high binding constants bind to the antigen and
Sephadex G-100 4-150 kDa are eluted later with a buffer with a high salt concentration
Sephadex G-200 5-600 kDa
Sephadex is a trademark of Pharmacia.
Immobilized Metal Ion Affinity Chromatography
Immobilized metal ion affinity chromatography (IMAC) of proteins
has the ability to differentiate a single histidine residue on the
surface of a protein, it can bind proteins with dissociation
constants of 10–5 to 10–7, and has had wide application in the field
of molecular biology for the rapid purification of recombinant
proteins. Since the first set of work was published describing the
Fig. Attachment of a protein to a bead of an affinity
immobilization of metal ions using a chelating agent covalently
column with a histidine tag. About 10 histidine
attached to a stationary support, to purify proteins, there have
residues were attached to the protein by genetic
been several modifications and adaptations of this technique over
engineering, e.g., by polymerase chain reaction (PCR)
the years. The traditional approach describes the use of
mutagenesis. The histidine residues strongly bind to
immobilized metal ions and in particular borderline Lewis metal
the bead made from a nickel chelate resin
ions such as Cu2+, Ni2+, and Zn2+, to purify proteins on the basis
of their histidine content. This application was further extended to
incorporating a hexa-histidine tail onto the protein and using the
tail as a purification handle in combination with a highly selective
stereochemistry in the form of a Ni-nitrilotriacetic acid (Ni-NTA)
complex for binding the purification handle. Yet another mode of
interaction involved in the IMAC of proteins was the mixed mode
interactions involving aspartate and/or glutamate surface residues
on proteins along with electrostatic interactions, again
independent of histidine interactions.
The traditional use of IMAC for proteins has been to select
proteins on the basis of their histidine content. The approach uses
a chelating agent immobilized on a stationary surface to capture a
metal ion and form an immobilized metal chelate complex (IMCC)
(see Fig). Generally, Cu2+, Ni2+, and Zn2+, have been used in this
mode but other metal ions such as Co2+, Cd2+, Fe2+, and Mn2+
have also been examined as the metal ions of choice. Histidine
selection by the IMCC exploits the preference of borderline.
Lewis metals with a pKa of 6.0, histidine will be able to donate
electrons effectively.

Fig. Refolding of expensive, poorly folding proteins:


Folding chaperones, also known as chaperonins, are
attached to the beads and the unfolded or misfolded
protein is rinsed through the column. The chaperone
interacts with the sample protein and catalyses its
folding into the correct conformation
Separation by affinity chromatography is based on a
biological property of macromolecules rather than on
a physical property. It is a highly sensitive technique
and, in principle, can give absolutely pure sample in a
single step. The technique requires that the
macromolecule be able to bind to a specific ligand
attached to an insoluble matrix under certain
conditions, and then detach itself under certain other
conditions. In other words the binding should be
specific and reversible. Fig: Histidine residues binding to an immobilized metal ion
affinity adsorbent.
Thin layer chromatography
In this case, the stationary phase is applied to a
glass, plastic or metal foil plate as a uniform, thin
layer and the sample is applied at the top edge of the
layer using a micro-pipette or syringe. This
technique can be used for partition chromatography,
adsorption chromatography and exclusion chromat-
ography. The advantage of this method is that a
large number of samples can be studied simulta-
neously. Also, it is quick and simple.

Paper chromatography Adsorption chromatography


This technique separates the sample into its components
In this technique, the cellulose fibres of a sheet of
based on the degree of their adsorption by the adsorbent and
special chromatographic paper act as a support or
on their solubility in the solvent used. Silicic acid (silica
the stationary phase (Figure below). There are
gel), aluminum oxide, calcium carbonate and cellulose may
generally two methods that are employed in paper
be used as the stationary phase in this method. The optimum
chromatography – the ascending method, and the
combination of adsorbent and eluting solvent will depend
descending method. In both of them the solvent is
upon the type of sample separation under consideration. For
placed at the bottom of a jar or tank so that the
example, hydroxyapatite (calcium phosphate) is used for
chamber is saturated with solvent vapor. The
DNA separation as this adsorbent can bind double stranded
chromatographic paper is held vertically inside the
DNA but not single stranded DNA or RNA. Normally any
tank. In the ascending method, the lower end of the
organic solvent can be chosen as the mobile phase, e.g.
paper dips into the solvent and the sample spot is
hexane, heptane, proponal, butanol etc.
applied just above the surface of the solvent. As the
solvent moves up the sheet of paper by capillary
action, the sample constituents move along with it Partition chromatography
and get separated. In the descending method, the There are two types of partition chromatography. (a) Liquid
upper end of the paper, where sample is applied, is -liquid chromatography: In this type of partition
held in a trough of solvent at the top of the tank, and chromatography, the material separation is achieved based
the rest of the paper is allowed to hang down, on its differing solubilities in two (immiscible) liquid
without touching the solvent at the bottom of the phases. The sample is dissolved in a relatively non-polar
tank. Again by capillary action (and gravity), the solvent and is passed through a column that contains
solvent moves down the sheet of paper, taking the immobilized water or some other polar solvent in a
sample along with it and separating the components. supporting matrix such as silica gel. When the sample
The components are detected by spraying the paper moves through the column it gets partitioned between the
with specific colouring reagents, for example, mobile phase and the immobile, polar phase. In the reverse
ninhydrin, which binds only to amino acids and phase liquid-liquid chromatographic method, the stationary
proteins. Sometimes fluorescent dyes are used. For phase is a non-polar substance like liquid paraffin supported
example, ethidium bromide is used with DNA by the matrix. High Performance Liquid Chromato-
samples. When the paper is subsequently examined graphy (HPLC) is a liquid-liquid partition chromatographic
under ultraviolet light, the position of the technique. It is widely used for the separation of biological
fluorescent or ultraviolet absorbing spots can be compounds either as a normal phase method, or as a reverse
seen. The corresponding compounds are identified phase method. In the latter, it is useful for separating polar
on the basis of their Rf values where compounds. (b) Counter current chromatography: This is
also partition chromatography with this major difference,
that there is no supporting matrix. The sample is
The value is the relative displacement of solute with equilibrated between two immiscible solvents (e.g.
respect to the solvent front and is a constant for a ethylacetate and water) in a test tube. The partition
given compound under standard conditions. Thus by coefficient is defined as:
looking up a table of values, it is possible to identify
the compound after separation.
After equilibration the upper phase is withdrawn and Chromatofocusing
transferred to a fresh volume of lower phase solvent
Chromatofocusing is a protein-separation technique that
in another tube. The original lower phase is added to
was introduced by Sluyterman and his colleagues
a fresh volume of upper phase solvent, and so on.
between 197’7 and 1981. Chromatofocusing combines
The process is repeated several times, yielding
the advantage of high-capacity ion exchange
several test tubes, half of them finally containing the
procedures with the high resolution of isoelectric
upper phase solvent, and half containing the lower
focusing into a single chromatographic focusing
phase solvent. A solute which is more soluble in
procedure. During chromatofocusing, a weak ion-
upper phase (K > 1) will concentrate in tubes
exchange column of suitable buffering capacity is
containing the upper phase solvent. If the solubility
equilibrated with a buffer that defines the upper pH of
is greater for lower phase (K < 1) then it will
the separation pH gradient to follow. A second
concentrate in tubes of the lower phase solvent.
“focusing” buffer is then applied to elute bound
Counter current chromatography is successfully used
proteins, roughly in order of their isoelectric (PI) points,
for cell organelle fractionation.
The pH of the focusing buffer is adjusted to a pH that
defines the lower limit of the pH gradient. The pH
Gas liquid chromatography (GLC) gradient is formed internally during isocratic elution
The two phases used are a liquid and a gas. The with a single focusing buffer; no external gradient
method is very sensitive and reproducible. The forming device is required. The pH gradient is formed
partition coefficient of a volatile sample between the as the eluting buffer (i.e., focusing buffer) titrates the
liquid and the gas phases, as it is carried through the buffering groups on the ion exchanger. Peak widths in
column by the carrier gas, may be largely different the range of 0.05 pH unit and samples containing
from unity. This feature is exploited to separate or several hundred milligrams of protein can be processed
purify the compound. The stationary or immobile in a single step. Chromatofocusing is therefore a
phase is generally nonvolatile and thermally stable at powerful analytical probe of protein surface charge, as
the temperature of the experiment. Usually organic well as an effective preparative technique for protein
compounds with high boiling point are coated on a isolation. The application of chromatofocusing to silica-
base to form the stationary phase. The base is based stationary phases for use in a high-performance
generally made up of celite (diatomaceous silica). mode has extended the utility of this technique.
The sample to be separated is packed in a narrow,
coiled, steel or glass tube and placed in an oven at an
elevated temperature. An inert gas like argon,
nitrogen or helium is passed through the tube. The
sample is volatilised and is carried away by the
mobile gas phase. It is passed over the solid phase
packed into another column, and as it does so, the
sample distributes itself between the gas and solid
phases.
Electrophoresis Moving boundary electrophoresis
Important biological molecules like peptides, proteins, In this method, the sample is dissolved in a buffer and
carbohydrates and nucleic acids have ionizable chemical placed in a cell. An additional quantity of the buffer is
groups and hence, under suitable conditions, exist in layered on top of it. When the electric field is applied,
solution as charged species. Even nonpolar molecules closely related molecules will tend to move as a band
can be made to exist as weakly charged species by since their charges are similar, and boundaries are
preparing phosphate, borate or similar derivatives. formed between groups of molecules with different
Molecules with similar charge may have different electrophoretic mobility. The separations can be seen
charge/mass ratios since their molecular weights could by direct optical observations. This method suffers
be different. Molecules with differing charges or from many experimental difficulties such as errors
differing charge/mass ratios would migrate at different due to convection. Hence it is not very useful as a
rates in an electric field. The separation of molecules by preparative technique.
utilizing these differences in rates of migration is known
as electrophoresis. If a molecule has a net charge q, the Zone electrophoresis
application of an electric field E will result in a force: In this method the mixture to be analyzed is applied as
a small spot or very thin band to the supporting
F= qE medium. When electric field is applied the molecules
move as distinct bands or zones. The molecules can be
This force will accelerate the particle in a fluid till a identified by staining, UV absorption etc. There are
steady state is reached when the frictional force is equal several different types of zone electrophoresis. We
and opposite to the applied force. If v is the steady state treat some of them below.
velocity then,
Fv = qE Low voltage electrophoresis
where f is the frictional force. Therefore The electrophoresis unit (see Figure below) consists
v= qE/F of a power pack, which supplies DC current across
For a spherical molecule with radius a and charge Ze two electrodes, buffer reservoirs, a support, and a
(where e is the charge on electron and Z the atomic transparent insulating cover. The low voltage power
number), pack can either give a constant voltage or a constant
V= Ze E/(6πηα) current and has an output of 0 to 500 V and 0 to 150
since q = Ze and where is the viscosity of the solution. mA. Either stainless steel or platinum electrodes are
The electrophoretic mobility u is defined as the velocity used. The supporting medium, which can be paper or
per unit electric field. cellulose acetate, must first be saturated with buffer
u= v/E and held on a sheet of insulating material like Perspex.
However, due to the presence of counter ions in any The sample is then applied as a small spot using a
aqueous solution, this equation does not completely micropipette. The location of the spot depends on the
account for the electrostatic mobility. These ions can nature of the molecular mixture present in the sample.
neutralize some of the charges on the macromolecule if For example, if there are molecules with opposite
they bind tightly to it. Alternatively unbound or loosely charges, then they will separate and move towards
bound ions can modify the ionic strength of the solution opposite electrodes and hence the sample must be
and thus alter the electric field that is felt by the applied at the centre. The two reservoirs on either side
polymer. In such a situation the electrophoretic mobility of the medium are isolated from the electrodes so that
for a spherical particle is modified by a factor K, where any pH change there does not affect the buffer. After
K is a function of the screening parameter which has application of the sample, power is switched on for
dimensions of L-1 (inverse of length). Therefore, the required amount of time. At the end of the
u= (ZeK)/(6πηα) experiment, the migration of the different constituents
Thus mobility will increase with increasing charge on of the mixture may be analyzed. The experiment may
the macromolecule. be performed in different ways. For example, instead
Electrophoresis procedures can be generally divided into of paper or cellulose acetate, thin layers of silica,
three categories: (1) Moving boundary electrophoresis. alumina or cellulose may be applied on to a glass plate
(2) Zone electrophoresis. (3) Continuous flow and used as the supporting medium. This technique is
electrophoresis. similar to TLC (thin layer chromatography) and is
known as thin layer electrophoresis (TLE).
High voltage electrophoresis
A disadvantage with low voltage paper electro- Cross-linker
phoresis is that diffusion processes may influence Most protocols use acrylamide and the cross-linker
the migration of the molecules so that it is not bisacrylamide (bis) for the gel matrix. TEMED
strictly according to the charges on them. One way (N,N,N¢,N¢-tetramethylethylenediamine) and ammonium
to overcome this is to resort to high voltages, of the persulfate are used to catalyze the polymerization of the
order of 10,000 volts. Such a voltage can produce acrylamide and bis. TEMED, a base, interacts with
potential gradients up to 200 volts/cm. If the ammonium persulfate at neutral to basic pH to produce free
separation between the electrodes is d metres and if radicals. The free radical form of ammonium persulfate
Vis the potential difference in volts then the initiates the polymerization reaction via the addition of a
potential gradient is V/d volts/metre. The force on vinyl group. Another crosslinker, BAC (bis-acrylylcyst-
the charged molecule is then Vq/d Newtons, where q amine) can be dissolved by β-mercaptoethanol. It is useful
is the charge in coulombs. The migration of the for nucleic acid electrophoresis. One other crosslinker,
molecules is caused by this force and is proportional piperazine diacrylamide (PDA), can replace bis-acrylamide
to it, and an increase in the potential gradient leads in isoelectric focusing (classical tube gel or flatbed gel)
to an increased rate of migration. The distance experiments.
migrated by the ions will depend on the voltage as
well as the amount of time for which it is applied.
This technique results in higher resolution and faster
separation. But because of the high voltage, it
generates considerable heat and the apparatus
requires cooling.
Initiator or Catalyt
Gel electrophoresis
TEEMED and APS
When applied to the separation of a mixture of
proteins, simple zone electrophoresis has limited
resolving power, due to the similarity in mobility of
the components. The molecular sieving property of
the gel helps in separating molecules that have
similar charge properties but differ in size and shape.
The gels can be run as horizontal slabs or as vertical
slabs. Gels are prepared in glass or Perspex
containers.

Figure: Polymerization of acrylamide


Will Your SDS Gel Accurately Indicate the Molecular
How Do You Control Pore Size?
Weight of Your Proteins?
Pore size is most efficiently and predictably
Estimation of the molecular weight of the protein of
regulated by manipulating the concentration of
interest, accurate to within 2000 to 5000 daltons, requires
acrylamide in the gel. Pore size will change with the
the protein band(s) to run within the middle two-thirds of
amount of crosslinker, but the effect is minimal and
the gel. This is illustrated in the graph of the log of the
less predictable. Note the greater impact of
molecular weight of a set of standard proteins vs. the
acrylamide concentration on pore size, especially at
relative mobility of each one . Note that the proteins with
the levels of crosslinker usually present in gels.
a relative mobility below 0.3 or above 0.7 fall off the
Practical experience with various ratios of
linear portion of the curve. Thus the most accurate
acrylamide : bis have shown that it is best to change
molecular weight values are obtained when the relative
pore size by changing the acrylamide concentration.
mobility of the protein of interest is between 0.3 and 0.7.
This means that if your protein doesn’t enter the gel very
Why Should You Overlay the Gel?
well, you must change the gel %T before you can get a
An overlay is essential for adequate resolution. If
good molecular weight value. The sample may require a
you do not overlay, the bands will have the shape of
different (better) solubilization procedure also.
a meniscus. Two closely spaced bands will overlap;
the middle of the top band will extend down between
the front and back of the bottom band. Overlaying
the gel during polymerization will prevent this
problem. Common overlays are best quality water,
the buffer used in the gel at a 1x dilution, and water-
saturated t-butanol. The choice is a matter of
personal preference.

Reproducible Polymerization:
Reproducible polymerization is one of the most
important ways to ensure that your samples migrate
as sharp, thin bands to the same location in the gel
every time. Attention to polymerization will also
help keep the background of your stained gels low. Figure : Log of the molecular weight (in daltons) of a
Acrylamide polymerization is affected by the protein versus the relative mobility. Reproduced with
amount of oxygen gas dissolved in the solution, the permission from Bio-Rad Laboratories.
concentrations and condition of the catalysts, the
temperatures and pH of the stock solutions, and the Straight % Gel or a Gradient Gel?
purity of the gel components. If you want to resolve proteins that are within a few
thousand daltons of each other in molecular weight,
Gel Percentage vs. Catalyst Concentration
then use a straight percent gel (the same concentration
of acrylamide throughout the gel). To get baseline
resolution for such proteins, that is, to get clear,
unstained space between bands, you may need to use a
longer gel. Mini gels have 6 to 8 cm resolution space.
WHICH GEL SHOULD YOU USE? SDS-PAGE, Large gels have 12 to 20cm space. The closer the
NATIVE PAGE OR I SOELECTRIC FOCUSING? bands are in molecular weight, the longer the gel must
The strategy you choose depends on your goal, of be.
course. If you want to determine the molecular weight A gradient gel is used to resolve a larger molecular
of your protein, use SDS-PAGE. If you want to weight range than a straight percent gel. A 10% gel
measure the isoelectric point of your protein, choose resolves proteins from 15 to 100kDa, while a 4% to
isoelectric focusing (IEF). For proteomics work, use 2- 20% gradient gel resolves proteins from 6 to 300kDa,
D electrophoresis (IEF followed by SDS-PAGE). although the restriction about good molecular weight
Native PAGE is used to assay enzyme activity, or other determination discussed above still holds. Accurate
biological activity, for example, during a purification molecular weights can be determined with gradient
procedure. Each kind of protein PAGE has issues to gels
consider, and these issues are addressed in the next
section.
Isoelectric Focusing: Location of Band of Interest
Isoelectric focusing (IEF) measures the Sample proteins move in a native gel as a function of
isoelectric point, or pI, of a protein. The main their charge as well as their mass and conformation,
problem for IEF is sample solubility, seen as and because of this, the location of the protein band of
interest may be difficult to determine. For instance, in
streaking or in-lane background on the stained
some buffer systems, BSA,at 64kDa, will move in
IEF gel, or horizontal streaking on a 2-D gel. front of soybean trypsin inhibitor, at 17kDa.The easiest
Sample solubilization should be way to detect the protein of interest is to determine
optimized for each new sample. At present its location by Western blotting. Alternatively, the
there are two kinds of IEF gels in use: gels protein’s location can be monitored by enzyme activity
formed with carrier ampholytes, and gels or bioassay, which usually requires elution from the
formed with acrylamido buffers, known as gel.
IPG gels (immobilized pH gradient gels).
How Can You Be Sure That Your Proteins Have
Sufficient Negative Charge to Migrate Well into a
Native PAGE Gel?
To determine this, it is useful to have some idea of the
pI of the protein of interest. The pH of the buffer
should be at least 2 pH units more basic than the pI of
the protein of interest. An alternative is to use an acidic
buffer system, and reverse the polarity of the
electrodes. This works well for very basic proteins.

Buffer Systems for Native PAGE


Buffer systems for native PAGE are either continuous
or discontinuous. Discontinuous buffer systems focus
the protein bands into thin fine lines in the stacking gel,
and these systems are preferred because they provide
superior resolution and sample volumes can be larger
and more dilute. In a discontinuous buffer system, the
buffers in the separating gel and stacking gel, and the
upper and lower tank buffers, may all be different in
concentration, molecular species, and pH. The reader
should initially try the standard Laemmli SDS-PAGE
buffer system without the SDS and reducing agent.
That buffer system is relatively basic, so most proteins
will be negatively charged and run toward the anode.
CRITICAL FOR SUCCESSFUL NATIVE For protein gels, the choice between continuous and
PAGE discontinuous buffer systems is usually made on the
basis of what works, and the pI of the protein(s) of
Sample Solubility interest.
Native PAGE is performed under conditions that don’t
denature proteins or reduce their sulfhydryl groups. POWER SUPPLY
Solubilizing samples for native PAGE is especially Macromolecules move through a polyacrylamide or
challenging because most nondenaturing detergents do not agarose gel because they carry a charge at the pH of
solubilize complex samples well, and the unsolubilized the buffer used in the system, and the voltage potential
proteins stick on the gel origin and bleed in, causing in- put across the cell by the power supply drives them
lane background. through the gel. This is the effect of the main voltage
potential, set by the power supply.
Constant Current or Constant Voltage—When and Why Are Sequencing Gels Electrophoresed under
Why? Constant Power?
The choice of constant current or constant voltage Sequencing gels are run under constant voltage or
depends on the buffer system, and especially on the constantpower, at a temperature between 50 and 55°C.
size of the gel. Historically constant voltage was used If constant voltage is used, then the voltage must be
because constant current power supplies were not changed during the run, after the desired temperature is
available. However, currently available programmable reached. If constant power is used, the power can be
power supplies, with constant voltage, constant current, set, and the voltage and current will adjust as the run
or constant power options, permit any power protocol proceeds, maintaining the elevated temperature
to be used as needed. Generally speaking, constant required for good band resolution. Elevated temperature
current provides better resolution because the heat in and the urea in the sequencing gel maintain the DNA in
the cell can be controlled more precisely (The higher a denatured condition.
the current, the higher the heat, and the poorer is the
resolution, due to diffusion of the bands.) However, IMPROVING RESOLUTION AND CLARITY
constant current runs will take longer than constant OF PROTEIN GELS
voltage runs (Table below).
How Can You Generate Reproducible Gels with
Perfect Bands Every Time?
High-quality, reproducible results are generated by
using pure, electrophoresis grade chemicals and
electrophoresis grade water, by preparing solutions the
same way every time and with exact measurement of
volumes, by correctly polymerizing your gels the same
way every time as discussed above, and by preparing
the samples so that they enter the gel completely,
without contaminating components that can degrade the
resolution. The most important factors for good band
resolution and clarity are correct sample preparation
and the amount of protein loaded onto the gel, and they
are discussed in greater detail below. Finally, the
detection procedure must be followed carefully, with
attention to detail and elapsed time.

What Procedures and Strategies Should Be Used to


Optimize Protein Sample Preparation?
Consider the cellular location of your protein of
interest, and attempt to eliminate contaminating
materials at the earliest stages of the purification. If it is
Why Are Nucleic Acids Almost Always Separated via a nuclear binding protein, first isolate the nuclei from
Constant Voltage? your sample, usually with differential centrifugation,
Nucleic acids are usually separated with a continuous and then isolate the proteins from the nuclei. If it is a
buffer system (the same buffer everywhere). Under these mitochondrial protein, use differential centrifugation to
conditions, the runs take the same time with constant isolate mitochondria (spin the cell lysate at 3000 x g to
voltage as with any other parameter held constant, and remove nuclei, then at 10,000 x g to bring down
the resolution is not improved using another parameter as mitochondria). If the protein is membrane bound, use a
constant. This is usually true for both agarose and PAGE step gradient of sucrose or other centrifugation medium
The use of continuous buffers in nucleic acid electro- to isolate the specific membrane of interest. For soluble
phoresis makes the gels easy to pour and to run. As with proteins, spin the cell lysate at 100,000 x g to remove
protein separation, small sample sizes must be utilized all cellular membranes and use the supernatant. Note
within continuous buffer systems, particularly when that nucleic acids are very sticky; they can cause
using vertical systems, to prevent bands from proteins to aggregate together with a loss of
overlapping. electrophoretic resolution. If you have smearing in your
sample, add 1mg/ml of DNase and RNase to remove
the nucleic acids.
Is the Problem Caused by the Sample or the
Some proteins are very difficult to solublize for electro-
Sample Buffer?
phoresis. Urea can be used, from 2 to 8M or 9.5M.
Sometimes it is difficult to determine whether the
Thiourea can be used at up to 2M; it greatly enhances
problem is in the sample or the sample buffer. Run the
solubility but cannot be used at higher concentration.
standard both with and without the sample buffer to
This is because above 2M, the urea, thiourea, or
determine this. It is best to prepare the sample buffer
detergent may precipitate out. The total urea concen-
without reducing agent—dithiothreitol (DTT), beta-
tration (urea + thiourea) cannot be above approximately
mercaptoethanol (BME), or dithioerythritol (DTE)—
7.0 M if thiourea is used with a bis gel due to these
freeze it into aliquots, and add the reducing agent to
solubility constraints.
the aliquot before use. All these reducing agents
evaporate readily from aqueous solution. Adding the
AGAROSE ELECTROPHORESIS
reducing agent fresh for each use means the reducing
agent will always be fresh and in full strength. Buffer
What Is Agarose?
components may separate out during freezing,
Agarose, an extract of seaweed, is a polymer of
especially urea, glycerol, and detergents. Aliquots of
galactose. The polymer is 1,3-linked (beta)-d-galacto-
sample buffer must be mixed thoroughly after
pyranose and 1,4-linked 3,6-anhydro-(alpha)-l-galacto-
thawing, to make sure the buffer is a homogeneous
pyranose. The primary applications are electrophoresis
solution.
of nucleic acids, electrophoresis of very large proteins,
and immunoelectrophoresis.
How Do You Choose a Detergent for IEF or Native
PAGE?
What Is Electroendosmosis (-Mr or EEO)?
Triton X-100 is often used to keep proteins soluble
-Mr is a measure of the amount of electroendosmosis
during IEF or native PAGE, but it may solubilize only
that occurs during electrophoresis with a particular
70% of the protein in a cell. SDS is the best
grade of agarose. Electroendoosmosis is the mass
solubilizer, but it cannot be used for IEF because it
movement of water toward the cathode, against the
imparts a negative charge to the proteins. During the
movement of the macromolecules, which is usually
IEF, it is stripped off the proteins by the voltage
toward the anode. High -Mr means high
potential, and the formerly soluble proteins precipitate
electroendosmosis. The mass flow of water toward the
in the IEF gel, resulting in a broad smear. Of course,
cathode is caused by fixed negative charges in the
SDS cannot be used in native PAGE because it
agarose gel (sulfate and carboxyl groups on the
denatures proteins very effectively. Some authors
agarose). Depending on the application,
state that SDS may be used in combination with other
electroendosmosis causes loss of resolution, or it can
detergents at 0.1% or less. It may help solubilize some
enable certain kinds of separations to occur, for
proteins when used this way. However, this is not
instance, during counter immunoelectrophoresis.
recommended, as the protein loads must remain low,
Applications for agarose preparations of different -Mr
and other problems may result.
values are shown in Table here.
Many non-ionic or zwitterionic detergents can be used
for IEF or native PAGE to keep proteins soluble. Agarose Preparations of Different -Mr Values
CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-
1-propanesulfonate) is most often used, as it is a very
good solubilizer, and is non-denaturing. It should be
used from 0.1% up to 4.0%. Another very effective
solubilizer is SB 3-10 (decyldimethylammonio-
propanesulfonate), but it is denaturing. Other
detergents, designed especially for IEF on IPG gels,
have recently been designed and used successfully.
The minimum detergent concentration for effective
solubilization must be determined for each sample.

What Other Additives Can Be Used to Enhance


Protein Solubility?
What Causes Nucleic Acids to Migrate at Unexpected What Causes Fuzzy Bands?
Migration Rates? 1.The sample might have been degraded by
Supercoiled DNA is so twisted about itself that it has a endogenous Dnase
smaller Stoke’s radius (hydrated radius), and moves faster 2.longer run at higher temperatures
than some smaller DNA fragments. If supercoiled DNA is 3.Samples loaded too high in the well (overloading)
nicked, it will unwind or start to unwind during the electro- 4.Poor-quality agarose
phoresis, and become entangled in the agarose. As this 5.Contaminated buffers (Should be autoclaved)
occurs, the DNA slows down its migration, and produces
unpredictable migration rates.

ELUTION OF NUCLEIC ACIDS AND PROTEINS FROM GELS


Table here summarizes the features, benefits and limitations of different elution strategies. DNA purification and elution.
DETECTION
Will the Choice of Stain Affect a
What Should You Consider before Selecting a Stain?
Downstream Application?
There are several factors to consider before selecting a stain, primary
This is an important question. Colloidal
among them the sensitivity needed. Table given below provide a
Coomassie and Sypro® Ruby can be used
general guide to stain sensitivity, and mention other considerations.
on 2-D gels when mass spectrometry is the
Common Protein Stains detection procedure. Certain silver stains
can also be used to stain samples for mass
spec analysis because of impro-vements in
the sensitivity of mass spectro-meters.
Sypro Red covers three orders of
magnitude, Coomassie covers two, and
silver stains provide coverage over one
magnitude. Not all silver stains give good
mass spectrometry results and those which
are used are not as good as Coomassie or
Sypro Ruby. For amino acid sequencing,
the gel is usually blotted to PVDF, stained
for the protein of interest, and then
sequenced. Immuno-detection or other
more sensitive methods can be used, but
usually the sequencing requires at least
1mg of protein. For this reason we suggest
that you stain your blot with Coomassie.
This does not interfere with sequencing.
Note that if you want to blot your gel after
staining, only rever-sible stains such as
copper stain and zinc stain can be used
with good success. If you stain your gel
with Coomassie or silver, the proteins are
fixed in the gel and are very difficult to
transfer to a membrane. Only copper or
zinc stains are recommended before
blotting a gel for immune detection.

How Much Time Is Required for the


Common Nuclic Acid Stains Various Stains?
The speed of staining is quite variable
depending on the quality of water, the
temperature, and how closely the staining
steps are timed. Gels stained with
Coomassie can be left in stain from 30
minutes to overnight, but longer staining
times will require much longer destaining
times, and more changes of destain
solution. Colloidal Coomassie may require
several days in the stain for optimum
sensitivity and uniformity of staining.
Silver stain must be timed carefully for best results. There are many silver staining protocols; most can be
completed in 1.5 to 4 hours. Both copper and zinc staining require only 5 to 10 minutes. The fluorescent stains
have various time requirements, usually from a few minutes to an hour at most. It is recommended that the
protocols for fluorescent staining be followed carefully for best
results.
Figure. Gel-Electrophoresis. Proteins of known molecular weight are used to
establish a standard curve that allows the MW of sample proteins to be determined.

This method can separate proteins which differ only by a


Will the Presence of Stain on Western-Blotted
single charge, resulting in a difference of pI-value of
Proteins Interfere with Subsequent Hybridization
0.001.
or Antibody Detection Reactions?
Isoelectric focussing (IEF) can be performed with native
Proteins can be detected on a blot after staining the
proteins or with proteins whose tertiary and secondary
blot with a general protein stain such as Coomassie
structure has been destroyed with (nonionic) caotropes
or colloidal gold, but the interference with
like urea.If after separation the IEF-gel is mounted across
subsequent immunodetection will be high. The
an SDS-PAGE-gel, the proteins which have already been
interference can be 50% or more, but this may not
separated by pI can be separated again by molecular
matter if the protein of interest is in high abundance.
weight.
Proteins which have been stained in the gel will not
transfer out of the gel properly, and it is unlikely that
an immuno detection procedure will be successful. It
is usual to run duplicate gels or run duplicate lanes
on the same gel and cut the gel in half, if you want to
both stain and blot the protein of interest.

Does Ethidium Bromide Interfere with the


Common Enzymatic Manipulation of Nucleic
Acids?
Ethidium bromide does not usually interfere with the
activities of most common DNA modifying
enzymes. However, ethidium bromide has been
shown to interfere with restriction endonucleases.

Isoelectric focusing and 2D-gel


electrophoresis
If the gel contains several thousand different buffer Figure : Isoelectric focussing. A pH-gradient is
molecules, with slightly different pKa-values, these established across the gel by electrophoresis at
buffers can be sorted by an electrical field according relatively low voltages (≈ 500V). The sample is
to pKa. This results in a continuous pH-gradient applied to this gradient and run at high voltages until
across the gel. If proteins are added to the gel, they all proteins have achieved their equilibrium position.
move in the pH-gradient until they reach their pI,
where they are uncharged and do no longer move. As
a result proteins are sorted by pI.
Proteomics
This “two-dimensional” electrophoresis can separate The method of choice for fractionating a large number
about thousand different proteins from a cell lysate. of proteins contained in a natural extract is two-
Comparing the protein expression profiles from cells dimensional gel electrophoresis (Figure below). This
of different developmental stages, or of healthy and technique separates proteins in a plane, first in one
diseased cells, can turn up proteins involved in direction, as a function of their isoelectric point2, then
developmental regulation or in disease processes. in the orthogonal direction, according to their
With luck such a protein may be a useful drug target. molecular weight. After staining, the result is a two-
Thus the field of proteomics (the proteome of a cell dimensional image consisting of a large number of
is the collection of all proteins it contains, like the spots corresponding to the constituent proteins. The
genome is the collection of all its genes) has attracted intensity of spot coloring with certain stains is
considerable attention in the pharmaceutical industry. approximately proportional to the quantity of protein
present.

Figure: 2-dimensional electrophoresis. A protein mixture is first separated on an IEF-gel, which is then
mounted across a Laemmli-gel. The second electrophoresis separates the bands of proteins with identical pI
by molecular weight. About 1000 different proteins can be distinguished on a 2d-gel of mammalian cells.

However, spot resolution may not be sufficient to


separate all the proteins; therefore the results
obtained using two-dimensional electrophoresis gels
are subject to problems of reproducibility and
artifacts. Recently, these problems have been
partially resolved by using highly standardized
protocols and high-precision techniques. Today, it is
possible to separate 2000 spots on a single gel.
Proteins that have extreme isolectric points are
under-represented (supplementary gels with an
extreme pH can partly remedy this), as are
particularly hydrophobic proteins, which are not Figure : Two-dimensional protein electrophoresis gel. Proteins
solubilized by the weak detergent used in the first contained in a natural extract are fractionated in a polyacryl-
separation. amide gel subjected to an electric field. They are separated first
according to their isoelectric point in a stabilized pH gradient
and a weak detergent (horizontal axis), then by their molecular
weight, in the presence of a strong ionic detergent (vertical
axis). Finally, they are stained in the gel to render them visible.
It is impossible to know at the outset which proteins
are present in a spot. Identification may be achieved CSIR Favorites
in part by referring to a database containing two-
dimensional gel electrophoresis results. Such
1. Explain Beer-Lambert Law.
databases exist, notably for bacteria, yeast, fruit fly,
2. Define spectroscopy. What are the different types
mouse, rat, and human proteins. When such
of spectroscopy?
information is not available in a database, and if the
3. Name the techniques that can be used for
sequences of the proteins are known, their positions
separating molecules of different sizes.
may sometimes be estimated by calculating their
4. What is the driving force in electrophoresis?
isoelectric points and molecular weights, providing
5. Addition of salts in an aqueous solution of protein
they have not undergone post-translational
causes the precipitation of protein. How?
modification.Otherwise, the spots must be removed
6. What is the basis of gel permeation
and the proteins they contain eluted from them. If
chromatography?
the sequence of a protein is known, it may be
7. What is the use of high salt concentration in
identified after mild hydrolysis, either by
hydrophobic interaction chromatography?
microsequencing or by mass spectrometry of the
8. Distinguish between absorption chromatography
polypeptides obtained. In rare instances, an eluted
and partition chromatography.
protein can be renatured and its biological activity
9. A sample of protein mixture having a pH 7.5 is
tested.
given along with CM cellulose and DEAE
cellulose. You are asked to separate a protein
Intracellular localization
of isoelectric point 5.4 from the mixture by ion
It is more difficult to evaluate the cellular
exchange chromatography. Which ion
localization of proteins on a large scale; however,
exchanger you will select, and why?
some attempts have been made to do so for yeast
10. Describe the principle and use of x-ray
proteins. All coding sequences have been fused to a
crystallography.
fluorescent reporter protein, such as green
11. What is meant by absorption spectroscopy?
fluorescent protein (GFP), each strain containing
What are the applications in biochemistry?
only one such fusion sequence. The strain collection
12. Explain the principle of mass spectroscopy and
includes at least one representative of each coding
its application in protein science.
sequence fused to GFP. The fluorescence is then
13. Define the following:
located in the cells of each strain, using a light
microscope equipped with a fluorescence device.
(a) Zonal centrifugation
This method is limited by the low resolution of the
(b) Amphoteric molecules
light microscope (0.3µm) compared with the size of
(c) Extrinsic fluorescence
the cell, as well as by localization artifacts
(d) Isoelectric pH
sometimes caused by the fused reporter protein, or
(e) Reverse phase chromatography
by over-expression.
(f) Isoelectric focusing
(g) Osmotic pressure
Protein–protein interactions
(h) Isopycnic centrifugation
The vast majority of proteins interact with other
(i) Ampholytes
proteins, either in a stable manner, in which case
they are known as a complex of polypeptide subunits,
14. Explain how osmotic pressure can be used for
or in a more or less transitory manner. All degrees of
the determination of molecular weight of a
stability are possible, and may be expressed in terms
solute.
of the half-dissociation time, or of the dissociation
15. Differentiate between a colorimeter and
constant, to which the half-dissociation time is
spectrophotometer.
inversely proportional. The variation in free energy
is proportional to the logarithm of the association/
dissociation equilibrium constant. Like the tertiary
structure of individual polypeptides, association
between polypeptides involves weak chemical bonds
or covalent disulfide bridges between two cysteine
residues. These interactions may be demonstrated in
various ways.
Light Microscopy Bright Field Microscopy
With a conventional bright field microscope, light from an
The light microscope, so called because it employs incandescent source is aimed toward a lens beneath the
visible light to detect small objects, is probably the most stage called the condenser, through the specimen, through
well-known and well-used research tool in biology.A an objective lens, and to the eye through a second
beginner tends to think that the challenge of viewing magnifying lens, the ocular or eyepiece. We see objects in
small objects lies in getting enough magnification. In the light path because natural pigmentation or stains absorb
fact, when it comes to looking at living things the light differentially, or because they are thick enough to
biggest challenges are, in order, obtaining sufficient absorb a significant amount of light despite being colorless.
contrast finding the focal plane obtaining good A Paramecium should show up fairly well in a bright field
resolution recognizing the subject when one sees it. The microscope, although it will not be easy to see cilia or most
smallest objects that are considered to be living are the organelles. Living bacteria won't show up at all unless the
bacteria. The smallest bacteria can be observed and cell viewer hits the focal plane by luck and distorts the image
shape recognized at a mere 100x magnification. They by using maximum contrast.
are invisible in bright field microscopes. These pages A good quality microscope has a built-in illuminator,
will describe types of optics that are used to obtain adjustable condenser with aperture diaphragm (contrast)
contrast, suggestions for finding specimens and control, mechanical stage, and binocular eyepiece tube.
focusing on them, and advice on using measurement The condenser is used to focus light on the specimen
devices with a light microscope. through an opening in the stage. After passing through the
specimen, the light is displayed to the eye with an apparent
Types of light microscopes field that is much larger than the area illuminated. The
The bright field microscope is best known to students. magnification of the image is simply the objective lens
Better equipped labs may have dark field and/or phase magnification (usually stamped on the lens body) times the
contrast optics. Differential interference contrast, ocular magnification.
Nomarski, Hoffman modulation contrast and variations The bright field condenser usually contains an aperture
produce considerable depth of resolution and a three diaphragm, a device that controls the diameter of the light
dimensional effect. Fluorescence and confocal beam coming up through the condenser, so that when the
microscopes are specialized instruments, used for diaphragm is stopped down (nearly closed) the light comes
research, clinical, and industrial applications. Other straight up through the center of the condenser lens and
than the compound microscope, a simpler instrument contrast is high. When the diaphragm is wide open the
for low magnification use may also be found in the image is brighter and contrast is low. Bright field
laboratory. The stereo microscope, or dissecting microscopy is best suited to viewing stained or naturally
microscope usually has a binocular eyepiece tube, a pigmented specimens such as stained prepared slides of
long working distance, and a range of magnifications tissue sections or living photosynthetic organisms. It is
typically from 5x to 35 or 40x. Some instruments useless for living specimens of bacteria, and inferior for
supply lenses for higher magnifications, but there is no non-photosynthetic protists or metazoans, or unstained cell
improvement in resolution. Such "false magnification" suspensions or tissue sections.
is rarely worth the expense.

Theory of microscopy

The resolving power of a microscope can be defined in


terms of the ability to see two neighboring points in the
visual field as distinct entities
Dark Field Microscopy
Dark field microscopy is a method which also creates
contrast between the object and the surrounding field. As
the name implies, the background is dark and the object
is bright. A annular stop is also used for dark field, but
the stop is now outside the field of view. Only light
coming from the outside of the beam passes through the
object and it cannot be seen directly. Only when light
from the stop is deflected and deviated by the object can
it be seen. This method also produces a great deal of
glare and therefore the specimen often appears as a
bright silhouette rather than as a bright object of which
much detail can be determined.
Principle
To view a specimen in dark field, an opaque disc is
placed underneath the condenser lens, so that only light
that is scattered by objects on the slide can reach the eye
(see figure below). Instead of coming up through the Phase Contrast Microscopy
specimen, the light is reflected by particles on the slide.
Most of the detail of living cells is undetectable in bright
Everything is visible regardless of color, usually bright
field microscopy because there is too little contrast between
white against a dark background. Pigmented objects are
structures with similar transparency and there is insufficient
often seen in "false colors," that is, the reflected light is
natural pigmentation. However the various organelles show
of a color different than the color of the object. Better
wide variation in refractive index, that is, the tendency of the
resolution can be obtained using dark field as opposed to
materials to bend light, providing an opportunity to disting-
bright field viewing.
uish them.
Dark field illumination is most readily set up at low
Principle
magnifications (up to 100x), although it can be used with
Highly refractive structures bend light to a much greater
any dry objective lens. Any time you wish to view
angle than do structures of low refractive index. The same
everything in a liquid sample, debris and all, dark field is
properties that cause the light to bend also delay the passage
best. Even tiny dust particles are obvious. Dark field is
of light by a quarter of a wavelength or so. In a light
especially useful for finding cells in suspension. Dark
microscope in bright field mode, light from highly refractive
field makes it easy to obtain the correct focal plane at
structures bends farther away from the center of the lens than
low magnification for small, low contrast specimens.
light from less refractive structures and arrives about a
Uses
quarter of a wavelength out of phase. Light from most
1. Initial examination of suspensions of cells such as
objects passes through the center of the lens as well as to the
yeast, bacteria, small protists, or cell and tissue fractions
periphery. Now if the light from an object to the edges of the
including cheek epithelial cells, chloroplasts,
objective lens is retarded a half wavelength and the light to
mitochondria, even blood cells (small diameter of
the center is not retarded at all, then the light rays are out of
pigmented cells makes it tricky to find them sometimes
phase by a half wavelength. They cancel each other when the
despite the color).
objective lens brings the image into focus. A reduction in
2. Initial survey and observation at low powers of pond
brightness of the object is observed. The degree of reduction
water samples, hay or soil infusions, purchased protist or
in brightness depends on the refractive index of the object.
metazoan cultures.
3. Examination of lightly stained prepared slides. Simplified optical pathway for phase
contrast microscopy.
1 = light source
2 = annular shaped light mask (condenser)
3 = condenser
4 = specimen
5 = background light
6 = light bent by the specimen
Bright field Dark field 7 = phase ring
8 = eyepiece with intermediate image
9 = eye
With this arrangement the specimen is concentrically
illuminated by the apex of a cone of light. The light Principle
beams which are diffracted by the specimen pass the Differential interference microscopy requires several optical
objective lens at various angles which are dependent on components, therefore it can be very expensive to set up.
the relative refractive index and the thickness of the Light from an incandescent source is passed through a
specimen. The other light components, corresponding to polarizer, so that all of the light getting through must vibrate
the background, pass through the phase ring in the in a single plane. The beam is then passed through a prism
objective which produces an additional phase that separates it into components that are separated by a very
difference. Thus, the phase differences between the small distance - equal to the resolution of the objective lens.
specimen, its details and the background are amplified The beams pass through the condenser, then the specimen.
in the final image, so that minimal differences in In any part of the specimen in which adjacent regions differ
refractive index are visible even in colorless specimens in refractive index the two beams are delayed or refracted
with a low contrast and thickness. differently. When they are recombined by a second prism in
Depending on the configuration and properties of the the objective lens there are differences in brightness
phase ring in the objective, the phase contrast corresponding to differences in refractive index or thickness
microscopy can be positive or negative. In positive in the specimen. Regions such as the edge of a cell or
phase contrast the specimen is visible with medium or nucleus are very distinct because the quality of the specimen
dark grey features, surrounded by a bright halo; the changes so much over a very short distance.
background is of higher intensity than the specimen. In One or more components of the system are adjustable to
negative phase contrast the background is darker and obtain the maximum contrast. When the contrast is
the specimen appears brighter, surrounded by a dark optimized one can obtain a very distinct image that appears
halo. three dimensional. The effect is very much like what you
The bright and dark halos are artifacts which are one of see when a subject is shadowed by a strong light coming
the major disadvantages of phase contrast; they are from one side, as with craters on the moon near the
especially prevalent in specimens inducing large phase terminator, namely the boundary between the sunlit portion
shifts. of the Moon's surface and the dark side.
Applications
Phase contrast is preferable to bright field microscopy
when high magnifications (400x, 1000x) are needed and
the specimen is colorless or the details so fine that color
does not show up well. Cilia and flagella, for example,
are nearly invisible in bright field but show up in sharp
contrast in phase contrast. Amoebae look like vague
outlines in bright field, but show a great deal of detail in
phase. Most living microscopic organisms are much
more obvious in phase contrast.
Interference Microscopy
The ideas behind high-resolution interferometry were
introduced by Tychinski, By combining an optical
microscope with an interferometer, it is possible to obtain
high-resolution maps of the phase of the optical field at
high magnification. As observed by Tychinski, structure
of the phase distribution inside the diffraction-limited
spot can be seen with such equipment. Fast phase
variations are produced by phase dislocations in the
optical field, and are readily observable using an
uses of this microscope involving live and unstained
interference microscope. In contrast to the amplitude, the
biological samples, such as a smear from a tissue culture or
phase is not governed by the classical resolution limit of
individual water borne single-celled organisms. Its resolution
the optical imaging system. Unfortunately, there is no
and clarity in conditions such as this are unrivaled among
trivial relation between the structure and the optical
standard optical microscopy techniques.The main limitation
phase field that it produces, thus making it difficult to
is requirement of a transparent sample of fairly similar
extract surface profile information without a priori
refractive index to its surroundings. This is unsuitable for
knowledge.
thick samples, such as tissue slices, and highly pigmented
cells.
Fluorescence Microscopy
A fluorescence microscope is a light microscope used to Biologists usually need to enhance and differentiate details
study properties of organic or inorganic substances using within living or fixed cells. To do this, special dyes are
the phenomena of fluorescence and phosphorescence applied. To increase the sensitivity of observation, dyes
instead of, or in addition to, reflection and absorption. The (fluorochromes) that fluoresce under invisible or visible
method of fluorescence microscopy is an excitatory light is radiation are used to stain and delineate the structures of
passed from above (or, for inverted microscopes, from interest. Sometimes, the inherent tendency of some cellular
below), through the objective and then onto the specimen components to fluoresce without additional staining may be
instead of passing it first through the specimen. (In the usefully employed. This autofluorescence can be seen in
latter case the transmitted excitatory light reaches the cellular components ranging from plant chlorophylls to
objective together with light emitted from the specimen). proteins in eye tissue. Unlike traditional biological staining
The fluorescence in the specimen gives rise to emitted (e.g., methylene blue and eosin for tissue differentiation),
light which is focused to the detector by the same due to the remarkable efficiency and detectability of
objective that is used for the excitation. A filter between fluorochromes fluorescing under UV or visible light, much
the objective and the detector filters out the excitation light smaller concentrations of these dyes (generally toxic) can
from fluorescent light. Since most of the excitatory light is be used. This translates into fewer side effects to living
transmitted through the specimen, only reflected excitatory cells with minimum disruption to their normal physiology.
light reaches the objective together with the emitted light Also, the biochemical workings of cells, such as calcium
and this method therefore gives an improved signal to ion transport, can be recorded in time-lapse mode using
noise ratio. A common use in biology is to apply fluorescence microscopy.
fluorescent or fluorochrome stains to the specimen in order
Fluorescent dyes are commonly used for the detection of
to image a protein or other molecule of interest.
live cells and key functional activities in a variety of cell-
based assays. The fluorescent dyes, calcein, acetoxymet-
hylester can be used as an insert Systems to label cells
when performing analyses such as tumor cell invasion,
endothelial cell migration, endothelial cell tubulogenesis,
and other cell-based assays.
Fluorescence Resonance Energy Transfer
Fluorescence resonance energy tranfer (FRET) allows the
study of molecular interactions in the lower nanometer
range. FRET offers a variety of donor/acceptor pairs. This
particular mechanism has become the basis for a useful
technique involving the study of molecular interactions
and associations at distances far below the lateral
resolution of the optical microscope.
What is difference between resolution, resolving power
and limit of resolution of microscope?
Resolution is the ability to tell two points apart as separate
points. If the resolving power of your lens is 2um that
The fluorochromes emit light of a given wavelength when means two points that are 2um apart can be seen as
excited by incident light of a different (shorter) separate points. If they are closer together than that, they
wavelength. To view this fluorescence in the microscope, will blend together into one point. The magnification is
several light filtering components are needed. Specific something different-the ability to make an object larger.
filters are used to isolate the excitation and emission If the resolving power of a microscope is poor, it will just
wavelengths of a fluorochrome. A dichroic beam splitter magnify a blurry object.
(partial mirror) reflects shorter wavelengths of light and Resolution, resolving power, and limit of resolution all
allows longer wavelengths to pass. A dichoric beam mean the same thing. They refer to the ability of a
splitter is required because the objective acts as both a microscope to distinguish between two objects.
condenser lens (excitation light) and objective lens
(emission light); therefore the beam splitter isolates the If a microscope have resolving power 0.3µm , it can
emitted light from the excitation wavelength. This epi- distinguish two points that are at least 0.3 µm (300 nm)
illumination type of light path is required to create a dark apart.
background so that the fluorescence can be easily seen.
Transmission Electron Microscope (TEM)
Electron Microscopy
The original form of electron microscope, the transmission
electron microscope (TEM) uses a high voltage electron beam
to create an image. The electrons are emitted by an electron
gun, commonly fitted with a tungsten filament cathode as the
electron source. The electron beam is accelerated by an anode
typically at +100keV (40 to 400 keV) with respect to the
cathode, focused by electrostatic and electromagnetic lenses,
and transmitted through the specimen that is in part
transparent to electrons and in part scatters them out of the
beam. When it emerges from the specimen, the electron beam
carries information about the structure of the specimen that is
magnified by the objective lens system of the microscope.
The spatial variation in this information (the "image") is
viewed by projecting the magnified electron image onto a
fluorescent viewing screen coated with a phosphor or
The first electron microscope prototype was built in scintillator material such as zinc sulfide. The image can be
1931 by the German engineers Ernst Ruska and Max photographically recorded by exposing a photographic film or
Knoll. An electron microscope is a type of microscope plate directly to the electron beam, or a high-resolution
that uses a particle beam of electrons to illuminate a phosphor may be coupled by means of a lens optical system
specimen and create a highly-magnified image.Electron or a fibre optic light-guide to the sensor of a CCD (charge-
microscopes have much greater resolving power than coupled device) camera. The image detected by the CCD may
light microscopes that use electromagnetic radiation be displayed on a monitor or computer.
and can obtain much higher magnifications of up to 2
million times, while the best light microscopes are Resolution of the TEM is limited primarily by spherical
limited to magnifications of 2000 times. Both electron aberration, but a new generation of aberration correctors have
and light microscopes have resolution limitations, been able to partially overcome spherical aberration to
imposed by the wavelength of the radiation they use. increase resolution. Software correction of spherical
The greater resolution and magnification of the electron aberration for the High Resolution TEM (HRTEM) has
microscope is because the wavelength of an electron; allowed the production of images with sufficient resolution to
its de Broglie wavelength is much smaller than that of a show carbon atoms in diamond separated by only 0.89
photon of visible light. The electron microscope uses ångström (89 picometers) and atoms in silicon at 0.78
electrostatic and electromagnetic lenses in forming the ångström (78 picometers) at magnifications of 50 million
image by controlling the electron beam to focus it at a times. The ability to determine the positions of atoms within
specific plane relative to the specimen. This manner is materials has made the HRTEM an important tool for nano-
similar to how a light microscope uses glass lenses to technologies research and development.
focus light on or through a specimen to form an image.
1. The "Virtual Source" at the top represents the electron gun,
producing a stream of monochromatic electrons.
2. This stream is focused to a small, thin, coherent beam by
the use of condenser lenses 1 and 2. The first lens (usually
controlled by the "spot size knob") largely determines the
"spot size"; the general size range of the final spot that strikes
the sample. The second lens (usually controlled by the
"intensity or brightness knob" actually changes the size of the
spot on the sample; changing it from a wide dispersed spot to
a pinpoint beam.
3. The beam is restricted by the condenser aperture (usually
user selectable), knocking out high angle electrons (those far
from the optic axis, the dotted line down the center)
4. The beam strikes the specimen and parts of it are
transmitted
5. This transmitted portion is focused by the objective lens
into an image.
6. Optional Objective and Selected Area metal apertures The display of the SEM maps the varying intensity of any of
can restrict the beam; the Objective aperture enhancing these signals into the image in a position corresponding to
contrast by blocking out high-angle diffracted electrons, the position of the beam on the specimen when the signal
the Selected Area aperture enabling the user to examine was generated. In the SEM image of an ant shown at right,
the periodic diffraction of electrons by ordered the image was constructed from signals produced by a
arrangements of atoms in the sample secondary electron detector, the normal or conventional
7. The image is passed down the column through the imaging mode in most SEMs. Generally, the image
intermediate and projector lenses, being enlarged all the resolution of an SEM is about an order of magnitude poorer
way than that of a TEM. However, because the SEM image relies
8. The image strikes the phosphor image screen and light on surface processes rather than transmission it is able to
is generated, allowing the user to see the image. The image bulk samples up to several centimetres in size
darker areas of the image represent those areas of the (depending on instrument design) and has a much greater
sample that fewer electrons were transmitted through depth of view, and so can produce images that are a good
(they are thicker or denser). The lighter areas of the representation of the 3D structure of the sample.
image represent those areas of the sample that more
electrons were transmitted through (they are thinner or
less dense)

1. The "Virtual Source" at the top represents the electron


gun, producing a stream of monochromatic electrons.
2. The stream is condensed by the first condenser lens
(usually controlled by the "coarse probe current knob").
This lens is used to both form the beam and limit the
amount of current in the beam. It works in conjunction
with the condenser aperture to eliminate the high-angle
electrons from the beam
3. The beam is then constricted by the condenser aperture
Scanning Electron Microscope (SEM) (usually not user selectable), eliminating some high-angle
Unlike the TEM, where electrons of the high voltage electrons
beam carry the image of the specimen, the electron beam
4. The second condenser lens forms the electrons into a
of the Scanning Electron Microscope (SEM) does not at
thin, tight, coherent beam and is usually controlled by the
any time carry a complete image of the specimen. The
"fine probe current knob"
SEM produces images by probing the specimen with a
focused electron beam that is scanned across a 5. A user selectable objective aperture further eliminates
rectangular area of the specimen (raster scanning). At high-angle electrons from the beam.
each point on the specimen the incident electron beam
6. A set of coils then "scan" or "sweep" the beam in a grid
loses some energy, and that lost energy is converted into
fashion (like a television), dwelling on points for a period
other forms, such as heat, emission of low-energy
of time determined by the scan speed (usually in the
secondary electrons, light emission (cathodolumine-
microsecond range.
scence) or x-ray emission.
7. The final lens, the Objective, focuses the scanning The STEM's use of SEM-like beam rastering simplifies
beam onto the part of the specimen desired. annular dark-field imaging, and other analytical techniques,
8. When the beam strikes the sample (and dwells for a but also means that image data is acquired in serial rather
few microseconds) interactions occur inside the sample
Sample preparation
and are detected with various instruments
9. Before the beam moves to its next dwell point these Materials to be viewed under an electron microscope may
instruments count the number of interactions and display require processing to produce a suitable sample. The
a pixel on a CRT whose intensity is determined by this technique required varies depending on the specimen and
number (the more reactions the brighter the pixel). the analysis required:
10. This process is repeated until the grid scan is finished Chemical fixation- for biological specimens aims to
and then repeated, the entire pattern can be scanned 30 stabilize the specimen's mobile macromolecular structure
times per second by chemical cross-linking of proteins with aldehydes such
Reflection Electron Microscope (REM) as formaldehyde and glutaraldehyde, and lipids with
osmium tetroxide. Samples are preserved by fixation with
In the Reflection Electron Microscope (REM) as in glutaraldehyde (Covalently cross-links proteins) first and
the TEM, an electron beam is incident on a surface, then by osmium tetroxide which Binds to and stabilizes
but instead of using the transmission (TEM) or lipid bilayers and proteins. Tissue dehydrated, permeated
secondary electrons (SEM), the reflected beam of with a polymerizing resin & sectioned into ultra-thin
elastically scattered electrons is detected. This sections. 50 – 100 nm thick (1/200 thickness of a cell).
technique is typically coupled with Reflection High Sections stained with electron-dense material (e.g uranyl
Energy Electron Diffraction (RHEED) and Reflection acetate) to achieve contrast. Tissue composed of atoms of
high-energy loss spectrum (RHELS). Another low atomic number (e.g. carbon, oxygen, nitrogen,
variation is Spin-Polarized Low-Energy Electron hydrogen). To make them visible impregnated with salts of
Microscopy (SPLEEM), which is used for looking at heavy metals.
the microstructure of magnetic domains.
Cryofixation – freezing a specimen so rapidly, to liquid
Scanning Transmission Electron Microscope nitrogen or even liquid helium temperatures, that the water
(STEM) forms vitreous (non-crystalline) ice. This preserves the
specimen in a snapshot of its solution state. An entire field
The STEM rasters a focused incident probe across a
called cryo-electron microscopy has branched from this
specimen that (as with the TEM) has been thinned to
technique. With the development of cryo-electron
facilitate detection of electrons scattered through the
microscopy of vitreous sections (CEMOVIS), it is now
specimen. The high resolution of the TEM is thus
possible to observe samples from virtually any biological
possible in STEM. The focusing action (and
specimen close to its native state.
aberrations) occur before the electrons hit the
specimen in the STEM, but afterward in the TEM. Dehydration – freeze drying, or replacement of water with
organic solvents such as ethanol or acetone, followed by
critical point drying or infiltration with embedding resins.
Embedding, biological specimens – after dehydration, A subclass of this is Focused ion beam milling, where
tissue for observation in the transmission electron gallium ions are used to produce an electron transparent
microscope is embedded so it can be sectioned ready for membrane in a specific region of the sample, for example
viewing. To do this the tissue is passed through a through a device within a microprocessor. Ion beam milling
'transition solvent' such as epoxy propane and then may also be used for cross-section polishing prior to SEM
infiltrated with a resin such as Araldite epoxy resin; tissues analysis of materials that are difficult to prepare using
may also be embedded directly in water miscible acrylic mechanical polishing.
resin. After the resin has been polymerised (hardened) the Conductive Coating – an ultrathin coating of electrically-
sample is thin sectioned (ultrathin sections) and stained conducting material, deposited either by high vacuum
then it is then ready for visualization. evaporation or by low vacuum sputter coating of the sample.
Embedding, materials - after embedding in resin, the This is done to prevent the accumulation of static electric
specimen is usually ground and polished to a mirror like fields at the specimen due to the electron irradiation
finish using ultra-fine abrasives. The polishing process required during imaging. Such coatings include gold,
must be performed carefully to minimize scratches and gold/palladium, platinum, tungsten, graphite etc. and are
other polishing artifacts that reduce image quality. especially important for the study of specimens with the
Sectioning – produces thin slices of specimen, scanning electron microscope. Another reason for coating,
semitransparent to electrons. These can be cut on an even when there is more than enough conductivity, is to
ultramicrotome with a diamond knife to produce ultrathin improve contrast, a situation more common with the
slices about 60 to 90 nm thick. operation of a FESEM (field emission SEM).
Staining – uses heavy metals such as lead, uranium or
tungsten to scatter imaging electrons and thus give contrast Applications
between different structures, since many (especially
biological) materials are nearly "transparent" to electrons 1) Diagnostic electron microscopy
(weak phase objects). Typically thin sections are stained 2) Cryobiology
for several minutes with an aqueous or acoholic solution of 3) Protein localization
uranyl acetate followed by aqueous lead citrate. 4) Electron tomography
Freeze-fracture or freeze-etch – a preparation method 5) Cellular tomography
particularly useful for examining lipid membranes and 6) Cryo-electron microscopy
their incorporated proteins in "face on" view. The fresh 7) Toxicology
tissue or cell suspension is frozen rapidly (cryofixed), then 8) Biological production and viral load monitoring
fractured by simply breaking or by using a microtome 9) Particle analysis
while maintained at liquid nitrogen temperature. The cold 10) Pharmaceutical QC
fractured surface (sometimes "etched" by increasing the 11) Structural biology
temperature to about –100 °C for several minutes to let 12) 3D tissue imaging
some ice sublime) is then shadowed with evaporated 13) Virology
platinum or gold at an average angle of 45° in a high 14) Vitrification
vacuum evaporator. A second coat of carbon, evaporated 15) Forensics
perpendicular to the average surface plane is often
performed to improve stability of the replica coating. The
specimen is returned to room temperature and pressure, Few questions
then the extremely fragile "pre shadowed" metal replica of 1. What is the significance of the N.A. number
the fracture surface is released from the underlying inscribed on the outer barrel of an objective?
biological material by careful chemical digestion with 2. What is meant by resolving power of an objective
acids, hypochlorite solution or SDS detergent. The still and how is that distinguished from resolution?
floating replica is thoroughly washed from residual 3. What is the relationship between numerical aperture
chemicals, carefully fished up on fine grids, dried then and brightness of an image? Between magnification
viewed in the TEM. and brightness of an image?
Ion Beam Milling – thins samples until they are 4. Can phase contrast objectives be used for regular
transparent to electrons by firing ions (typically argon) at brightfield observation?
the surface from an angle and sputtering material from the 5. When a 40x objective is used, the image may appear
surface. worse than with a 20x objective. Why?
Electromagnetic Spectrum
Ionizing Radiation Visible Nonionizing Radiation
Infrared
Ultraviolet Near Far Radar
X Rays
FM
Gamma Rays TV
Short wave
Cosmic Rays Broadcast Power
Transmission

10-14 10-12 10-10 10-8 10-6 10-4 10-2 1 102 104 106 108
Wavelength in Meters

1010 108 106 104 102 1 10-2 10-4 10-6 10-8 10 -10 10-12 10-14

High Energy - Electron Volts Low

Radioactivity A common unit of activity is the megabecquerel (MBq),


where 1 MBq = 106 dps.
Roentgen: Discoverer of X-rays 1895
An earlier unit of activity, the curie (Ci) is defined as
Becquerel: Discoverer of Radioactivity 1896 1Ci = 3.7 × 1010 dps
Multiples of the curie are the picocurie (10−12 Ci),
Curies: Discoverers of Radium and Polonium 1900-1908
nanocurie (10−9 Ci), microcurie
Rutherford: Discoverer Alpha and Beta rays 1897
(10−6 Ci), millicurie (10−3 Ci), kilocurie (103 Ci), and
megacurie (106 Ci). The becquerel and the curie are
Radioactivity can be described mathematically without related by 1 Bq = 1 dps = 2.7 × 10−11 Ci. The activity
reference to the specific mode of decay of a sample of of a radioactive sample per unit mass (e.g., MBq/mg) is
radioactive atoms. The rate of decay (the number of atoms known as the specific activity of the sample.
decaying per unit time) is directly proportional to the The rutherford (Rf) was once proposed as a unit of
number of radioactive atoms N present in the sample: activity, where 1 Rf = 106 dps.
∆N/∆t = −λN
where ∆N/∆t is the rate of decay. The constant λ is the
decay constant of the particular species of atoms in the
sample, and the negative sign reveals that the number of
radioactive atoms in the sample is diminishing as the
sample decays. The decay constant can be expressed as –
(∆N/∆t )/N, revealing that it represents the fractional rate
of decay of the atoms. The value of λ is characteristic of
the type of atoms in the sample and changes from one
nuclide to the next. Units of λ are (time)−1. Larger values
of λ characterize more unstable nuclides that decay more
rapidly.
The rate of decay of a sample of atoms is termed the
activity A of the sample (i.e., A = ∆N/∆t ). A rate of decay
of 1 atom per second is termed an activity of 1
becquerel(Bq). That is,

1Bq = 1 disintegration per second (dps)


WHY ATOMS BECOME RADIOACTIVE

The nuclei of many atoms are stable. In general, it is these


atoms that constitute ordinary matter. In stable nuclei of
lighter atoms, the number of neutrons is about equal to the
number of protons. A high level of symmetry exists in the
placement of protons and neutrons into nuclear energy
levels similar to the electron shells constituting the
extranuclear structure of the atom. The assignment of
nucleons to energy levels in the nucleus is referred to as the
“shell model” of the nucleus. For heavier stable atoms, the
number of neutrons increases faster than the number of
protons, suggesting that the higher energy levels are spaced
more closely for neutrons than for protons. The number of
neutrons (i.e., the neutron number) in nuclei of stable atoms
is plotted in Figure below as a function of the number of
protons (i.e., the atomic number). Above Z = 83, no stable
forms of the elements exist, and the plot depicts the
neutron/proton (N/Z) ratio for the least unstable forms of
the elements (i.e., isotopes that exist for relatively long
periods before changing).
Unstable Stability curve
This process can be either positron decay or electron capture
and yields an atomic number of Z –1 and an unchanged mass
number A. The path from X to R represents negatron decay
in which a neutron is trans-formed into a proton, leaving the
progeny with an atomic number of Z + 1 and an unchanged
mass number A. In the path from R to S, the constant Z and
constant A signify that no change occurs in nuclear
composition. This pathway is termed an isomeric transition
between nuclear isomers and results only in the release of
energy from the nucleus through the processes of gamma
emission and internal conversion.
Nuclei that have an imbalance in the N/Z ratio are
positioned away from the stability curve. These
unstable nuclei tend to undergo changes within the
nucleus to achieve more stable configurations of
neutrons and protons. The changes are accompanied
by the emission of particles and electromagnetic
radiation (photons) from the nucleus, together with the
release of substantial amounts of energy related to an
increase in binding energy of the nucleons in their
final nuclear configuration. These changes are
referred to as radioactive decay of the nucleus, and
the process is described as radioactivity. If the number
of protons is different between the initial and final
nuclear configurations, Z is changed and the nucleus
is transmuted from one elemental form to another.
The various processes of radioactive decay are Alpha Decay: Alpha decay is a decay process in which
summarized in Table 1-1. greater nuclear stability is achieved by emission of 2
protons and 2 neutrons as a single alpha (α) particle (a
TYPES OF RADIOACTIVE DECAY nucleus of helium) from the nucleus. Alpha emission is
The process of radioactive decay often is described by confined to relatively heavy nuclei such where 2He4
a decay scheme in which energy is depicted on the represents the alpha particle. The sum of mass numbers
vertical ( y) axis and atomic number is shown on the and the sum of atomic numbers after the transition equal
horizontal (x) axis. A generic decay scheme is the mass and atomic numbers of the parent before the
illustrated in Figure. The original nuclide (or “parent”) transition. In α decay, energy is released as kinetic energy
is depicted as Z X A, and the product nuclide (or of the α particle, and is sometimes followed by energy
“progeny”) is denoted as element P , Q, R, or S released during an isomeric transition resulting in
depending on the decay path. In the path from X to P , emission of a γ ray or conversion electron. Alpha
the nuclide gains stability by emitting an alpha (α) particles are always ejected with energy characteristic of
particle, two neutrons and twoprotons ejected from the the particular nuclear transition.
nucleus as a single particle. In this case, the progeny
nucleushas an atomic number of Z − 2 and a mass
number of A−4 and is positioned atreduced elevation An alpha transition is depicted in the margin, in which the
in the decay scheme to demonstrate that energy is parent 226Ra decays directly to the final energy state
released as thenucleus gains stability through (ground state) of the progeny 222Rn in 94% of all
radioactive decay. The released energy is referred to as transitions. In 6% of the transitions, 226Ra decays to an
the transition energy. In the path from X to Q, the intermediate higher energy state of 222Rn, which then
nucleus gains stability through the process in which a decays to the ground state by isomeric transition. For each
proton in the nucleus changes to a neutron. of the transition pathways, the transition energy between
parent and ground state of the progeny is constant. In the
example of 226Ra, the transition energy is 4.78 MeV.
Beta Decay
Nuclei with an N/Z ratio that is above the line of RADIOACTIVE EQUILIBRIUM
stability tend to decay by a form of beta (β ) decay Some progeny nuclides produced during
known as negatron emission. In this mode of decay, a radioactive decay are themselves unstable
neutron is transformed into a proton, and the Z of the
and undergo radioactive decay in a
nucleus is increased by 1 with no change in A. In this
manner, the N/Z ratio is reduced, and the product continuing quest for stability. For example,
226
nucleus Ra decays to 222Rn, which, in turn, decays
is nearer the line of stability. Simultaneously an by alpha emission to 218Po.When a radio-
electron (termed a negative beta particle or negatron) is active nuclide is produced by radioactive
ejected from the nucleus together with a neutral mass
decay of a parent, a condition can be reached
less particle, termed a neutrino (actually an
“antineutrino” in negatron decay), that carries away the inwhich the rate of production of the progeny
remainder of the released energy that is not accounted equals its rate of decay. In this condition,
for by the negatron. The neutrino (or antineutrino) the number of progeny atoms and therefore
seldom interacts with matter and is not important to the progeny activity reach their highest level
applications of radioactivity in medicine
and are constant for a moment in time. This
constancy reflects an equilibrium condition
known as transient equilibrium because it
exists only momentarily. In cases in which a
shorter-lived radioactive progeny is produced
by decay of a longer-lived parent, the activity
curves for parent and progeny intersect at the
moment of transient equilibrium. This
intersection reflects the occurrence of equal
Gamma Emission and Internal Conversion
activities of parent and daughter at that
Gamma rays are high-energy electromagnetic radiation particular moment. After the moment of
that differ from x rays only in their origin: Gamma rays transient equilibrium has passed, the progeny
are emitted during transitions between isomeric energy activity decays with an apparent half-life
states of the nucleus, whereas x rays are emitted during
equal to that of the longer-lived parent.
electron transitions outside the nucleus. Gamma rays
and other electromagnetic radiation are described by
their energy E and frequency ν, two properties that are NATURAL RADIOACTIVITY AND
related by the expression DECAY SERIES
E= hν, where h = Planck’s constant (h = 6.62 × 10−34 J- Most radionuclides in nature are members of
sec). The frequency ν and wavelength λ of
one of three naturally occurring radioactive
electromagnetic radiation are related by the expression
ν = c/λ, where c is the speed of light in a vacuum. decay series. Each series consists of a
sequence of radioactive transformations that
Internal conversion is a competing process to gamma begins with a long-lived radioactive parent
emission for an isomeric transition between energy and ends with a stable nuclide. In a closed
states of a nucleus. In a nuclear transition by internal
environment such as the earth, intermediate
conversion, the released energy is transferred from the
nucleus to an inner electron, which is ejected with a radioactive progeny exist in secular
kinetic energy equal to the transferred energy reduced equilibrium with the long-lived parent, and
by the binding energy of the electron. decay with an apparent half-life equal to that
of the parent
All naturally occurring radioactive nuclides decay by Radioactive nuclides are also produced as a result of
emitting either alpha or negative beta particles. Hence, nuclear fission. These nuclides can be recovered as
each transformation in a radioactive series changes the fission byproducts from the fuel elements used in
mass number by either 4 or 0 and changes the atomic nuclear reactors. Isotopes such as 90Sr,99Mo,131I, and
number by −2 or +1. 137Cs can be recovered in this manner. Fission-
The uranium series depicted in Figure 1-8 begins with produced nuclides (fission byproducts) are often
the isotope 238U and ends with the stable nuclide 206Pb. mixed with other stable and radioactive isotopes of the
The parent and each product in this series have a mass same element, and cannot be separated chemically as
number that is divisible by 4 with remainder of 2; the a solitary radionuclide.13As a consequence, fission
uranium series is also known as the 4n + 2 series. The byproducts are less useful in research and clinical
naturally occurring isotopes 226Ra and 222Rn are medicine than are radionuclides that are produced by
members of the uranium series. The actinium (4n + 3) neutronor charged- particle bombardment.
series begins with 235U and ends with 207Pb, and the
thorium (4n ) series begins with232Th and ends with SUMMARY
208Pb. Members of the hypothetical neptunium (4n+ 1)
• Radioactive decay is the consequence of nuclear
series do not occur in nature because no long-lived instability.
parent is available. Fourteen naturally occurring • Negatron decay occurs in nuclei with a high n/p
radioactive nuclides are ratio.
not members of a decay series . These nuclides, all with • Positron decay and electron capture occur in nuclei
relatively long half-lives, are 3H, 14C, 40K, 50V, with a low n/p ratio.
87Rb,115In,130Te,138La,142Ce,144Nd,147Sm,176Lu, 187Re,
• Alpha decay occurs with heavy unstable nuclei.
192
and Pt. • Isomeric transitions occur between different energy
states of nuclei and result in the emission of γ rays and
ARTIFICIAL PRODUCTION OF
conversion electrons.
RADIONUCLIDES
• The activity A of a sample is
Radioactive isotopes with properties useful in
A = Aoe−λt
biomedical research and clinical medicine may be
where λ is the decay constant (fractional rate of
produced by bombarding selected nuclei with neutrons
decay).
and high- energy charged particles. Nuclides with
• The half-life T1/2 is the time required for half of a
excess neutrons that subsequently decay by negatron
radioactive sample to decay.
emission are created by bombarding nuclei with
• The half-life and the decay constant are related by
neutrons in a nuclear reactor or from a neutron
T1/2=0.693/λ.
generator. Typical reactions are
13 C + n → C + isomeric transition • The common unit of activity is the becquerel (Bq),
6 0 6 with 1 Bq = 1 disintegration/second.
15P+10n→3215P+ isomeric transition
• Transient equilibrium may exist when the progeny
Useful isotopes produced by neutron bombardment
nuclide decays with a T1/2/T1/2 parent.
include3H,35S,51Cr,60Co,99Mo, 133Xe, and 198Au.
• Secular equilibrium may exist when the progeny
Because the isomeric transition frequently results in
nuclide decays with a T1/2/T1/2 parent.
prompt emission of a gamma ray, neutron
• Most radioactive nuclides found in nature are
bombardment often is referred to as an (n, γ) reaction.
members of naturally occurring decay series
The reaction yields a product nuclide with an increase
in A of 1 and no increase in Z .

1. Natural oxygen contains three isotopes with atomic masses in amu of 15.9949, 16.9991, and 17.9992
and relative abundances of 2500:1:5. Determine to three decimal places the average atomic mass of
oxygen.
2. Determine the energy required to move an electron from the K to the L shell in tungsten and in
hydrogen, and explain the difference.
3. How many MBq of132I (T1/2=2.3 hours) should be ordered so that the sample activity will be 500 MBq
when it arrives 24 hours later?
4. . How many atoms and grams of 90Y are in secular equilibrium with 50 mCi of 90Sr?
5. If a radionuclide decays for an interval of time equal to its average life, what percentage of the original
activity remains?
Measurement of Dose The sensitivity of the system depends on the voltage
When alpha or beta particles, or gamma radiation, pass applied between the electric plates. Since alpha
through matter, they form ions. They accomplish this particles are significantly easier to detect than beta
by knocking electrons from the orbits of the molecules particles, it requires lower voltage to detect the high
they pass through. We can monitor the ionization effect energy alpha particles. In addition, alpha particles will
by allowing the radiation to pass through dry air and penetrate through the metal casing of the counter tube,
measuring the numbers of ions formed. This is most whereas beta particles can only pass through a quartz
often done by designing a chamber with an electrical window on the tube. Consequently, ionization chambers
charge capacitance, allowing the radiation to pass are most useful for measuring alpha emissions. High-
through the chamber and monitoring the amount of energy beta emissions can be measured if the tube is
capacitance discharge caused by the formation of ions. equipped with a thin quartz window and the distance
The device is a Geiger-Mueller Counter and has many between the source of emission and the tube is minimal.
variations. The ionizing ability is measured in A modification of the basic ionization chamber is the
roentgens, and a roentgen is the number of ionizations pocket dosimeter. This device is a capacitor, which is
necessary to form one electrostatic unit (esu) in 1 cc of charged by a base unit and which can then be carried as
dry air. Since the roentgen is a large unit, dosages for a portable unit. They are often the size and shape of a
cell research use are normally divided into milli- pen and can thus be carried in the pocket of a lab coat.
roentgens (mR). When exposed to an ionizing radiation source, the
Curies measure the amount of radioactive decay, and capacitor discharges slightly. Over a period of time, the
roentgens measure the amount of radiation transmitted charge remaining on the dosimeter can be monitored
through matter, over distance. Neither unit is useful in and used as a measure of radiation exposure. The
determining biological effect, since biological effect dosimeters are usually inserted into a reading device
implies that the radiation is absorbed by the tissues that that is calibrated to convert the average exposure of the
are irradiated. The rad (radiation absorbed dose) is a dosimeter directly into roentgens or rems. Since the
unit of absorbed dose and equals 100 ergs absorbed in 1 instrument works by discharging the built-up charge,
gram of matter. and the charge is on a thin wire in the center of the
dosimeter, it can be completely discharged by the
Detection of Radioactivity flexing of that wire, as it touches the outer shell upon
Ionization chambers. The most common method of impact. When later read for exposure, the investigator
measuring radiation exposure is the use of an will be informed that they have been exposed to
ionization chamber. Among the more common forms of dangerously high levels of radiation, since there will be
ionization chambers are the Geiger-Müller counter, no charge left in the dosimeter. Besides causing great
scintillation counter, and pocket dosimeter. consternation with the radiation safety officer, and a
The chambers are systems composed of 2 electrical good deal of paper work, it also causes some unrest
plates, with a potential established between them by a with the investigator. The dosimeters should be used in
battery or other electrical source. In effect, they a location where they cannot impact any other objects.
function as capacitors. The plates are separated by an Since the dosimeters normally lack the fragile and
inert gas, which will prevent any current flow between vulnerable quartz windows of a Geiger tube, and carry
the plates. When an ionizing radiation enters the lower voltage potentials, they are used for the
chamber, it induces the formation of an ion, which in measurement of x-ray and high energy gamma
turn is drawn to one of the electrical plates. The radiation, and will not detect beta emissions.
negative ions are drawn to the anode (+ plate), while
the positive ions are drawn to the cathode (– plate). As beta
the ions reach the plates, they induce an electric current
to flow through the system attached to the plates. This insulator coated with Ag or graphite “cathode”
is then expressed as a calibrated output, either through
the use of a digital or analog meter, or as a series of Ar+ Ar+ Ar+
Ar0  Ar+ + e- e- e- e-
clicks, by conversion of the current through a speaker.
Tungsten (W) wire “anode”

non absorbed beta


Photographic Film The light emitted from the crystal is then measured.
Low-energy emissions are detected more conveniently Gamma counters are usually smaller than liquid
through the use of a film badge. This is simply a piece scintillation counters, but are limited to use with
of photographic film sandwiched between cardboard gamma emittors.
and made into a badge, which can be pinned or clipped Modern scintillation counters usually combine the
onto the outer clothing of the investigator. They can be functional capabilities of both liquid scintillation and
worn routinely and collected on a regular basis for direct gamma counting. Since all use of radioactive
analysis. When the film is exposed to radiation, it materials, and particularly the expensive counting
causes the conversion of the silver halide salts to devices, is subject to local radiation safety regulations,
reduced silver (exactly as exposure of the film to the specific details of use must be left to institutional
light). When the film is developed, the amount of discretion. Under no circumstances should radioactive
reduced silver (black) can be measured and calibrated materials be used without the express supervision of
for average exposure to radiation. This is normally the radiation safety officer of the institution, following
done by a lab specializing in this monitoring. Because all specific institutional guidelines and manufacturer
of the simplicity of the system, its relatively low cost, directions for the instrument used.
and its sensitivity to nearly all forms of radiation, it is .
the primary means of radiation exposure monitoring of Electrodes
personnel. Anode
Scintillation Counters
For accurate quantitative measurement of low-energy 
beta emissions and for rapid measurement of gamma e−
emissions, nothing surpasses the use of scintillation
counters. Since they can range from low- to high
energy detection, they are also useful for alpha
emissions. Photocathode Dynodes
Scintillation counters are based on the use of light-
emitting substances, with a radioactive source (liquid
Photomultiplier tube, sensitive light meter
scintillation counter), the radiation strikes the
scintillant molecule, which will then fluoresce as it re-
emits the energy. Thus, the scintillant gives a flash of Solute
light for each radiation particle it encounters. The
counter then converts light energy (either as counts of •Solutes (or fluors) exhibit properties which in many
flashes, or as an integrated light intensity) to an respects are just the opposite of those of solvents.
electrical measure calibrated as either direct counts or •They tend to decay rapidly mainly through the emission
counts per minute (CPM). If the efficiency of the of light photons, thus having a high quantum fluorescent
system is known (the percentage of actual radioactive yield.
decays that result in a collision with a scintillant), then •Solutes that directly absorb the excitation energy of the
disintegrations per minute (DPM) can readily be solvent are also known as primary solutes.
calculated. DPM is an absolute value, whereas CPM is •Secondary solutes were added to ampilify the primary
a function of the specific instrument used. emissions.
Low-energy beta emissions can be detected with •Secondary solutes were also complex organic compounds
efficiencies of 40% or better with the inclusion of the with the ability to absorb the decay energy of the primary
scintillant directly into a cocktail solution. Alpha solute and rapidly emitting it at a longer wavelength,
emissions can be detected with efficiencies in excess shifting the overall signal to a wavelength more easily
of 90%. Thus, with a liquid scintillation counter, very detectable by photomultiplier tubes.
low doses of radiation can be detected. This makes it •As more sensitive photomultiplier tubes were constructed,
ideal for both sensitivity of detection and safety. secondary solutes became unnecessary. However, they
If the system is modified so that the scintillant is a may still be used to improve counting efficiency, as both
crystal placed outside of the sample chamber (vial), the shorter and longer wavelengths can be detected.
then the instrument becomes a gamma counter.
Gamma emissions are capable of exiting the sample
vial and entering into a fluorescent crystal.
Photomulitiplier tube (PMT)
Positron Emission Tonography (PET)
•The emitted light causes the emission of
photoelectrons from the PMT which are multiplied PET is a technique used in clinical medicine and
by the PMT into a measurable electrical pulse. biomedical research to create images that show
•The amplitude of the pulse is proportional to the anatomical structure as well as how certain tissues
number of photons which interact in the PMT. are performing their physiological function. Functional
•The pulse height at the output of the PMT is imaging is the major.
proportional to the energy of the beta particle in the
sample. How does PET work?
•These pulse can be analyzed to provide the energy •The radioisotopes used in PET have very short half
of the beta particle and the rate of beta emission in lives compared to conventional nuclear medicine
the sample. radioisotopes (in the order of minutes).
•Also possible to count very low energy gamma •PET radioisotopes emits a positron (a positively
emitters by LSC since most of the gammas are charged electron) in the process of decay. When this
absorbed in the counting solution. positron collides with an electron, the 2 particles
annihilate each other, and produce 2 photons traveling
Quenching in opposite directions.
A reduction in the total photon output of a •Two detectors positioned opposite one another can be
sample resulting from a reduction in energy used to detect the event.
transfer efficiency. •Many such events are collected in computer memory,
4 basic types of quenching •and are used to make up a 3 dimensional image of the
1. Impurity : strong inorganic acids, oxidizing original distribution of tracer within the patient.
agent, some organic compounds
2. Color : chlorophyll, hemoglobin
3. Dilution : insufficient number of flour molecules
4. Absorption : heterogeneous samples.

Gamma Scintillation Detector


•Scintillators can be made of a variety of materials,
depending on the intended applications.
•The most common scintillators used in gamma-
raydetectors which are made of inorganic
materials are usually an alkali halide salt, such as
NaI or CsI.
•To help these materials do their job, a bit of • The most common radiopharmaceutical or tracer
impurity is often added. This material is called an used in clinical PET imaging is 2- 18F-deoxy-D-
'activator‘. Thallium and sodium are often used for glucose (FDG).
this purpose. • This is transported into the cell like normal
•So one often sees detectors described as NaI(Tl), glucose, but cannot be metabolized after initial
which means it is a sodium iodide crystal with a metabolism, and is trapped within the cell.
thallium activator, or as CsI(Na), which is a cesium • The greater the uptake of the FDG in the cell,
iodide crystal with a sodium activator. the more metabolically active it is.
•TI and Na is used to shift the wavelength of the
photon emitted by the excited molecule to a value Detector
which is not absorbed by the crystal. • Scintillation crystal of Bismuth germanate
•In a solid-state sense, a gamma-ray interacting (BGO) to capture the annihilate photon.
with the crystal moves electrons from the valence • PMT to amplify the signal generated by the BGO
band (by ionization of the molecules) to the annihilation photon capture and convert it into an
conduction band. electric signal.
• Detection/Coincidence circuitry to assure that the
photon which caused the signal is actually a
annihilation photon
AUTORADIOGRAPHY
The site of synthesis of cellular molecules may be
The process of localizing radioactive materials detected by feeding cells a radioactive precursor
onto a cell is known as autoradiography. 3H for a short period and then fixing the cells. During
(tritium) is used in cell analysis because it is a this pulse labeling, radioactivity is incorporated at
relatively weak beta emitter (thus making it safer to the site of synthesis but does not have time to move
handle) and, more significantly, can be localized from this site. The site of utilization of a particular
within cell organelles. 14C and 32P are also used, molecule may be detected by chase labeling. Cells
but are more radioactive, require significantly more are exposed to a radioactive precursor, radio-
precautions in handling, and are inherently less activity is then washed or diluted away, and the
capable of resolving intracellular details. They are cells allowed to grow for a period of time. In this
used at the tissue or organ level of analysis. case, radioactivity is incorporated at the site of
Radioactive isotopes can be incorporated into synthesis, but then has time to move to a site of
cellular molecules. After the cell is labeled with utilization in the cell.
radioactive molecules, it can be placed in contact 3H-thymidine can be used to locate sites of
with photographic film. Ionizing radiations are
synthesis and utilization of DNA. Thymidine, the
emitted during radioactive decay and silver ions in
deoxyribose nucleoside of thymine, can be
the photographic emulsion become reduced to
purchased with the tritium label attached to the
metallic silver grains. The silver grains not only
methyl group of thymine. Thymidine is specifically
serve as a means of detecting radioactivity but,
incorporated into DNA in Tetrahymena. Some
because of their number and distribution, provide
organisms can remove the methyl group from
information regarding the amount and cellular
thymine, and incorporate the uracil product into
distribution of the radioactive label.
RNA.
The process of producing this picture is called
Even in this case, RNA would not be labeled
autoradiography and the picture is called an
because the tritium label would be removed with
autoradiogram..
the methyl group. Methyl-labeled thymidine,
The number of silver grains produced depends on
therefore, serves as a very specific label for DNA.
the type of photographic emulsion and the kind of
This is known as pulse labeling, after which the
ionizing particles emitted from the cell. Alpha
cells are washed free of the radioactive media. All
particles produce straight, dense tracks a few
remaining radioactivity would be due to the
micrometers in length. Gamma rays produce long
incorporation of the thymidine into the
random tracks of grains and are useless for
macromolecular structure of DNA. The cells will
autoradiograms. Beta particles or electrons produce
be fixed, covered with a photographic emulsion,
single grains or tracks of grains. High-energy beta
and allowed to develop.
particles (such as those produced by 32P) may
During this time, the activity emanating from the
travel more than a millimeter before producing a 3H will expose the photographic emulsion, causing
grain. Low-energy beta particles (3H at 14 °C)
the presence of reduced silver grains immediately
produce silver grains within a few micrometers of
above the location of the radioactive source DNA).
the radioactive disintegration site, and so provide
Thus, it will be possible to localize the newly
very satisfactory resolution for autoradiography.
synthesized DNA, or that which was in the S phase
of mitosis during the time period of the pulse
labeling.
Radiation will hit silver grains in emulsion and expose them

Expose to film
or emulsion

Isotope will emit


radiation (usually beta)

Incubate tissue with


radioactive ligand
Radioimmunoassay (RIA)
The amount of hot hormone is typically high enough to
Determination of Hormone Concentration in the saturate all of the antibody molecules attached to the
Plasma
bottom of the tube. Because the interaction between the
The concentration of most hormones in the blood is
antibody and the hormone involves very strong non-
very low; from the low picomolar (10−12) to the
covalent forces, the contents of the tube can be
high nanomolar (10−9 M) range. This very low
discarded without disturbing the hormone molecules
concentration presented serious challenges to early
that are bound to the antibody molecules in the bottom
investigators who were interested in the study of
of the tube. The tube can now be counted in a gamma
hormones. This is because traditional analytical
counter to obtain a counts per minute (CPM). This
means of measurement for steroids and proteins did
value represents the total count, and represents a
not allow detection in the physiological range of
situation when all of the antibody binding sites are
hormone levels. Therefore, the initial tests of
occupied by the hot hormone. We give the label of B0
hormone presence or absence included bioassays.
to this CPM measurement.
Bioassays involved the injection of tissue extracts
into experimental animal models followed by
Summary of Requirements for RIA
observation of some specific anticipated phenotype.
1. Highly specific antibody against the hormone of
For example, long before sensitive laboratory
interest. In modern commercial kits, these are
pregnancy tests were developed, urine samples
covalently attached to the bottom of tube in a way that
from suspected pregnant women were administered
the antibody binding site is still available to bind
to female African clawed frogs (Xenopus laevis).
hormone.
Induction of egg laying by the animal was
2. Radio labeled hormone of interest. The hormone is
indicative of the presence of human chorionic
typically labeled with 125I. Thankfully, this also comes
gonadotropin (hCG). This is a hormone that is
in the commercial kit.
secreted by the placenta.
3. Known amounts of cold hormone in order to obtain a
The technique is called radioimmunoassay (RIA),
standard curve. These known standards are also
so named because it combines the high specificity
provided with commercial kits.
of immunological methods with the high sensitivity
4. Sample tubes that do not have antibody attached to
of radiotracer methods.
their bottom. These are in every respect identical to the
RIA analysis can allow sensitivity down to the
ones containing the antibody, but of course, lack the
femtomolar range (10-15 M)! Therefore, RIA is said
antibody. These will be used to determine non-specific
to have very high sensitivity (i.e., detection at very
binding of the hormone to the walls of the experimental
low concentrations). In addition, because specific
tubes.
antibodies are used (they are carefully screened
during antibody selection), these antibodies react
only with the hormone of interest. Thus, RIA is
said to have very high specificity (i.e., the antibody
does not cross-react with other hormones).
For this work, Yalow received the Nobel Prize in
Physiology or Medicine in 1977, thus, becoming
the first woman to receive this prize. She was also
the first woman to receive the Lasker Prize, which
is the highest honor bestowed on a scientist in the
U.S. This prize is often said to be the ”American
Nobel”.
Principles of RIA
RIA works based on competition for binding
between non-labeled (”cold”) and isotope labeled
(”hot”) hormone for its specific antibody.
Typically, a fixed amount of the antibody is
attached to the bottom of a tube. Then a fixed
amount of hot hormone is added to the tube.
SPECTROPHOTOMETRY
Both are based on a simple design, passing light of a
known wavelength through a sample and measuring
Absorption spectroscopy, also referred to as UV-
the amount of light energy that is transmitted. This is
Visible (UV-Vis) spectroscopy, is one of the
accomplished by placing a photocell on the other side
simplest techniques of optical spectroscopy. The
of the sample. All molecules absorb radiant energy at
technique is most frequently employed in life
one wavelength of another. Those that absorb energy
science laboratories as a method to determine
from within the visible spectrum are known as
concentration or to monitor changes in states (such
pigments. Proteins and nucleic acids absorb light in the
as DNA melting) or chromophore environment
ultraviolet range. The following figure demonstrates
(such as receptor binding).
the radiant energy spectrum with an indication of
Absorption spectroscopy can be used to evaluate
molecules, which absorb in various regions of that
molecules that undergo electronic transitions
spectrum.
excited by ultraviolet (UV) and visible light (190–
The design of the single-beam spectrophotometer
800 nm). Biologically relevant chromophores in
involves a light source, a prism, a sample holder, and a
this region include the peptide bond (210 nm),
photocell. Connected to each are the appropriate
nucleic acid bases (250–260 nm), aromatic amino
electrical or mechanical systems to control the
acid side chains (260–280 nm), heme (400 and
illuminating intensity, the wavelength, and conversion
600 nm), and flavin (450 nm).
of energy received at the photocell into a voltage
fluctuation. The voltage fluctuation is then displayed
A spectrophotometer measures the relative
on a meter scale, is displayed digitally, or is recorded
amounts of light energy passed through a
via connection to a computer for later investigation.
substance that is absorbed or transmitted. We will
use this instrument to determine how much light of
(a) certain wavelength(s) is absorbed by (or
transmitted through) a solution. Transmittance (T)
is the ratio of transmitted light to incident light.
Absorbance (A) = – log T. Absorbance is usually
the most useful measure, because there is a linear
relationship between absorbance and
concentration of a substance. This relationship is
shown by the Beer-Lambert
law:
Absorption Spectrophotometers
A = ebc
A UV-Vis spectrophotometer is a device that measures
where
the amount of light absorbed by the chromophore as a
e = extinction coefficient (a proportionality
function of the wavelength of the electromagnetic
constant that depends on the absorbing species)
radiation. Conventional spectrophotometers consist of
b = pathlength of the cuvette. Most standard
the following: a light source (deuterium lamp for the UV
cuvettes have a 1-cm path and, thus, this can be
region: 190–350 nm and tungsten lamp for the
ignored
wavelength region above 350 nm); a collimator, for
c = concentration.
focusing the beam of light; a monochromator, for wave-
A spectrophotometer or calorimeter makes use of
length selection; a sample compartment; and a detector.
the transmission of light through a solution to
Conventional spectrophotometers have been largely
determine the concentration of a solute within the
replaced by diode array spectrophotometers, which have
solution. A spectrophotometer differs from a
an advantage of speed over the former. A diode array
calorimeter in the manner in which light is
spectrophotometer uses reverse optics, that is, the
separated into its component wavelengths. A
polychromatic light passes through the sample first and
spectrophotometer uses a prism to separate light
is then dispersed onto the diode array. The array
and a calorimeter uses filters.
comprises a series of photodiode detectors juxtaposed on
a silicon chip, with each diode designed to measure a
finite but narrow band of the spectrum. Thus, the entire
spectrum can be collected in a matter of seconds. The
short exposure to the incident light can also minimize
photodecomposition of samples.
Common Applications

1. Concentration Determination
This is a direct application of the Beer–Lambert law.
If the absorption coefficient for a molecule is known,
then the concentration of the molecule in solution can
be obtained by taking an absorption spectrum of the
solution and solving the equation:

If a literature value for the absorption coefficient is


used, it is important to know the buffer concentration,
concentration of any additives, and the pH of the Fig. : Absorption spectra of l-tyrosine below and above
solution. Differences in solution variables can affect an the pKa. The absorption maximum shifts from 275 nm
absorption coefficient. An absorption coefficient is in acidic to 294 nm in basic medium with a concomitant
always reported with the wavelength and the increase in the absorption intensity.
solvent/buffer solution. For instance, the two commonly
Small changes in absorption spectra are most easily
used absorption coefficients for measurement of
observed by difference spectroscopy. An absorption
concentration of anticancer drug paclitaxel for
spectrum of the unperturbed chromophore, such as a
laboratory purposes are ε228 nm, ethanol 2.79 x104 M-1
ligand in the absence of the receptor, is recorded. The
cm-1, ε273 nm, DMSO 1.70 x 103 M-1 cm-1 . Absorption
absorption spectrum of an identical concentration of the
coefficients for proteins are frequently encountered as
ligand in the presence of the receptor is then recorded,
percent solution absorption coefficients (ε%), which has
and the first (unperturbed) spectrum is subtracted from
units of (g/100 ml)-1cm-1. Absorption coefficients for
the second (perturbed) spectrum to yield the difference
DNA and RNA are frequently expressed as (g/L)-1 cm-1.
spectrum. Such measurements can be repeated with
Absorption coefficient data for nucleic acid polymers
varying concentrations of one of the components until
are also expressed in terms of O.D. units at 260 nm. (1
saturation in the signal change is reached. These data can
O.D. unit is equivalent to an absorbance of 1.0.) For
then be used to construct a binding curve.
example, an O.D. unit of 1 at 260 nm for dsDNA
corresponds to a concentration of 50 mg/ml. The most 3. Turbidity Measurements
critical aspect of this procedure is that the concentration Figure in the next page illustrates another function of an
of the original stock solution must be very accurate and absorption spectrophotometer, which is to indirectly
the subsequent dilutions must be carefully performed. detect light scattering. Particles in a solution will scatter
light in a manner dependent on the size of the particle
2. Ligand–Receptor Interactions
and the wavelength of the light. The wavelength
Absorption spectra can be sensitive to the environment of
dependence of the turbidity of a solution of biological
the chromophore, and this sensitivity may be exploited to
macromolecules can be used to estimate the size and
evaluate ligand–receptor interactions, particularly when
shape of the macromolecule. Since turbidity is directly
the ligand absorbs light in a region of the spectrum that is
proportional to absorbance, measurements performed on
different from that of the receptor. Environmental
the absorption spectrophotometer can provide
conditions that affect the ionization state of a
information about molecular weight and dimensions and
chromophore can result in large changes in the absorption
the concentrations of particles or macromolecules in the
maximum and molar absorptivity. For example, the
solution. For example, bacteria scatter light as if they
absorption spectrum of tyrosine at pH above and below
were small particles, and suspensions of bacteria at
the pKa of the phenol is shown in Fig. below. Ionization
sufficient concentration will appear turbid. Measurement
of the phenol results in 18 nm shift in the lowest energy
of turbidity (apparent absorption) is common laboratory
absorption maximum and an increase in the molar
practice to monitor bacterial growth and estimate
absorptivity. A shift in an absorption band to longer
bacteria concentration. Reactions that proceed with
wavelength is a shift to lower energy, and is also called a
protein aggregation or polymer formation may be
red shift or a bathochromic shift. A shift in an absorption
monitored by measuring turbidity changes.
band to shorter wavelength is a shift to higher energy, and
is also called a blue shift or a hypsochromic shift.
Fluorescence Spectroscopy
Fluorescence spectroscopy is one of the most widely
used optical techniques in biochemistry and cell
biology. Highly sensitive and tremendously versatile,
fluorescence spectroscopy can be found in virtually all
areas of life science research. The common instruments
encountered in life science laboratories are steady-state
spectrofluorimeter and fluorescence plate reader. This
chapter will be limited to the types of information
available from the basic versions of these instruments.
Even with these limitations, the coverage here is far
from comprehensive.

Fluorescence
Absorption of a photon of appropriate energy causes an
electronic transition from ground state to an excited
state. Once a molecule is in an electronically excited
Fig. Difference spectrum for hydrazone formation state, the energy must eventually be dissipated for the
reaction as a function of time. molecule to return to its ground state.

4. Microplate Reader Spectrophotometers


The use of plate readers for measuring optical
properties of multiple samples is increasingly common
in life science. Microtiter plates are commonly
available in 6-, 12-, 24-, 48-, 96-, 384-, and 1536-well
formats, and a majority of the commercial instruments
will read 96- and 384-well plates. A significant
difference between absorption data collected in plates
compared to those collected in a standard spectro-
photometer is that the path length in the plate is a
function of the sample volume, while the path length
when measured in spectrophotometer is physically
defined by the dimensions of the cuvette regardless of
Fig. Simplified Jablonski diagram depicting the
the sample volume. Since the absorbance of a solution
excitation of an electron by absorption of a photon
is a function of the path length, it is necessary to use
(hnA) to higher electronic states S1 or S2. The
identical sample volumes in each well in a plate and/or
electron returns to the ground electronic state, S0,
know how the plate reader responds to samples of
from the lowest vibrational state of S1. Fluoresce-nce
varying volumes. There are commercially available
is observed with the release of photon (hnF). The
plate readers that can correct the output data for
dotted and solid arrows in the diagram represent non-
variations in sample volume.
radiative and radiative electronic transitions,
The most common application using microtiter plates
respectively.
are assays involving multiple samples of similar
composition and identical volumes, such as colorimetric
The absorption of a photon occurs very quickly (10–15
assays for cytotoxicity or enzyme-linked immuno-
sec) from the lowest vibrational level of the ground
sorbent assays (ELISAs) using alkaline phosphatase-
state (S0) to higher electronic and vibrational states
conjugated secondary antibodies. Many protocols that
(e.g., S1, S2). The electron relaxes quickly from
require absorption measurements on multiple samples
higher energy states (10–10 to 10–12 sec) to the lowest
are much easier to perform using plate readers than
vibrational level of the first excited state (S1).
conventional spectrophotometers.
This is known as internal conversion. In the Vibrational relaxation of the excited state results in the
absence of photochemical reactions, there are emitted photon originating from the lowest vibrational
two general paths for loss of excited state level of the excited state. Emission of a photon returns the
energy: radiative and nonradiative. Loss of fluorophore to various vibrational levels within the
energy in a radiative pathway involves release of ground state. The vibrational energy levels of the ground
a photon (Fig. on previous page). When the and excited state are about equally spaced, therefore, the
photon comes from the first excited singlet state absorption and emission spectra appear to be reflected
to the ground state, the light released is through a mirror plane. An excitation spectrum is also a
fluorescence. An electron can also undergo plot of the number of photons emitted at a particular
intersystem crossing, that is, move to an excited wavelength; however, in this case, the emission wave-
triplet state from the excited singlet state. The length is held constant and the excitation wavelength is
return of the electron from this triplet state to the varied. The shape of an excitation spectrum of a molecule
ground state may be accompanied by release of is generally the same as its absorption spectrum.
a photon. This emission is referred to as phos- Normally, the shape of an excitation or emission
phorescence and will not be discussed in this spectrum is constant regardless of excitation wavelength,
section. although the overall intensity will vary depending on the
Excited state energy may also be dissipated by absorption coefficient at the excitation wavelength.
nonradiative paths (without emission of a
photon) through mechanisms such as release of 2. Stokes’ Shift
heat, interactions with solvent molecules, The emission maximum of a fluorophore is observed at a
collisions with other molecules, or resonance lower energy (longer wavelength) than the absorption
energy transfer to another chromophore. maximum (Fig. below). This is a consequence of the loss
of vibrational energy in the excited state before emission
Fluorophores of the photon (Fig. last page). The energy difference
A fluorophore may be endogenous to the between the absorption maximum (nA) and the emission
biological system, such as an aromatic amino maximum (nF) is the Stokes’ shift. The Stokes’ shift is
acid residue in a protein. Endogenous or expressed in wave numbers (cm-1). The magnitude of the
intrinsic fluorophores frequently absorb and emit Stokes’ shift of a fluorophore is a function of its
in the UV region. Visible region fluorophores molecular structure and, for many fluorophores, the
are generally exogenous, and may be introduced nature of its surroundings. Environmental effects on the
to a biological system through the use of Stokes’ shift of a fluorophore can be useful to probe some
fluorescently labeled antibodies, chemical aspects of biological systems.
labeling, or expression of green fluorescent
protein or one of its variants. Exogenous or
extrinsic fluorophores can be designed to absorb
and emit virtually any frequency of light,
including near infrared radiation. Both
endogenous and exogenous fluorophores may be
characterized by the following experimental
observation.

1. Excitation and Emission Spectra


An emission spectrum is a plot of number of
photons emitted at a particular wavelength when
the molecule is irradiated at a single wavelength
in an absorption band. In general, the shape of
an emission spectrum is a mirror image of the
absorption spectrum. Absorption of a photon
primarily occurs from the lowest vibrational
level of the ground state to multiple vibrational Fig. Absorption and emission spectrum of 8-anilino-1-
levels within the excited state. naphthalenesulfonic acid (ANS) in dimethyl sulfoxide.
3. Fluorescence Lifetime A fluorophore with a quantum yield of 1.0 emits all
The lifetime of a fluorophore is the average time the absorbed photons as fluorescence. Relative
molecule spends in the excited state. Loss of excited ‘‘brightness’’ of fluorophores can be assessed by
state energy is due to radiative and nonradiative multiplying the absorption coefficient at the
processes. Therefore, lifetime (t) is the inverse of the excitation wavelength by the fluorescence quantum
sum of the rate constant for radiative emission yield. A comparison of a molecule’s fluorescence
(fluorescence, kr) and the rate constants for all intensity with the emission intensity of a fluorophore
nonradiative dissipation of excited state energy whose quantum yield is available in the literature is
(knr): another common way to express relative quantum
yield.

5. Fluorescence Anisotropy
Fluorophores absorb photons that have their electric
The lifetime of a single electronic transition is
dipoles aligned parallel to the transition moment of
characterized by a single exponential decay of
the fluorophore. When freely diffusing fluorophores
fluorescence and is the time required for the fraction
are excited with polarized light, the emitted light will
of the population of molecules in the excited state to
normally be depolarized as a result of rotational
decrease by a factor of 1/e, or ~ 37%:
diffusion during the lifetime of the excited state. The
extent of depolarization is assessed by determining
the intensity of light emitted parallel (Ik) and
perpendicular (I┴) to the excitation light. The
In the above equation, t is the time, t is the fluorescence anisotropy (r) is calculated from these
fluorescence lifetime, F0 is the initial fluorescence at measurements by the following equation:
t= 0. Fluorescence lifetimes are measured by either
pulse fluorometry or phase-modulated fluorometry,
both of which are beyond the scope of this chapter. It
may be noted that the fluorescence lifetime is not
directly affected by the energy or intensity of the light
Applications of anisotropy measurements include
emitted. Thus, fluorophores that have similar spectral
determination of equilibrium binding constants for
characteristics can be distinguished if their lifetimes
ligand–receptor interactions, particularly if the
are different. Discrimination between fluorescence
fluorescence of the ligand is monitored.
lifetimes is the basis for the technique known as
fluorescence lifetime imaging microscopy.
C. Fluorescence Instrumentation
4. Quantum Yield Two types of instruments frequently available in life
The quantum yield (f) of a fluorophore is the ratio of science laboratories are fluorescence. Spectrophoto-
the number of photons emitted as fluorescence to the meters have some resemblance to absorption spectro-
number of photons absorbed by the molecule: photometers: both have light sources and accessories to
control the energy of light incident on the sample,
which is typically in a quartz cuvette. In an absorption
spectrophotometer, the detector is in a straight path to
the light source, but in a fluorescence spectrophoto-
meter, the detector is at a right angle to the light source.
Fluorescence spectrophotometers usually have two
The quantum yield can also be expressed in terms of monochromators, hence the wavelength of light that
the fluorescence lifetime: hits the sample and the detector can be modulated
independently. These instruments can collect two types
of spectra: excitation spectra and emission spectra.
Emission spectra are the more common. The sample is
irradiated with light of a particular energy, selected
with the excitation monochromator. The light emitted
from the sample passes through the emission. Mono-
chromator, which is usually scanned over a range
sufficient to collect data from the entire emission band.
General layout of a fluorimeter

Sample

Tunable light source


(laser, LED, lamp+ monochromator) Spectral dispersion
apparatus
Beamsplitter (filters, monochromator)

Exc. Mono
To the sample
Detector

Measuring Emission and Excitation Spectra


It is always a good idea to take an absorption spectrum of
IRef the sample that will be examined by fluorescence
I P L : I E x c / I R e f  I E x c /( A I E x c )  c o n s t spectroscopy prior to recording fluorescence spectra.

1. Collecting Emission Spectra


Absorption Versus Fluorescence
The most common steady-state fluorescence spectrum is the
The absorption intensity of a molecule in solution will
emission spectrum. To collect the spectrum, a cuvette
be the same regardless of the absorption spectrophoto-
containing the fluorophore is placed in the instrument and
meter used. The same cannot be said for fluorescence
the solution is equilibrated to the desired temperature. The
intensity of the same solution. Absorption intensity is
excitation monochromator is adjusted to the wavelength
defined as the ratio of photons transmitted per photons
chosen for excitation. This is frequently the absorption
absorbed, whereas fluorescence intensity is proporti-
maximum for the fluorophore, but another wavelength
onal to photons emitted. The number of photons
within the absorption band may also be chosen. The
emitted will depend on the number of photons
emission monochromator is set to collect a range of
incident on the sample, which will depend on
wavelengths. The lower limit should be a value greater than
instrumental parameters such as lamp intensity and slit
the excitation wavelength to avoid collecting stray
width. With the exception of quantum yield excitation light. The upper limit should be at a wavelength
measurements, fluorescence spectra are not ratioed, so beyond the end of the emission band. An initial scan of the
the absolute value of fluorescence intensity from the emission spectrum is performed, and the wavelength of the
same sample is not necessarily constant from day to maximum emission intensity is noted. The intensity should
day even on the same instrument under the same be compared to the acceptable range provided with the
experimental conditions. Fluorescence spectra are instrument documentation. The intensity of the emission can
therefore shown with arbitrary units in the legend on be adjusted by methods described in the instrument
the y-axis, although other identifying legends may be documentation, which frequently consists of increasing or
employed (number of photons, fluorescence intensity, decreasing the slits on the excitation or emission
etc.). Unlike absorption spectrum, the intensity of monochromator until a suitable value is achieved. The
fluorescence is largely influenced by the temperature. sample is scanned again using the adjusted slits and
Fluorescence quantum yields are sensitive to tempera- wavelength limits.
ture because the processes by which excited state A blank spectrum of the solution components without the
energy is dissipated (vibrations, collisions) are fluorophore must also be collected using the same
affected by temperature to a greater extent than the parameters employed for the emission spectrum. The blank
process by which the excited state is formed. emission spectrum is subtracted from the sample emission
Therefore, the solution in the cell compartment should spectrum to yield the emission spectrum of the fluorophore.
be controlled at a constant temperature, even when the
spectra are obtained at ‘‘room temperature.’’
Emission spectra may be corrected for fluctuations in
the emission intensity due to features of emission
monochromator and emission photomultiplier tube.

2. Collecting Excitation Spectra


The procedure for collecting an excitation spectrum
is similar to the procedure for collecting emission
spectra, except that the emission wavelength is held
constant and the excitation wavelength region is
scanned. A major difference is that excitation spectra Fig. Fluorescence of a sample is observed at right angle to
must be corrected to be meaningful because the the excitation. (A) The excitation at the front and back of
intensity of light from the excitation source varies the cuvette is same, so no inner filter effect is observed. (B)
with wavelength. The standard method for correcting For a concentrated solution, the excitation of the sample in
excitation spectra is to use a quantum counter in a the cuvette is not uniform and hence lesser amount of
reference channel. The specific procedure for a emission is observed. Inner filter correction needs to be
particular instrument should be found in the performed for this sample. (C) The cuvette has a smaller
instrument manual. excitation path (2 mm) and the emission path is 10 mm.
The use of this dual path length cuvette decreases the inner
Common Experimental Problems and Their filter effect.
Solutions
Most steady-state fluorescence experiments are
2. Secondary Absorption Effect
straightforward to perform. There are, however, some
A secondary absorption effect occurs when a component in
trivial sources of error that are frequently
the sample absorbs the emission light. This is less
encountered by amateur researchers. ‘‘Trivial’’ is
frequently encountered, and typically occurs when more
used in the sense of simple, not unimportant.
than one chromophore is in the sample. The secondary
absorption effect will depend on the concentration of the
1. Inner Filter Effect
species, absorption coeffcient of the acceptor (A), and
Emission spectra are collected assuming that the
quantum yield of the donor (D). The absorption spectrum
same intensity of light hits the front and the back of
of the sample should be examined to ensure that there is
the sample (see Fig.).When the sample absorbs the
little to no absorptivity in the spectral region in which the
excitation light strongly, the intensity of light
emission data are collected. There is no satisfactory way to
diminishes as it passes through the cell. The emission
correct emission spectra for secondary absorption effects,
intensity emanating from the back of the cell is less
so they should be avoided. Secondary absorption effects
than that emanating from the front of the cell (see
can often be eliminated by sample dilution.
Fig.). Therefore, the overall emission intensity will be
lower in a sample with higher absorption at the
3. Photobleaching
excitation wavelength. If the absorption of the sample
Molecules in the excited state can also undergo photo-
is 0.05 units or less over the effective path of the
chemical reactions. If these reactions lead to products with
sample cell, then no inner filter effect is observed. If
different emissive properties, the emission intensity in the
the absorption of the sample is >0.05, then a linear
sample will appear to decrease over time. For most
relationship between concentration of fluorophore
fluorophores, this process is irreversible. Photobleaching
and emission intensity cannot be assumed.
has been used in fluorescence microscopy as fluorescence
Two ways of managing the inner filter effect are
recovery after photo bleaching (FRAP). In in vitro assays,
avoiding it or correcting for it.If possible, avoiding an
however, photobleaching is normally undesirable. Photo
inner filter effect is the better choice. A simple way
bleaching is decreased by limiting the amount of time a
to decrease the light absorbed by the sample is to
fluorophore is exposed to excitation energy. If supplies and
move the excitation wavelength to a region of the
instrumentation permit, the effect of bleaching on a sample
band with lower molar absorptivity. Correction for an
can be minimized by employing relatively large volumes in
inner filter eVect can be done using the equation:
the cuvette and stirring the cuvette during data acquisition.
(Many instruments have magnetic cell stirrers included or
as optional accessories.)
4. Light Scattering
the quencher, and KD is the Stern–Volmer constant for
The presence of particles and bubbles may result in
dynamic quenching.
the scattering of light; these should be removed
A plot of F0/F against [Q] gives KD as the slope. A
from a sample whenever possible. A Raman band
linear Stern–Volmer plot is usually indicative of a
from the solvent may be observed in an emission
single class of fluorophores, all equally accessible to
scan. In biological systems, most commonly
the quencher.
employed solvent is water and the Raman signal is
In static quenching, a non-fluorescent complex is
observed 3600 cm-1 lower in energy than the
formed between the fluorophore and the quencher. The
excitation wavelength. Therefore, for an excitation
quenching equation then becomes:
wavelength of 280 nm, the Raman peak will be
observed at 311 nm. When the sample fluorescence
is intense, the contribution of the Raman band is
negligible. Identifying a peak as a scatter rather than
a fluorescence peak can be accomplished by where Ks is the association constant for the formation
changing the excitation wavelength and rescanning of the complex in static quenching.
the solution. If the peak is fluorescence, the Note that the form of the two equations is the same. In
emission intensity of the peak should change, but order to determine whether a process is due to static or
the wavelength of the emission maximum will not. dynamic quenching, additional experiments must be
If it is a scatter peak, the emission maximum will performed. One simple method is to repeat the
change as the excitation wavelength changes, and quenching experiment at higher temperature. The slope
the intensity of the peak will remain approximately of the plot F/F0 versus [Q] should increase if the
the same. For example, the water Raman peak will process is dynamic quenching and decrease if the loss
move from 311 to 362 nm if the excitation of fluorescence is due to a static quenching mechanism.
wavelength is changed from 280 to 320 nm.

Fluorescence Quenching Environmental Effects on Fluorescence


When the fluorescence intensity of a fluorophore is Absorption and emission spectra of fluorophores with
decreased by its interaction with its environment, this characteristic will be little affected by
the fluorescence is said to be ‘‘quenched.’’ environment; that is, the absorption and emission
Collisional encounters with other molecules in maxima will be similar whether the fluorophore is in
solution that result in deactivation of the excited an aqueous environment or in an apolar pocket of a
state result in collisional quenching. Contact protein. These are called environmentally insensitive
between the fluorophore and the quencher is fluorophores. Environmentally insensitive probes are
required for collisional quenching to occur, particularly useful in imaging. By contrast, when the
therefore, measurements of collisional quenching difference in electron density distribution is
can be useful for determining the accessibility of a pronounced, the absorption and emission spectra can
fluorophore on a biological macromolecule. be severely affected by the molecule’s milieu. These
There are two common types of collisional are referred to as environmentally sensitive
quenching: dynamic quenching and static fluorophores. Fluorophores that are environmentally
quenching. In the former, the quencher collides sensitive can also be used for imaging but are
with the fluorophore in its excited state, dissipating frequently used as sensors or to monitor ligand–
its energy without release of a photon. Dynamic receptor interactions.
quenching of fluorescence is described by the Many of the environmentally sensitive fluorophores
Stern–Volmer equation: used as biological probes have an excited state that is
more polar than the ground state. The effect of
changing the polarity of the environment for such a
probe is illustrated in the next Fig. An apolar
where F0 and F are the fluorescence intensities in environment will stabilize the ground state but
the absence and presence of quencher, destabilize the excited state. The energy difference
respectively. kq is the bimolecular quenching between the two states will increase as the polarity of
constant, t0 is the lifetime of fluorophore in the the solvent decreases. Thus, the absorption and
absence of quencher, [Q] is the concentration of emission maxima will shift to shorter wavelength
(higher energy) A more polar environment will
stabilize the excited state and destabilize the ground
Fluorescence Resonance Energy Transfer
Fluorescence resonance energy transfer (FRET or simply
referred to as RET) occurs when a molecule in its excited
state transfers energy to another molecule through dipole–
dipole interactions, without the appearance of a photon
(see Fig.). The transfer is highly dependent on the
distance between the D and A species. The efficiency of
energy transfer (E) is described by the following equation:

where R is the distance between the D and A and R0 is the


Forster distance, which is the distance at which energy
transfer is 50% efficient. The sharp dependence of RET
efficiency on distance means that energy transfer between
Fig. Jablonski diagram depicting the influence of D and A will be observed only when the pair is within a
solvent on fluorescence of the fluorophore. In this limited range of distances. Here we illustrates the
representation, the dipole moment of the fluorophore relationship between R0 and RET efficiency. A D–A pair
in excited state is greater than the ground state (µE > with a Forster distance of 20 Å will undergo transfer with
µG), and hence a polar environment stabilizes the 15% efficiency at 27 Å and 85% efficiency at 15 Å ; thus,
excited state better. in order for energy transfer to be readily observed, the D
and A need to be within about 12 Å of one another. The
state, and thus the absorption and emission maxima dynamic range is larger when the Forster distance is
will shift to longer wavelength (lower energy). The larger: a D–A pair with a Forster distance of 60 Å will
nature of the environment of a fluorophore in a undergo transfer with 15% efficiency at 80 Å and 85%
biological system can therefore be assessed by efficiency at 45 Å. A good rule of thumb is that the D–A
examining the Stokes’ shift of the fluorophore as a distance (R) should be within a factor of 2 of R0 for
function of solvent and comparing those data to the reliable distance measurements.
Stokes’ shift of the fluorophore in the biological
system. The quantum yield of environmentally
sensitive fluorophores also may be affected by the
environment. The relationship between environment
and quantum yield is less defined than the relationship
between fluorophore environment and Stokes’ shift.
Many of the environmentally sensitive fluorophores
routinely used in biological systems undergo an
increase in quantum yield when the polarity of the
environment is decreased. Since receptor sites are
typically less polar than the medium on the exterior of
the binding site, the increase in fluorescence intensity
Fluorescence resonance energy transfer (FRET or RET)
can be used to quantitatively assess ligand–receptor
between a donor (D) and acceptor (A). The dipole–dipole
interactions
interaction of the electron in the D excited state with the
A few examples of environmentally sensitive
A, results in A excitation.
fluorophores are shown in the Fig. Some environ-
mentally sensitive probes have structural features that
will be affected in a specific way by the environment,
and therefore such molecules can be used as sensors.

The efficiency of resonance energy transfer is determined


by the distance between the D–A pair. The figure
represents D–A pair Forster distance, R0, of: 20 Å (—),
40 Å (. . .), 60 Å (– –), and 80 Å (– –).
Infrared Spectroscopy
In wave numbers, the mid IR range is 4000–400 cm–1.
Theory
An increase in wave number corresponds to an increase
An important tool of the organic chemist is Infrared
in energy. As you will learn later, this is a convenient
Spectroscopy , or IR. IR spectra are acquired on a
relationship for the organic chemist.
special instrument, called an IR spectrometer. IR is
Infrared radiation is absorbed by organic molecules and
used both to gather information about the structure
converted into energy of molecular vibration. In IR
of a compound and as an analytical tool to assess the
spectroscopy, an organic molecule is exposed to
purity of a compound.
infrared radiation. When the radiant energy matches the
energy of a specific molecular vibration, absorption
The Electromagnetic Spectrum
occurs. A typical IR spectrum is shown below. The
Infrared refers to that part of the electromagnetic
wave number, plotted on the X-axis, is proportional to
spectrum between the visible and microwave
energy; therefore, the highest energy vibrations are on
regions. Electromagnetic spectrum refers to the
the left. The percent transmittance (%T) is plotted on
seemingly diverse collection of radiant energy, from
the Y-axis. An absorption of radiant energy is therefore
cosmic rays to X-rays to visible light to microwaves,
represented by a “trough” in the curve: zero
each of which can be considered as a wave or
transmittance corresponds to 100% absorption of light
particle traveling at the speed of light.
at that wavelength.
These waves differ from each other in the length and
frequency, as shown in next figure. (Refers to
classical Einstein equations). Wavelength, λ is the
length of one complete wave cycle. It is often
measured in cm (centimeters). Wavelength and
frequency are inversely related and it is interesting to
note that energy is directly proportional to frequency
and inversely proportional to wavelength.
The IR region is divided into three regions: the near,
mid, and far IR. The mid IR region is of greatest
practical use to the organic chemist. This is the
region of wavelengths between 3 x 10–4 and 3 x 10–3
cm. Chemists prefer to work with numbers which are
easy to write; therefore IR spectra are sometimes
reported in µ m, although another unit, ν (nu bar or
wave number), is currently preferred.

Figure: The IR regions of the electromagnetic spectrum.

Band intensities can also be expressed as absorbance (A).


Absorbance is the logarithm, to the base 10, of the reciprocal of
the transmittance:
A = log 10 (1/T)
Note how the same spectrum appears when plotted as T and
when plotted as A figure below,

Figure: The electromagnetic spectrum


Figure : The IR spectrum of octane, plotted as transmission
(left) and absorbance (right)..
As illustrated in the spectrum of octane, even simple
organic molecules give rise to complex IR spectra.
Both the complexity and the wave numbers of the
peaks in the spectra give the chemist information
about the molecule. The complexity is useful to
match an experimental spectrum with that of a On the other hand, carbon dioxide, CO2, is linear and
known compound with a peak-by-peak correlation. hence has four fundamental vibrations (see Figure below).
To facilitate this analysis, compilations of IR spectra The asymmetrical stretch of CO2 gives a strong band in
are available, most well-known of which are those the IR at 2350 cm–1. The two scissoring or bending
by Sadtler and Aldrich vibrations are equivalent and therefore, have the same
The wave numbers (sometimes referred to as freque- frequency and are said to be degenerate , appearing in an
ncies) at which an organic molecule absorbs IR spectrum at 666 cm–1. The symmetrical stretch of CO2
radiation give information on functional groups is inactive in the IR because this vibration produces no
present in the molecule. Certain groups of atoms change in the dipole moment of the molecule. In order to
absorb energy and therefore, give rise to bands at be IR active, a vibration must cause a change in the dipole
approximately the same frequencies. The chemist moment of the molecule. (The reason for this nvolves the
analyzes a spectrum with the help of tables which mechanism by which the photon transfers its energy to the
correlate frequencies with functional groups. The molecule, which is beyond the scope of this discussion).
theory behind this relationship is discussed in the Only two IR bands (2350 and 666 cm–1) are seen for
next section on molecular vibrations. carbon dioxide, instead of four corresponding to the four
Molecular Vibrations fundamental vibrations. Carbon dioxide is an example of
There are two types of molecular vibrations, why one does not always see as many bands as implied by
stretching and bending. It is known fact that a our simple calculation. In the case of CO2, two bands are
molecule as having rigid bond lengths and bond degenerate, and one vibration does not cause a change in
angles, as when you work with your molecular dipole moment. Other reasons why fewer than the
model sets. This is not the actual case, since bond theoretical number of IR bands are seen include: an
lengths and angles represent the average positions absorption is not in the 4000–400 cm–1 range; an
about which atoms vibrate. A molecule consisting of absorption is too weak to be observed; absorptions are too
n atoms has a total of 3 n degrees of freedom, close to each other to be resolved on the instrument.
corresponding to the Cartesian coordinates of each Additional weak bands which are overtones or
atom in the molecule. In a nonlinear molecule, 3 of combinations of fundamental vibrations are observed.
these degrees are rotational and 3 are translational .
and the remaining correspond to fundamental
vibrations; in a linear molecule, 2 degrees are
rotational and 3 are translational. The net number of
fundamental vibrations for nonlinear and linear
molecules is therefore:

The stretching and bending vibrations for the important


organic group, –CH2, are illustrated in Figure next
page. (The 3n–6 rule does not apply since the –CH2
group represent only a portion of a molecule.) Note that
bending vibrations occur at lower frequencies than
Calculation reveals that a simple molecule such as corresponding stretching vibrations. Both the stretching
propane, C3H8, has 27 fundamental vibrations, and and bending vibrations of a molecule as illustrated in
therefore, you might predict 27 bands in an IR the above figures can be predicted mathematically, at
spectrum! The fundamental vibrations for water, H2O , least to a useful approximation, especially using
are given in Figure below. Water, which is nonlinear, computers. The mathematics of stretching vibrations
has three fundamental vibrations. will be sketched in the following section. An
understanding of these vibrations can help even the
beginning student correlate high and low frequencies in
an IR spectrum.
Figure : Stretching and bending vibrational modes for a CH2 group.

Stretching Vibrations
single bond 5 x 105 dyne/cm
double bond 10 x 105 dyne/cm
triple bond 15 x 105 dyne/cm
As the mass of the atoms increases, the vibration
frequency decreases. Using the following
mass values:
C, carbon 12/6.02 x 1023
H, hydrogen 1/6.02 x 1023

Figure : Energy curve for a vibrating spring (left) and


energy constrained to quantum mechanical model (right).

However, vibrational motion is quantized: it must follow the


rules of quantum mechanics, and the only transitions which are
allowed fit the following formula:
E = (n + 1/2)hν
where ν is the frequency of the vibration n is the quantum
number (0, 1, 2, 3, . . . ) Figure: Energy curve for an anharmonic oscillator
The lowest energy level is E0 = 1/2 hν, the next highest is E1= (showing the vibrational levels for a vibrating bond).
3/2 hν. According to the selection rule, only transitions to the
next energy level are allowed; therefore molecules will absorb ν for a C–H bond is calculated to be 3032 cm–1. (Try
an amount of energy equal to 3/2 – 1/2 hν or hν. This rule is this calculation!) The actual range for C–H
not inflexible, and occasionally transitions of 2 hν, 3 hν, or absorptions is 2850–3000 cm–1. The region of an IR
higher are observed. These correspond to bands called over- spectrum where bond stretching vibrations are seen
tones in an IR spectrum. They are of lower intensity than the depends primarily on whether the bonds are single,
fundamental vibration bands. double, or triple or bonds to hydrogen. The
A molecule is not just two atoms joined on a spring, of course. following table shows where absorption by single,
A bond can come apart, and it cannot be compressed beyond a double, and triple bonds are observed in an IR
certain point. A molecule is actually an anharmonic oscillator. spectrum. You should try calculating a few of these
As the interatomic distance increases, the energy reaches a values to convince yourself that the Hooke’s law
maximum, as seen in the next Figure. Note how the energy approximation is a useful one.
levels become more closely spaced with increasing interatomic
distance in the anharmonic oscillator. The allowed transitions,
hν, become smaller in energy. Therefore, overtones can be
lower in energy than predicted by the harmonic oscillator
theory. The following formula has been derived from Hooke’s
law. For the case of a diatomic molecule,
Although a useful approximation, the motion of two
atoms in a large molecule cannot be isolated from
the motion of the rest of the atoms in the molecule.
In a molecule,two oscillating bonds can share a
common atom. When this happens, the vibrations of
the two bonds are coupled. As one bond contracts,
f is the force constant of the bond (dyne/cm)
the other bond can either contract or expand, as in
asymmetrical and symmetrical stretching. In general,
Equation 7 shows the relationship of bond strength and atomic when coupling occurs, bands at different frequencies
mass to the wave number at which a molecule will absorb IR are observed, instead of superimposed bands as you
radiation. As the force constant increases, the vibrational might expect from two identical atoms in a bond
frequency (wave number) also increases. The force constants vibrating with an identical force constant. In the case
for bonds are: of the –CH2 group in Figure 15.6, you note there are
two bands in the region for C—H bonds: 2926 cm–1
and 2853 cm–1.
General Uses
• Identification of all types of organic and many types of
inorganic compounds
• Determination of functional groups in organic materials
• Determination of the molecular composition of surfaces
• Identification of chromatographic effluents
• Quantitative determination of compounds in mixtures
• Nondestructive method
• Determination of molecular conformation (structural isomers)
and stereochemistry (geometrical
isomers)
• Determination of molecular orientation (polymers and
solutions)

Common Applications
• Identification of compounds by matching spectrum of
unknown compound with reference
spectrum (fingerprinting)
• Identification of functional groups in unknown substances
• Identification of reaction components and kinetic studies of
reactions
• Identification of molecular orientation in polymer films
• Detection of molecular impurities or additives present in
amounts of 1% and in some cases as
low as 0.01%
• Identification of polymers, plastics, and resins
• Analysis of formulations such as insecticides and copolymers
Circular Dichroism
The near-UV CD bands of proteins (310–255 nm)
Circular dichroism (CD) is an excellent method for
derive from Trp, Tyr, Phe, and Cys and reflect the
the study of the conformations adopted by proteins
tertiary, and occasionally quaternary, structure of the
and nucleic acids in solution. Although not able to
protein. Although several amino acid side chains
provide the beautifully detailed residue-specific
(notably Tyr, Trp, Phe, His, and Met) absorb light
information available from nuclear magnetic
strongly in the far-UV region of the spectrum (below
resonance (NMR) and X-ray crystallography, CD
250 nm), the most important contributor here is the
measurements have two major advantages: they can
peptide bond (amide chromophore), with n →π* and
be made on small amounts of material in
π→π* transitions at ~220 and ~190 nm, respectively.
physiological buffers and they provide one of the
The far-UV CD bands of proteins reflect the secondary
best methods for monitoring any structural
structure of the protein (α-helix, β-sheet, β-turn, and
alterations that might result from changes in
unordered content). In the case of nucleic acids and
environmental conditions, such as pH, temperature,
oligonucleotides, the aromatic bases are the principal
and ionic strength. This chapter describes the
chromophores, with absorption beginning at around
important basic steps involved in obtaining reliable
300 nm and extending far into the vacuum UV region.
CD spectra: careful instrument and sample
The electronic transitions of the ether and hydroxyl
preparation, the selection of appropriate parameters
groups of the sugars begin at 200 nm, but their
for data collection, and methods for subsequent data
intensity is much weaker than that of the bases, and the
processing. The principal features of protein and
electronic transitions of the phosphate groups begin
nucleic acid CD spectra are then described, and the
further still into the vacuum.
main applications of CD are discussed. These
include: methods for analyzing CD data to estimate
Although CD spectroscopy generally provides only
the secondary structure composition of proteins,
low resolution structural information, it does have two
methods for following the unfolding of proteins as a
major advantages. First, it is extremely sensitive to
function of temperature or added chemical
changes in conformation, whatever their origin, and
denaturants, the study of the effects of mutations on
second, an extremely wide range of solvent conditions
protein structure and stability, and methods for
is accessible to study with relatively small amounts of
studying macromolecule–ligand and macromolecule
material. The principal applications of CD
–macromolecule interactions.
spectroscopy in the study of biomolecules are,
CD, the differential absorption of left- and right-
1. The estimation of protein secondary structure
handed circularly polarized light, is a spectroscopic
content from far-UV CD spectra.
property uniquely sensitive to the conformation of
2. The detection of conformational changes in proteins
molecules, and so has been very widely used in the
and nucleic acids brought about by changes in pH, salt
study of biomolecules. CD often provides important
concentration, and added co-solvents and the structural
information about the function and conformation of
analysis of recombinant native proteins and their
biomolecules that is not directly available from
mutants
more conventional spectroscopic techniques, such
3. Monitoring protein or nucleic acid unfolding brought
as fluorescence and absorbance. The experimentally
about by changes in temperature or by the addition of
measured parameter in CD is the difference in
chemical denaturants (such as urea and guanidine
absorbance for left- and right-handed circularly
hydrochloride)
polarized light, ΔA (= AL − AR). Because CD is an
4. Monitoring protein–ligand, protein–nucleic acid,
absorption phenomenon, the chromophores that
and protein–protein interactions e. Studying (in
contribute to the CD spectrum are exactly the same
favorable cases) the kinetics of macromolecule–
as those contributing to a conventional absorption
macromolecule, macromolecule–ligand interactions
spectrum. In order to show a CD signal, a
(particularly slow dissociation processes), and the
chromophore must be either inherently chiral
kinetics of protein folding reactions. The general
(asymmetric) or must be located in an asymmetric
principles of the most common kinetic methods .
environment. It is the interaction between the
chromophores in the chiral field of the protein that
introduces the perturbations leading to optical
activity.
Instrumentation
CD instruments are commercially available from The CD spectra of membrane proteins are often
several sources: A Peltier system for temperature recorded in detergent solubilized form in order to
control and thermal ramping is an invaluable avoid artifacts arising from differential light scattering
accessory, particularly for studies of the thermal and absorption flattening. The far-UV CD spectra of
unfolding of proteins and nucleic acids. The only proteins (260–178 nm) are intense, and relatively
other significant requirement is for a set of high small amounts of material are required to record them.
quality quartz cuvettes with good far-UV Because all peptide bonds contribute to the observed
transmission with path lengths ranging from 0.1 to spectrum, the amount of material required (measured
10 mm. Cuvettes should always be cleaned in mg/ml) is effectively the same for any protein. The
immediately after use in order to avoid the buildup near-UV CD spectra of proteins are generally more
of hard-to-remove protein deposits. than an order of magnitude weaker than the far-UV
CD spectra. Recording them therefore requires more
Instrument Care and Calibration concentrated material and/or longer optical path
The CD instrument should always be purged with lengths.
high-purity, oxygen-free, nitrogen (generally run at
3–5 L/min) for at least 20 min before starting the Determination of Sample Concentration
light source and throughout the measurements. If Accurate sample concentrations are absolutely
oxygen is present, it may be converted to ozone by essential for the analysis of far-UV CD spectra for
the far-UV light from the high-intensity arc, and secondary structure content and whenever one wishes
ozone will damage the expensive optical surfaces. to make meaningful comparisons between different
Higher nitrogen flow rates will generally be protein or nucleic acid samples. We routinely
necessary for measurements made at very short determine protein and nucleic acid concentrations
wavelengths. The calibration of the instrument using absorption spectroscopy. When the extinction
should be checked periodically. Although several coefficient is known, the concentration can be
CD standards are available, the one used most calculated with considerable accuracy. The absorption
frequently is d10 camphorsulfonic acid (d10-CSA). spectrum should ideally be recorded with temperature
The exact concentration (C) of a solution of d10- control and careful attention should be given to correct
CSA in water (at 2.5 mM) should be determined baseline subtraction, especially when buffers
from an absorption spectrum (using ε285 = 34.5 M-1 containing reducing agents are being used. Highly
cm-1). This measurement also provides a useful scattering samples should always be clarified by low-
check on the wavelength calibration of the speed centrifugation or filtration prior to concentration
instrument, although this can also simply be done determination. If the spectrum still shows significant
by scanning with a holmium oxide filter in the light light scattering, that is, significant background
path and monitoring the voltage on the instrument’s absorption above 315 nm, a correction must be
photomultiplier. applied.

Sample Preparation Data Collection


All samples should, of course, be of the highest Having chosen the appropriate sample concentration
possible purity. For example, the weak near-UV and cuvette path length for the measurement, the user
signals of proteins can be swamped by the strong will need to select suitable instrument settings.
signals from relatively small levels of Consideration should also be given to the selection of
contaminating nucleic acids. The absorption an appropriate temperature for the measurement.
spectrum of the sample should always be checked
to see if and where this absorbance limit is going to A. Wavelength Range
be exceeded. In far-UV measurements the Far-UV spectra of proteins should generally be
absorbance of the sample itself is generally rather scanned from 260 nm to the lowest attainable
small, and the major problems arise from wavelength. This low-wavelength limit will depend
absorption by buffer components, almost all of largely on the composition of the buffer being used.
which will limit far-UV penetration to some extent. Near-UV spectra are routinely scanned over the range
The majority of simple buffer components will 340–255 nm for proteins and from 340 nm to the
generally permit CD measurements to below 200 lowest attainable wavelength for nucleic acids.
nm.
B. Scanning Speed and Time Constant
In the case of analogue instruments, the product of the
scanning speed (nm min-1) and the time constant (sec)
should be less than 20 nm min-1 sec. If the instrument
where L is the path length (in cm), CM is the molar
uses a response time (equal to three time constants),
concentration, Cmg/ml is the concentration in mg/ml, and
then the product of scanning speed and response time
MRW is the mean residue weight (molecular weight
should be less than 60 nm min-1 sec. If significantly
divided by the number of residues). Although large
higher values are used, there will be potentially
globular proteins generally have a mean residue weight
serious errors in both the positions and intensities of
of approximately 111, the actual value must always be
the observed CD bands. Good S/N ratios can therefore
calculated in order to avoid potentially large errors in
be achieved either by averaging multiple fast scans
the calculated intensities. Calculating far-UV intensities
recorded with a short time constant or by recording a
is almost invariably done on a per residue basis in order
small number of slow scans with a long time constant.
to facilitate comparison between proteins and peptides
The choice is largely one of personal preference.
with different molecular weights. Near-UV CD
intensities should generally be reported on a molar
C. Spectral Bandwidth
rather than a per-residue basis because only four of the
The spectral bandwidth is generally set to 1 nm, but it
amino acid side chains contribute to the CD signals in
may occasionally be necessary to use lower values in
this region. In the case of nucleic acids, the CD
order to resolve fine structure in near-UV spectra of
intensities can be calculated using the base, base pair,
proteins. Increasing the spectral bandwidth will reduce
or molar concentrations.
the noise by increasing light throughput, but it should
CD intensities are also sometimes reported as molar
always be 2 nm or less in order to avoid distorting the
ellipticity ([θ]M) or mean residue ellipticity ([θ]mrw),
spectrum.
which may be directly calculated as: [θ] and Δε values
may be interconverted using the relationship [θ]=3298
D. Temperature Control
Δε.
It is good practice to always record CD spectra with
temperature control. This is particularly important for
the far-UV CD spectra of proteins, which often show
quite pronounced temperature dependence, even
outside the range of any thermally induced unfolding
B. Spectral Characteristics
of the protein. These small changes in the signal from
1.Near-UV Spectra of Proteins
the folded protein with temperature reflect a true
Near-UV CD bands from individual residues in a protein
change in conformation and are not simply due to
may be either positive or negative and may vary
changes in the optical properties of a helix or strand.
dramatically in intensity, with residues that are producing
The changes, which are often linear with temperature,
the strongest signals. Knowledge of the position and
are probably due to fraying of the ends of a helix or to
intensity of CD bands expected for a particular
changes in helix–helix interactions.
chromophore is helpful in understanding the
observed near-UV CD spectrum of a protein and the
Data Processing and Spectral Characteristics
principal characteristics of the four chromophores are
Data Processing
therefore summarized,
The first step is to subtract the baseline scan from the
i). Phenylalanine has sharp fine structure in the range 255–
sample scan. All spectra should have been collected
270 nm with peaks generally observed close to 262 and
with a starting wavelength that gives at least 15–20 nm
268 nm (ΔεM = ± 0.3 M-1 cm-1).
at the start of the scan where there should be no signal.
ii). Tyrosine generally has a maximum in the range 275–
After baseline subtraction this region should be, and
282 (ΔεM = ± 2.0 M-1 cm-1), possibly with a shoulder
generally is, flat, but the signal may not be zero. The
some 6 nm to the red.
spectrum should be converted to the desired units. In
iii). Tryptophan often shows fine structure above 280 nm
the case of proteins, the observed CD signal, S in
in the form of two Lb bands [one at 288 to 293 and one
millidegrees (Note: 1 millidegree =32,980 ΔA), is
some 7 nm to the blue, with the same sign ((ΔεM = ± 5.0
generally converted to either the molar CD extinction
M-1 cm-1)] and a La band (around 265 nm) with little fine
coefficient (ΔεM) or to the mean residue CD extinction
structure (ΔεM = ± 2.5 M-1 cm-1).
coefficient (Δε MRW) using:
iv). Cystine CD begins at long wavelength (>320 nm) APPLICATIONS
and shows one or two broad peaks above 240 nm (ΔεM
= ± 1.0 M-1 cm-1). 1. Secondary Structure Content of Proteins
The estimation of protein secondary structure content
2. Far-UV Spectra of Proteins from far-UV CD spectra is one of the most widely
Far-UV CD spectra of proteins depend on secondary used applications of CD. several recent experiments,
structure content and simple inspection of a spectrum particularly those using site-directed mutagenesis, have
will generally reveal information about the structural shown that aromatic residues can make surprisingly
class of the protein. The characteristic features of the large contributions to the far-UV CD spectra of some
spectra of different protein classes may be summarized proteins and this obviously has serious implications for
as follows. secondary structure estimation. Some useful programs
i). All α-proteins show an intense negative band with are available for the analysis of far-UV CD data of
two peaks (at 208 and 222 nm) and a strong positive proteins these include K2D; and CDNN. On the other
band (at 191–193 nm). The intensities of these bands hand three very popular programs (SELCON,
reflect α-helical content. Δεmrw values for a totally CDSSTR, and CONTIN/LL)provided with several
helical protein would be of the order of −11M-1cm-1 (at reference sets with different wavelength ranges are
208 and 222 nm) and +21 M-1cm-1(at 191–193 nm). available on the Internet.
ii The spectra of regular all-β-proteins are significantly
weaker than those of all-α-proteins. These spectra 2. Detecting Altered Conformation
usually have a negative band (at 210–225 nm, Δεmrw : The stability of any particular secondary structure
−1 to −3.5 M-1cm-1) and a stronger positive band (at element in a protein or nucleic acid will depend on
190–200 Δεmrw : 2 to 6M-1cm-1) . several different factors . Changes in the pH and ionic
iii). Unordered peptides and denatured proteins have a strength of the solution will generally affect
strong negative band (at 195–200 nm, Δεmrw : −4 to −8 interactions between charged side chains in proteins,
M-1cm-1 ) and a much weaker band (which can be either and this can lead to significant changes in secondary
positive or negative) between 215 and 230 nm (Δεmrw : and/or tertiary structure. Such changes are readily
+0.5 to −2.5 M-1cm-1 ). studied using CD. Similarly, change in structure with
iv). α+β and α/β proteins almost always have spectra increasing temperature can be determined by
dominated by the α-helical component and therefore measuring the CD spectrum in the solvent at room
often show bands at 222, 208, and 190–195 nm. In temperature and comparing it with the spectrum
some cases, there may be a single broad minimum measured in an aqueous buffer at particular
between 210 and 220 nm because of overlapping α- temperature.
helical and β-sheet contributions.
3. Changes Accompanying Complex Formation
3. Nucleic Acids Protein–protein and protein–nucleic acid interactions
Because the aromatic bases themselves are planar, they are often accompanied by changes in the intrinsic CD
do not possess any intrinsic CD signals; it is the of one or both of the components owing to changes in
presence of the sugars that creates the asymmetry which secondary structure and/or the environment of
leads to the small CD signals of the monomeric aromatic groups. Such changes may also be caused by
nucleotides. Likewise, the stacking of the bases in the the binding of small ligands, such as drugs and metal
different polymeric forms results in the close contact ions, to macromolecules, and in certain cases the
and electronic interactions that produce the intense CD ligand itself may change its optical activity or become
signals of the nucleic acids and oligonucleotides. CD is optically active.
sensitive to secondary structure because the precise
nature of these interactions determines the shape of the 4. Structural Analysis of Recombinant Native
spectrum. The principal conformational forms of the Proteins and Their Mutants
nucleic acids are the A- and B-forms. In neutral When working with mutant proteins, it is, of course, a
aqueous buffers at moderate salt concentrations, DNA good practice to test for any effect of the mutation on
is usually in the B-form, while RNA adopts the A-form. the general conformation of the protein and, here
These conformations have characteristic CD spectra again, CD provides a convenient means of doing this
that depend on base composition and somewhat on with the limited amounts of material that are
sugar type. Variation in spectral shape with base sometimes available. A significant difference in shape
composition is, of course, significantly more important between the far-UV CD spectra of the wild-type and
with short oligonucleotides. mutant proteins can be an indication that the mutation
has produced some change in the secondary structure.
5. CD in the Study of Protein Stability
Unfolding of macromolecules is generally studied by NMR Spectroscopy
using an optical method (absorbance, fluorescence, or
CD) or by using diVerential scanning calorimetry
NMR is a versatile tool and it has applications in wide
(DSC). CD is very widely employed in the study of
varieties of subjects in addition to its chemical and
protein stability because unfolding is almost
biomedical applications, including material and quantum
invariably accompanied by major changes in both the
computing. In 939 Rabi et al. First detected unclear
near and far-UV CD spectra. The free energy of
magnetic resonance phenomenon by applying r.f. energy
unfolding is determined by monitoring an appropriate
to a beam of hydrogen molecules in the Stern-Gerach
CD signal as a function of the concentration of a
set up and observed measurable deflection of the beam.
chemical denaturant (urea or guanidine hydrochloride)
In 1991 and 2002 Nobel prize in Chemistry was awarded
or as a function of temperature.
to Richard Ernst and Kurt Wuthrich respectively for their
contribution in NMR spectroscopy.
6. Determination of Equilibrium Dissociation
Before going to NMR, we should know what are N, M,
Constants
and R ?
As noted in the previous section, the interaction of a
Nucleus: Nuclear spin and Nuclear magnetic moments.
macromolecule with another macromolecule or small
Magnetic: The Nucleus in a Magnetic Field include,
molecule is often associated with a change in the CD
Nuclear Zeeman effect & Boltzmann distribution.
signal of one or both of the components We will do
Resonance: When the nucleus meet the right magnet and
this with reference to the simplest possible binding
radio wave.
model, formation of a 1:1 complex according to the
Nuclear spin:
following scheme.
Nuclear spin is the total nuclear angular momentum
quantum number. This is characterized by a quantum
number I, which may be integral, half-integral or 0. Only
nuclei with spin number I≠0 can absorb/emit
The primary requirement for a direct titration, as with electromagnetic radiation. The magnetic quantum number
any spectroscopic method, is that the sum of the spectra mI has values of –I, -I+1, … ..+I . ( e.g. for I=3/2, mI=
of the components (A and B) should be different from –3/2, –1/2, 1/2, 3/2),
that of the complex (AB) at some wavelength. 1. A nucleus with an even mass A and even charge Z→
nuclear spin I is zero
Summary Example: 12C, 16O, 32S à No NMR signal
In summary, we hope to have shown that CD remains 2. A nucleus with an even mass A and odd charge Z →
an excellent technique for studying the structures of integer value I
proteins and nucleic acids in solution. CD Example: 2H, 10B, 14N → NMR detectable
measurements allow one to quickly determine if a 3. A nucleus with odd mass A → I=n/2, where n is an odd
protein is folded and, if so, to characterize its secondary integer
structure content. Such measurements therefore enable Example: 1H, 13C, 15N, 31P → NMR detectable
one to compare the structures of different mutants of a
protein, and of different samples of a protein obtained Nuclear magnetic moments
from different species or using different expression Magnetic moment m is another important parameter for a
systems. The great sensitivity of CD to changes in nuclei µ = γI (h/2Ω).
conformation also makes it an excellent technique for I: spin number; h: Plank constant;
studying the effects of changes in environmental γ: gyromagnetic ratio (property of a nuclei).
conditions (such as pH, temperature, and ionic
strength), for determining protein stability using either Precession and the Larmor frequency
chemical or thermal denaturation studies, and (in •The magnetic moment of a spinning nucleus processes
appropriate cases) for determining equilibrium with a characteristic angular frequency called the Larmor
dissociation constants. One final remark is worth frequency w, which is a function of r and B0
making. It has been emphasized the unfortunate fact Remember µ = γI (h/2π) ?
that CD measurements are often severely compromised Angular momentum dJ/dt= µ x B0
by inappropriate experimental design or by a lack of Larmor frequency ω= γ B0
attention to important key aspects of instrument Linear precession frequency,
calibration and sample characterization. We hope that v= ω/2π = γ B0/2π
this chapter shows how reliable CD data can be
obtained, analyzed, and understood.
Nuclear Magnetic Resonance Spectrometer
Quantum mechanics tells us that, for net absorption
How to generate signals?
of radiation to occur, there must be more particles in
the lower-energy state than in the higher one. If no B0: Magnet
net absorption is possible, a condition called B1: Applied small energy
saturation. When it’s saturated, Boltzmann
distribution comes to rescue:

where P is the fraction of the particle population in


each state, T is the absolute temperature,
k is Boltzmann constant 1.381x10-28 JK-1

What happen before irradiation


Before irradiation, the nuclei in both spin states are
Example: At 298K, what fraction of 1H nuclei in 2.35 processing with characteristic frequency, but they are
T field are in the upper and lower states? (m= −1/2 : completely out of phase, i.e., randomly oriented around
0.4999959 ; m=1/2 : 0.5000041 ) the z axis. The net nuclear magnetization M is aligned
The difference in populations of the two states is only statically along the z axis (M= Mz, Mxy=0).
on the order of few parts per million. However, this
difference is sufficient to generate NMR signal.
Anything that increases the population difference
will give rise to a more intense NMR signal.

Nuclear Magnetic Resonance


For a particle to absorb a photon of electromagnetic
radiation, the particle must first be in some sort of
uniform periodic motion . If the particle “uniformly What happen during irradiation
periodic moves” (i.e. precession) When irradiation begins, all of the individual nuclear
at vprecession, and absorb erengy. magnetic moments become phase coherent, and this
phase coherence forces the net magnetization vector M to
The energy is E=hvprecession process around the z axis. As such, M has a component
in the x, y plan, Mxy=Msinα. a is the tip angle which is
determined by the power and duration of the
For I=1/2 nuclei in B0 field, the energy gap
electromagnetic irradiation.
between two spin states:
ΔE=rhB0/2π

The radiation frequency must exactly match the


precession frequency
What happen after irradiation ceases
After irradiation ceases, not only do the population of
the states revert to a Boltzmann distribution, but also
This is the so called “ Nuclear Magnetic
the individual nuclear magnetic moments begin to lose
RESONANCE”!!!!!!!!!
their phase coherence and return to a random
arrangement around the z axis. (NMR spectroscopy
record this process!!). This process is called “relaxation
process”. There are two types of relaxation process :
T1(spin-lattice relaxation) & T2(spin-spin relaxation)
B1(the irradiation magnet, current induced)
(1) Induce energy for nuclei to absorb, but still spin at
w or vprecession,
Ephoton=hvphoton=DE=rhB0/2π=hvprecession
And now, the spin jump to the higher energy ( from
m= 1/2m → m= – 1/2).

NMR Parameters
Chemical Shift
The chemical shift of a nucleus is the difference
between the resonance frequency of the nucleus and a Nuclear Overhauser Effect (NOE)
standard, relative to the standard. This quantity is The NOE is one of the ways in which the system (a certain
reported in ppm and given the symbol delta, spin) can release energy. Therefore, it is profoundly related
d = (n - nREF) x106 / vREF to relaxation processes. In particular, the NOE is related to
In NMR spectroscopy, this standard is often exchange of energy between two spins that are not scalarly
tetramethylsilane, Si(CH3)4, abbreviated TMS, or 2,2- coupled (JIS = 0), but have dipolar coupling.
dimethyl-2-silapentane-5-sulfonate, DSS, in The NOE is evidenced by enhancement of certain signals in
biomolecular NMR. The good thing is that since it is a the spectrum when the equilibrium (or populations) of other
relative scale, the d for a sample in a 100 MHz magnet nearby are altered. For a two spin system, the energy
(2.35 T) is the same as that obtained in a 600 MHz diagram is as following:
magnet (14.1 T).

Electron surrounding each nucleus in a molecule serves


to shield that nucleus from the applied magnetic field.
This shielding effect cause the DE difference, thus,
W represents a transition probability, or the rate at which
different v will be obtained in the spectrum
certain transition can take place. For example, the system in
equilibrium, there would be WII and WIS transitions, which
represents single quantum transitions.

NOE could provide information of distance between two


atoms: NOE / NOEstd = rstd6/r 6
Thus, NOE is very important parameter for structure
Beff=B0-Bi where Bi induced by cloud electron
determination of macromolecules
Bi = sB0 where s is the shielding constant
Beff=(1-s) B0
vprecession= (rB0/2π) (1-s)
s=0 à naked nuclei
s >0 à nuclei is shielded by electron cloud
s <0 à electron around this nuclei is withdraw
, i.e. deshielded
NMR Parameters employed for determining protein Solid-state NMR spectroscopy
structure Main article: Solid-state nuclear magnetic resonance
This technique complements biopolymer X-ray
1. Chemical Shift Indices: Determining secondary
crystallography in that it is frequently applicable to
structure.
biomolecules in a liquid or liquid crystal phase, whereas
2. J-coupling: Determine dihedral angles. (Karplus
crystallography, as the name implies, is performed on
equation)
molecules in a solid phase. Though nuclear magnetic
3. Nuclear Overhauser Effect (NOE): Determine inter-
resonance is used to study solids, extensive atomic-level
atomic distances (NOE µ R-6)
biomolecular structural detail is especially challenging
4. Residual dipolar coupling: Determine bond
to obtain in the solid state. There is little signal
orientations.
averaging by thermal motion in the solid state, where
.5. Relaxation rates (T1, T2 etc): Determine
most molecules can only undergo restricted vibrations
macromolecular dynamics.
and rotations at room temperature, each in a slightly
Multi-dimensional NMR Spectroscopy different electronic environment, therefore exhibiting a
The use of pulses of different shapes, frequencies and different NMR absorption peak. Such a variation in the
durations in specifically-designed patterns or pulse electronic environment of the resonating nuclei results
sequences allows the spectroscopist to extract many in a blurring of the observed spectra—which is often
different types of information about the molecule. only a broad Gaussian band for non-quadrupolar spins
Multi-dimensional nuclear magnetic resonance in a solid- thus making the interpretation of such
spectroscopy is a kind of FT NMR in which there are at "dipolar" and "chemical shift anisotropy" (CSA)
least two pulses and, as the experiment is repeated, the broadened spectra either very difficult or impossible.
pulse sequence is varied. In multidimensional nuclear
magnetic resonance there will be a sequence of pulses Applications
and, at least, one variable time period. In three Medicine: The use of nuclear magnetic resonance best
dimensions, two time sequences will be varied. In four known to the general public is magnetic resonance
dimensions, three will be varied. imaging for medical diagnosis and MR Microscopy in
There are many such experiments. In one, these time research settings, however, it is also widely used in
intervals allow (amongst other things) magnetization chemical studies, notably in NMR spectroscopy such as
transfer between nuclei and, therefore, the detection of proton NMR, carbon-13 NMR, deuterium NMR and
the kinds of nuclear-nuclear interactions that allowed phosphorus-31 NMR. Biochemical information can also
for the magnetization transfer. Interactions that can be be obtained from living tissue (e.g. human brain tumors)
detected are usually classified into two kinds. There are with the technique known as in vivo magnetic
through-bond interactions and through-space resonance spectroscopy or chemical shift NMR
interactions, the latter usually being a consequence of Microscopy.
the nuclear Overhauser effect. Experiments of the
nuclear Overhauser variety may be employed to Chemistry: By studying the peaks of nuclear magnetic
establish distances between atoms, as for example by resonance spectra, chemists can determine the structure
2D-FT NMR of molecules in solution. of many compounds. It can be a very selective
Although the fundamental concept of 2D-FT NMR was technique, distinguishing among many atoms within a
proposed by Professor Jean Jeener from the Free molecule or collection of molecules of the same type
University of Brussels at an International Conference, but which differ only in terms of their local chemical
this idea was largely developed by Richard Ernst who environment. See the articles on carbon-13 NMR and
won the 1991 Nobel prize in Chemistry for his work in proton NMR for detailed discussions.
FT NMR, including multi-dimensional FT NMR, and
especially 2D-FT NMR of small molecules. Multi- Non-destructive testing: Radio waves and static
dimensional FT NMR experiments were then further magnetic fields easily penetrate many types of matter
developed into powerful methodologies for studying and anything that is not inherently ferromagnetic. For
biomolecules in solution, in particular for the example, various expensive biological samples, such as
determination of the structure of biopolymers such as nucleic acids, including RNA and DNA, or proteins,
proteins or even small nucleic acids. can be studied using nuclear magnetic resonance for
Kurt Wüthrich shared (with John B. Fenn) in 2002 the weeks or months before using destructive biochemical
Nobel Prize in Chemistry for his work in protein FT experiments. This also makes nuclear magnetic
NMR in solution. resonance a good choice for analyzing dangerous
samples.
Mass spectrometry a). Sector mass spectrometer
Mass spectrometry is an incredibly important
analytical technique for the identification of molecules
by way of measuring their mass-to-charge ratios, m/z,
in the ionized state. It is particularly useful for the
detection and analysis of traces of macromolecules
down to less than 1 pg (10–12 g). The general design
of a mass spectrometer comprises sample injector,
sample ionizer, mass analyzer and ion detector (Fig.
1). First the s ample is injected into the ionizer which
ionizes sample molecules. Then sample ions are
analyzed and detected. To prevent collisions with gas
molecules, sample ionizer, mass analyzer and ion
detector are generally operated in vacuum.

Fig. 1: General design of a mass spectrometer

The ion separation power of mass spectrometers is


described by the resolution, R, defined as:

where m and Δm are the ion mass and mass difference


between two resolvable peaks in the mass spectrum,
respectively. R typically ranges between 100 and
Fig. 2 Single magnetic or electric sector mass
500,000.
spectrometer with a single channel (a) and multi-
channel (b) detector, respectively. Ions leaving the
Principles of operation and types of spectrometers
ion source are accelerated and passed through the
sector in which the electric or magnetic field is applied
According to their mass analyzer designs, there are
perpendicular to the direction of the ion movement. The
five important types of mass spectrometers (MS): (a)
field bends the ion flight path and causes ions with
magnetic and/or electric sector MS (Figs. 2 and 3),
different m/z to travel on different paths. In scanning
(b) quadrupole MS (Fig. 4), (c) ion trap MS (Fig. 5),
mass analyzers (a) the electric or magnetic field
(d) time-of-flight MS (Figs. 6–8), and (e) Fourier
strength is varied and only one mass detected at a time.
transform MS (Fig. 9). Time-of-flight mass
In non-scanning mass analyzers (b) all masses are
spectrometers (TOFs) often are less expensive than
recorded simultaneously within a limited mass range
other types of mass spectrometers and have,
with the help of a multichannel detector.
compared to quadrupole MS and many sector MS,
the advantage of recording the masses of all ions
injected into the analyzer without scanning,
contributing to a high sensitivity. TOFs usually have
a smaller mass range and resolving power than
Fourier transform mass spectrometers (FTMS).

Fig. 3. Advanced virtual image ion optics with high transmission


in a benchtop single sector mass spectrometer.
b). Quadrupole mass spectrometer For a length, lTOF, the time of flight, t, is:

For example, for lTOF = 0.1 m, z = e = 1.602× 10–19 C, m


= 10 kDa = 10 kg /6.0221× 1023, and V = 100 V we obtain
t = 72 µs. A mass resolution of 1 Da requires in this
example a time resolution of 3.6 ns. Unfortunately, not all
ions start to move at the same time and not all ions have
the same velocity. The differences in velocity are called
Fig. 4. Quadrupole mass spectrometer. The ion beam is chromatic aberration. Due to the chromatic aberration
accelerated to a high velocity by an electric field and and the differences in the starting time, the requirements
passed through the quadrupole mass analyzer comprising for a very high resolution are hard to meet in the simple
four metal rods. DC and AC potentials are applied to the design of a linear TOF (Fig. 6). In reflectron TOFs (Figs.
quadrupole rods in such a way that only ions with one mass- 7–8), the ion optics reverses the flight direction of the ions
to-charge ratio (m/z) can pass though the analyzer at a time. and reduces chromatic aberration.
To scan different m/z, DC and AC potentials are varied
c). Ion trap mass spectrometer

Fig. 5 Ion trap mass analyzer. With the help of


different radio frequency signals applied to the ring
electrode and the endcaps, all ions are trapped in the cavity Fig. 6 Linear time-of-flight mass spectrometer (TOF)
and then sequentially ejected according to their m/z with matrix-assisted laser desorption ionization
(MALDI). The linear configuration of TOFs represents
the simplest implementation of the time-of-flight
d). Time-of-flight mass spectrometer
technique. The typical mass range lies between 0 and 100
In time-of-flight mass spectrometers, a uniform starting
kDa, and the typical mass resolution m/Δm is 300–2000
time of ions is caused, e.g., by a pulse of an ionizing
laser (Fig. 6) or a voltage pulse to an electric shutter (Fig.
7). After passing through the accelerating potential
difference, V, the kinetic energy, E, of an ion with the
charge, z, mass, m, and velocity, v, is:

Fig. 7 A simple reflectron time-of-flight mass spectrometer.


The reflector enhances mass-spectrometric resolution: it
increases the time of flight and can focus ions. Here a voltage
pulse at the shutter electrode causes a uniform starting time of
the ions
Ionization, ion transport and ion detection
Common methods of ionization are electrospray (Fig. 10),
MALDI (Fig. 6), electron bombardment, ion
bombardment, and chemical ionization. Ions are mainly
guided by electrostatic lenses and quadrupole or octopole
ion guides (Fig. 11). With the exception of FTMS, the ion
signals emerging from the mass analyzer of the MS are
commonly detected with an electron multiplier (Fig. 12). In
FTMS the cyclotroning ions are indirectly detected by
measuring and Fourier transforming the voltage signal they
induce into receiver electrodes.

Fig. 8 A reflectron time-of-flight mass spectrometer


with orthogonal ion inlet (e.g., BioTOF II from Bruker
Daltonik, Bremen, Germany)
Fig. 10: Electrospray ionization method. Analyte solutions
e). Fourier transform mass spectrometer delivered by liquid chromatography or a syringe pump are
sprayed through the narrow, heated capillary leading into
the mass spectrometer. A voltage of typically 200 V – 5 kV
is applied between capillary and orifice in front of the
electrostatic lenses. Ions form in vacuum by evaporation of
the analyte solution of charged droplets.

Fig. 11: High frequency octopole ion guide for the injection
of ions into an ion trap MS. Compared with a quadrupole
ion guide, it enables a higher precision of guidance

Fig. 9. Principle of operation of a Fourier transform mass


spectrometer (FTMS), also called “ion cyclotron
resonance mass spectrometer” (a) Ions are injected into
the analyzer cell of the spectrometer. The magnetic field
forces the thermal ions on orbits with small radii that Fig. 12: Electron multiplier for a MS. The first dynode
depend on their mass-to-charge ratio. (b) An applied converts the ion current into an electron current. Further
radio frequency pulse resonantly moves the ions to dynodes amplify the electrons by a total factor of typically
higher orbits. (c) The radio-frequency signal generated 103–108, largely dependent on the electron accelerating
by the cyclotroning of the ions is measured and Fourier- voltage between the dynodes, the number of dynodes, and
transformed. The striking characteristic of FTMS is the the dynode composition. The last dynode is connected with
high resolution, R, typically in excess of 100,000 an ammeter (not shown)
3.1.7 Ion fragmentation spectrometry with chromatographic methods (e.g., Fig.
Significant enlargement of the information content of 16). The resolution in two dimensions greatly enhances
spectra is achieved by fragmenting the sample, e.g., in a the analyzability of complex mixtures with a large
collision chamber (Fig. 13) or a helium containing cavity number of components. For example, ion exchange
of an ion trap mass analyzer (Fig. 5). chromatography on a crude cell extract with a resolution
of 100 combined with mass spectrometry with a
resolution of 10,000 can result in a total resolution of
almost 1,000,000 for small and medium-sized soluble
cellular proteins for which both methods are often
largely independent from each other. Buffer interference
which is occasionally observed in MS can usually be
prevented by increasing the sample concentration,
decreasing the buffer concentration, or changing the
buffer (Fig. 18).

Fig. 13: High resolution sector MS with a collision


chamber
Combination with chromatographic methods
For the study of highly complex systems, such as
complete cells, MS is often combined with chromato-
graphic methods, such as HPLC (high pressure liquid
chromatography; Fig. 14), FPLC (fast performance
liquid chromatography) and gas chromatography (GC;
Figs. 15 & 16). The two types of connectors between
chromatography and MS shown in Figs. 14 and 17 are
both applicable for HPLC and FPLC. Two-dimensional
spectra are obtained through combination of mass
Fig. 15: GC/MS. The combination of mass spectrometry
with gas chromatography can greatly enhance the
resolution of complex samples

Fig. 16: Example of a two-dimensional GC/MS spectrum

Fig. 14: Double sector MS in combination with HPLC


4. protein identification
5. protein purity control
6. peptide sequencing
7. proteome analysis
8. Protein folding investigations
9. protein conformational studies
10. protein-protein interaction comparisons and the
11. search for extraterrestrial life

Especially for the analysis of highly complex biological


systems, such as bacterial spores, the combination
pyrolysis-MS (PyMS) is extraordinary useful. In this
method the sample is partially decomposed in its
Fig. 17: FPLC/MS connector. In several stages the components prior to mass spectrometric analysis. The
solvent is removed from the analyte solution by mass spectra of pyrolyzed biological systems may
application of dry nitrogen and vacuum. The quadrupole contain more than 100 lines, enabling a very sensitive
ion guide leads the ions to the mass analyzer of the mass differentiation of different samples. PyMS is used for the
spectrometer detection of bacteria, bacterial spores and viruses and the
differentiation between different species of bacteria and
viruses, for the analysis of forensic samples and the
personal identification of humans and for
biotechnological applications. PyMS spectra may be
analyzed by using neuronal networks. Ion trap mass
spectrometers are particularly suitable for the pyrolysis-
MS identification of biological agents since they can
directly measure multiple fragmentation.

Fig. 18: Mass spectrogram of barstar, the 89-residue


inhibitor of the ribonuclease barnase. Left: a number of
side-peaks indicate the binding of buffer ions to the highly
charged protein. Right: a measurement with a lower buffer
concentration and higher protein concentration at a pH
closer to the pI of the protein yields a cleaner mass
spectrogram
Biophysical applications
A considerable interest in the fast point detection of toxic
and non-toxic biological materials, such as certain
bacterial strains, viruses, and proteins led to the
development of portable mass-spectrometric biological
detectors. The sensitivity of some of these detectors is
better than 1 biological agent particle per liter of air at a
detection time of less than 3 minutes. Fig. Mass spectrogram of the 10,212-Da protein
Further important biophysical applications of MS are, barstar. This protein preparation contains a fraction
1.detection of mutations in DNA or DNA sequencing with a molecular weight 131 Da higher than expected.
2.detection of mutations and post-translational This is due to an N-terminal methionine which is not
modifications of recombinant proteins properly cleaved after protein synthesis
3.diagnosis of diseases
Fig. Sequencing of the peptide SDLHQTLKKELALPEYYGENLDALWDCLTG by
proteolytic digestion and mass spectrometry. This peptide corresponds to the helix1-helix2
peptide of the protein barstar. In this example with only three proteases, only some parts of the
peptide sequence can unambiguously be identified

Fig. Peptide sequencing using Edman degradation and mass spectrometry:


reaction products of sequential degradation are mass-spectrometrically identified