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CHROMATOGRAHY
 

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Classification of chromatography

A. Mode of Separation

„ Adsorption (NPC, LSC)– separates molecules based on polarity, least polar eluting first

„ Partition - Separates molecules based on combination of solubility parameters, partition


coefficients, and polarity, most polar eluting first.

„ Ion exchange – Separates molecules on basis of molecular charge

„ Size exclusion (GPC) – separation based on molecular size, largest eluting first

„ Affinity – based on affinity with ligand

B. Basis of Mobile Phase

„ Liquid Chromatography – LLC, LSC

„ Gas chromatography – GLC, GSC

Two common approaches are used to bring the mobile phase and stationary phase into contact. In column
chromatography, the stationary phase is placed in a narrow column through which the mobile phase moves under
the influence of gravity or pressure. The stationary phase is either a solid or a thin, liquid film coating on a solid
particulate packing material or the column’s walls. In planar chromatography the stationary phase coats a flat
glass, metal, or plastic plate and is placed in a developing chamber. A reservoir containing the mobile phase is
placed in contact with the stationary phase, and the mobile phase moves by capillary action.

                                Thin layer chromatography (TLC)

TLC is solid-liquid chromatography. Adsorbent or solid phase (polar) which does not dissolve in the associated
liquid phase or mobile phase (nonpolar) e.g. Silica Gel (SiO2) & Alumina (Al2O3) which are polar and eluent is
liquid phase (hexane + ethyl acetate). Test substances are spotted onto a TLC plate coated with the adsorbent and
allowed to elute up the plate by capillary action. The More polar substances bind strongly to the adsorbent and
elute SLOWER while less polar substances bind weakly to the adsorbent and elute FASTER as the stationary
phase is polar. The strength of interactions between the adsorbent and eluting components vary
approximately in this order:

Salt formation > coordination > H-bonding > dipole-dipole > van der Waals

(More Polar) (Less Polar)

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solvent front

component B

dS
dB

component A

dA
origin

A given coompound will always


a travel a fixed distancce relative to thhe distance thee solvent travells; this ratio is called
the Rf (Rettentension facctor)

Rf of compponent A = dA / dS or distan
nce travelled by
b analyte froom origin / distance travellled by solventt from
origin

Rf of compponent B = dB / dS

The Rf of a compound can’t


c be greatter than 1 but it can be 1 in some cases likke if analyte is having more affinity
a
for the sollvent or it is co
ompletely not absorbed on the
t stationary phase
p as the annalyte is highlly non polar annd the
stationary phase
p in TLC is
i polar. So both the solvent and
a the analytee will move to the same distaance on TLC pllate.

The efficieency of thin la


ayer plate is exxpressed by itss number of th
heoretical plattes (N) and pllate height (H)).

N = 16 (dA / w) 2 dA is the
t distance traavelled by the analyte and w is width of thee spot.

H = dA / N here N is nu
umber of theoreetical plates

K ` = 1 – Rf or dS – dA K ` is capacity faactor for the annalyte, dS is thee distance traveelled by solvennt.

Rf dA

Thin layerr plate preparration

Slurry of SP
S phase is usu
ually prepared in water or chhloroform is appplied to a glasss, plastic or fooil plate generaally 20
cm squaree with the help
p of the platee spreader whhich gives a unniform thin layyer. The thicknness of the sluurry is
important as
a for the analytical separaations the thick
kness of the SP
S must be 0.225 mm while for the prepaarative
separation
ns it may be up
u upto 2 mm
m. Binding ageent calcium su
ulphate (gypssum, 10-15%) is incorporateed into
the slurry in
i order to faciilitate the adheesion of adsorbbent on plate. So
S once the sluurry layer has been
b prepared then
t it
is dried too 1100C for 30
0 mins in oven
n and this proocess is called
d as activation
n of the adsorrbent as water in the

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slurry has to be removed otherwise it will block the adsorption (e.g. in silica gel when used as SP, the active part
for adsorption are silanols or Si- OH grp which undergoes reversible H- bonding with the analyte become
deactivated in presence of water). So the heating of SP activate the adsorbent by evaporation of water molecules.
Sometimes silica become highly active and the polar compounds get strongly retained on it, so then we have to
deactivate the silica. So water is playing an important role for activation and deactivation of adsorbent in SP.

After the plate has been dried than sample is applied to the plate 2-2.5 cm above from the edge of plate either with
the aid of capillary or micropipette or microsyringe as a uniform spot.

A TLC developing chamber or jar contains a filter paper wrapped over its inside wall boundary, a covering lid and
solvent mixture or mobile phase. When filter paper is completed wetted with mobile phase (MP move into the filter
paper due to capillary action) indicate that chamber equilibrated with the solvent. After the chamber is
equilibrated with MP then lid is taken off and the TLC plates are placed vertically in the TLC chamber and again
lid is placed. (Applied analyte spots must be above from the level of the MP in the TLC chamber, otherwise the
analyte may dissolve in the MP resulting in sample loss) The MP move due to capillary action in SP which is porous
in nature and separates the compounds on the basis of polarity. Now the analyte which have stronger affinity for SP
i.e. polar retained while nonpolar analyte move along with the nonpolar mobile phase and there retention factor is
calculated with respect to distance travelled by the mobile phase. Generally MP for TLC is hexane & ethyl acetate.
We start with higher % of nonpolar hexane (say 95%) and lower % of polar ethyl acetate (say 5%). In this
concentration range mostly nonpolar analytes goes along with the hexane and occur at the top of the TLC plate
(most nonpolar) but polar compounds retained on SP and they are separated by increasing % of ethyl acetate from 5-
6%, 6- 7%, 7- 20 % etc for polar compounds as they get retained on TLC plate and not showing any distance
travelled from the origin line. As the % of the polar mobile phase (ethyl acetate) is increased polar compounds starts
to move from the origin line or sample application line and attained a particular distance with particular
concentration of the solvent. So the distance travelled by the analyte is dependent upon the % of nonpolar and
polar MP in the solvent mixture.

Nowadays precoated silica plates in which silica is absorbed on aluminium plate are available supplied by Merck.

Detection of compounds on TLC plates:

1. Ultraviolet Light — some organic compounds illuminate or fluoresce under short-wave UV light

2. Iodine Vapor — forms brown/ yellow complexes with organic unsaturated compounds and get added to
double bonds

3. Fluorescent — compounds fluoresce when placed under UV light. Other can be thin layer adsorbents
which contain a fluorescent dye so that when the plate is placed under UV light, the separated compounds
show blue, black , green areas against fluorescent background. E.g. zinc silicate

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4. Silver Nitrate Spray (for Alkyl Halides)—dark spots form upon exposure to light

5. Sulfuric Acid Spray (50%) + Heat (1100 C)—permanent charred spots and showing brown spots
(Universal method of detection)

6. Ninhydrin reagent - for detection of amino acids and peptides

7. Autoradiography – if the compound is radiolabelled

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Co
olumn Ch
hromatogrraphy

In column chromatograp
phy three way equilibrium beetween samplee, adsorbent, soolvent occurs. A solvent is chosen
c
that gives Rf about 0.35. Elution of com
mpound is deteccted by using TLC.
T

A. Column
C pac
cking can be done
d as Slurryy packing and Dry packing.

A column is prepared by
y placing a smaall plug of glasss wool in the bottom
b of the cylindrical glaass column, folllowed
by a small layer of sand. The column is
i then packed with the solid adsorbent phaase (silica gel).. Slurry of adsorbent
in solvent is prepared with the same soolvent to be lateer used in sepaaration. The sluurry is carefullly and slowly poured
p
into the coolumn after it is partially fillled with solveent in order to prevent disturrbance of the sand. The solvvent is
allowed to drain as the siilica packs tighhtly. Once the solvent
s just barrely becomes leevel with the silica
s (without drying
d
it!), anotheer small layer of
o sand is appliied carefully without
w disturbinng the silica.

B. Sample
S app
plication
1. Dissolv
ved in mobile phase (~ 25%
% solution for viscous
v sample)

2. Dry loa
ad - Adsorptioon on silica (1:2 ratio)

3. Neat - non viscous saample

The compoound mixture is dissolved inn a minimum amount


a of solvvent (same as in the columnn) and very carrefully
added to thhe top of the column using a Pasteur pipeette. After alloowing the com
mpound to adsoorb into the coolumn,
solvent is continually
c add
ded to the top of
o the column until
u each bandd resolves and is carefully collected.

With coloored substancces, the bandss may be direectly observed and collectted as they ru
un off the coolumn.
However, with colorlesss compounds,, the developm
ment can be observed by coollecting manyy small fractioons of
the elutingg solvent and testing
t each by
b thin layer ch
hromatograph
hy under UV lamp.
l

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C. Adsorbents

Stationary phases used in order of polarity includes Alumina (highest Polarity), Magnesium oxide, Carbon,
Silica, Starch, Cellulose (lowest Polarity). The most commonly used adsorbent in column chromatography is silica.
It is available in different particle size and shapes. Greater the particle size, lower is the surface area, lower the
number of theoretical plates, lower is the efficiency of the column. Lower the particle size, higher is the
efficiency of column.

Generally the particle size of silica for column chromatography: 60-200 µm

For Thin Layer chromatography (TLC) pre coated plates: 10 µm

For High Performance Liquid Chromatography (HPLC): 3-10 µm

Stationary phases (adsorbents) may be acidic or basic. Silica is acidic – has preferential ability to retain amines and
other basic compounds. Alumina and magnesia are basic – they preferentially retain acidic compounds.

Adsorption – a boundary reaction between dissolved substance and solid substance, weak and reversible interactions
(weak bonds) between two phases. The interactions includes Dipole Interactions (Bonding between two atoms of
different electronegativity), Hydrogen bridge bonds (Electrostatic bonds between hydrogen of one molecule and
strongly electronegative element of the other molecule (F,O, N,S), Pi-Complex (Adduct of electrophilic species
with C-C double bond).

D. Activity of Stationary Phase

Adsorbents require activation before use. Activity of silica is due to free silanol groups Si – OH grp. Addition of
water results in loss of activity and too much heating results in loss of activity. Exposure to moisture increases
physically adsorbed water on surface and decreases interaction between solute and silanol. So silica activated by
heating at 110oC for 30 min as at this temp as adsorbed water present on surface is removed and max number of
silanols are present. Heating silicas above 2000C leads to conversion of silanol groups to siloxane groups (Si-O-
Si). This decreases the ability of surface to retain polar materials. Alumina – heat at 200oC for 4h (active), too
long heating at high temperature (1000oC) results in total loss of activity. If fresh silica or alumina is too active,
deactivation is done by adding water.

Brockman Activity Scale for Alumina

1. Grade I – most active, heating at 350oC for few hrs

2. Grade II – 2-3 % water added

3. Grade III – 5-7 % water added

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4. Grade IV – 9-11 % water added

5. Grade V ~ 15 % water added (least active)

The activity can be tested by relative adsorbtivity of azodyes

– Azobenzene – least adsorbed

– p-phenylazophenol – most strongly adsorbed

Silica gel

Silica gel is totally Porous, narrow pore size distribution, wide choice of pore size and particle size. Silica gel is
manufactured by releasing silicic acid from a strong solution of sodium silicate by hydrochloric acid.

Na2SiO3 +H2O + 2HCl Si (OH)4 + 2NaCl

The matrix of the primary silica gel particle consists of a core of silicon atoms joined together with oxygen atoms by
siloxane bonds (silicon-oxygen-silicon bonds). Sodium silicate is prepared by heating sand at high temperature
in contact with sodium carbonate, initially silicic acid is released which condenses with itself with elimination
of water to form dimers, trimers and eventually polymeric silica. The polymer grows, forms polymer aggregates
and then polymer spheres (primary silica particles) of few Angstrom in dia. The primary particles continue to grow
until surface silanols on adjacent primary particles condense with elimination of water; this condensation causes
silica to gel.

The structure of Silica gel

The matrix of primary silica particles consist of core of silicon atoms joined together with oxygen atoms by siloxane
bonds. On the surface of each primary particle some residual, uncondensed hydroxyl groups from the original
polymeric silicic acid remain. These residual hydroxyl groups confer upon silica gel its polar properties. These
hydroxyl groups react with the silane reagents to form bonded phases. The silica surface is quite complex and
contains more than one type of hydroxyl group, and strongly bound or chemically adsorbed water and loosely bound
or physically adsorbed water.
OH
OH
OH
OH
OH Si
Si O
Si O
Si O O
Si O O
O O
O O
O Si Si
O O
Si
Si O O O
Si O O
O O
   O O
Si
Si O
O
O
O
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Three types of silanols –

Free silanols – low concentration, strong binding to basic solutes, gives broad and tailing peaks

Fully hydroxylated – high concentration of geminal and

Associated silanols – most favored (less acidic)

The disadvantage of silica gel includes it start to dissolves at pH 8 due to breakage of siloxane bond. Purity of
silica gel is important. Metal contaminants can complex with chelating solutes – lead to tailing peaks or even
complete retention

Alumina – Alumina is a network of aluminium and oxygen atoms. Active points are both the Al3+ centers and the
connecting O2- atoms. Natural alumina is basic, however it is also available in basic or neutral forms.
Chemisorption (Irreversible binding) more probable in alumina mainly when analyte contain COOH grp, so
used only for specific purposes.

Elution pattern in normal phase column i.e. stationary phase is polar and MP is

nonpolar

Normal Phase

Alkanes olefins aromatics organic halogen compounds sulfides ethers


nitro compounds esters/aldehydes/ketones alcohols/amines sulfones
sulfoxides amides carboxylic acids

In NP Chromatography polarity of the mobile phase determines the elution time. Solvents in order of increasing
polarity –

Eluotropic Series

Pentane/hexane <pet ether <cyclohexane <xylene <toluene <diethyl ether <chloroform <dichloromethane
<THF <acetone <dioxane <acetonitrile <methanol <water

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Reversed Phase Chromatography (RPC)

Stationary phase is nonpolar while mobile phase is polar. It is based on the partitioning of the solute between two
liquid phases. The less polar the solute, greater attraction, longer retention. It is liquid- liquid chromatography. The
stationary phase is liquid which is supported or chemically treated with solid spot. Mechanism of retention by
interaction of the stationary phases non-polar hydrocarbon chain with non-polar parts of the sample molecules.
Commonly used MP are methanol or acetonitrile / water or buffer (sometimes with additives of THF or dioxane)
which are polar. Stationary phase includes n-octadecyl (RP-18), n-octyl (RP-8), ethyl (RP-2), and phenyl.

C18 silica is the most widely used reverse phase stationary material.

-Si-OH + Cl-Si (CH3)2-R → Si-O-Si (CH3)2-R; R = octadecyl, octyl

This give bonded phase chromatography in which the normal phase silica which is polar is chemically
modified to nonpolar stationary phase. These Octadecyl- sterically hindered so all silanols do not react which
results in some free silanol grps which may cause peak tailing in HPLC as they strongly bind to polar analyte and
retards its passage from the columns. So to prevent it second silanization carried with small silylating reagent, such
as trimethylchlorosilane to remove residual silanols (End capping).

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A: Reaction of surface silanol with chlorodimethylsilane

B: Reaction of surface silanol with trifunctional silane

C: Reaction of surface silanol with trifunctional alkoxysilane

Reacting silanol with chlorodimethylalkyl silanes or chloroalkoxy silanes – transform polar stationary phase to
non polar stationary phase, chain length varying.

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HPLC (HIGH PERFORMANCE LIQUID CHROMATOGRAPHY)


HPLC is a reverse phase liquid chromatography. HPLC is used for thermally labile, high molecular weight,
peptides, non volatile compounds. Almost any compound that can be retained by a column can be separated by a
column. HPLC separations have been achieved based on differences in polarity, size, shape, charge, specific
affinity for a site, stereo, and optical isomerism.
Pressure is 1000-6000 psi, for these double reciprocating pumps are used which produce pulse free stream of mobile
phase. It can be operated via both isocratic and gradient elution. Particle size is generally 5 µm for analytical
columns. Generally a guard column is there before main column which have particle size 30 µm to filter the
impurities. Sample is introduced to column by either by auto sampler which uses injection needle, take sample from
sample vial placed in the sample rack which is a part of HPLC unit or the sample can be introduced manually via
injection in the column.

HPLC system has following component

1. Mobile reservoir
2. Reciprocating pumps to build the pressure (1000 psi – 6000 psi)
3. Auto sampler or injection system
4. Column placed in oven
5. Detector
6. Sample collector

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An HPLC typically includes two columns: an analytical column responsible for the separation and a guard column.
The guard column is placed before the analytical column, protecting it from contamination.

Guard Columns Two problems tend to shorten the lifetime of an analytical column. First, solutes binding
irreversibly to the stationary phase degrade the column’s performance by decreasing the available stationary phase.
Second, particulate material injected with the sample may clog the analytical column. To minimize these problems,
a guard column is placed before the analytical column. Guard columns usually contain the same particulate packing
material and stationary phase as the analytical column, but are significantly shorter and less expensive; a length of
7.5 mm and a cost one-tenth of that for the corresponding analytical column is typical. Because they are intended to
be sacrificial, guard columns are replaced regularly.

Detectors used in HPLC

1. Ultraviolet visible spectrophotometer (most common)


2. Fluorescence detectors
3. Electrochemical detectors (amperometric and coulometric)
4. HPLC-Mass spectrophotometer
5. Refractive index detectors

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olution.kailey@gm
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Chromatographic terms:
t
Poor 
resolution  Shou
ulder  
Baseeline drift  
     

Column dead time, retention time

t0 = colu
umn dead time = timee an unretardeed compound needs to passs the column, tR = retentionn time

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Capacity
y factor (K
K):

If th
he substance is not retained by t
the statiionary p
phase,
e.g. tR = tm, the capacity
y factor is k' = 0.

Selectiv
vity (α):

Resolution:

…….. For Gaussian pea


…… ak

R = ¼ × (α-1) × √N
√ × K' / 1+ K' ------------------For non
n Gaussian peak

Plate nu
umber and plate heigh
ht

The numb
ber of theoretical plates is prroportional to the
t column lenngth L. The lonnger a column, the more theooretical
plates.

emter Curv
Van Dee ve reflects the relationship beetween theoretiical plate heighht H and the linnear velocity u of the
mobile phaase. Since the plate
p height H is a measure for
fo the band brooadening of thee injection peaak of a substancce, the
path of th
he H(u) curve of a separationn column is off great interestt for determininng the chromatographic condditions
for practicaal applications.

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Eddy diffu
usion (A) The band broadeniing as a result of
o the Eddy diiffusion is creaated by the vaarious flow paaths of
individuall sample moleecules through
h the column. In the ideal case,
c the samplle molecules shhould travel thhrough
the columnns as a group or
o band. But siince several molecules
m are deelayed by obsttacles or deviatte from the path, the
band broaddens during Ed
ddy diffusion, independent of
o the flow speeed u of the mobile
m phase. We
W receive a parallel
p
straight linne to the u-axis for term A.

Longitudinal diffusion (B) Length diffusion effectss a dilution off sample moleecules in direcction of the coolumn
axis only at
a very low flo
ow speed of thhe mobile phase. In the rangge of the usuall flow speeds for chromatoggraphic
separationss length diffusiion seldom plaays an importannt part.

Mass tran
nsfer into porres in stationaary phase (C) The dominannt factor of bannd broadening is the movem
ment of
sample moolecules betweeen mobile phasse and stationaary phase. it Reelates to the kinnetics of the mass
m transfer beetween
mobile andd stationary ph
hase. Moleculees which are completely
c in the mobile ph
hase always arre slightly aheead of
the peak center.
c

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Gas chromatography

The compound separated with gas chromatography must be volatile or can be derivatized so that become
volatile and it must be thermally stable. Carrier gas either Nitrogen, Helium, hydrogen are used as mobile phase.
It can be Gas solid chromatography (GSC) or Gas liquid chromatography (GLC). In GLC mobile phase is a gas and
the stationary phase is a liquid coated either on a solid packing material or on the column’s walls.

It is most valuable for the compounds of relatively low polarity or nonpolar. The polar compounds which
contain carboxylic acids, alcohols or primary amines as functional grps badly tailed due to interactions of
these functional grps with stationary phase. To overcome this problem pyrolysis GC or photolysis is done.

In pyrolysis GC the sample for analysis is heated to very high temp. (8000C) for an extremely short time then
the thermally destroyed samples are swept into gas chromatograph.

A stationary phase of high boiling point liquid like silicone grease is supported on an inert granular solid like
diatomaceous earth or celite. This celite or diatomaceous earth or celite has one problem as it also have free
hydroxyl grps which can cause support sample interaction. So these hydroxyl grp must be modified by silanization
agent like trichlorosilane. More recently, solid supports made from glass beads or fluorocarbon polymers have
been introduced. These supports have the advantage of being more inert than diatomaceous earth. The main
criteria for selecting a stationary phase are that it should be chemically inert, thermally stable, of low volatility, and
of an appropriate polarity for the solutes being separated. The stationary phase is often high boiling point organic
compounds which includes polyethylene glycols, methyl phenyl siloxane, squalene and esters of adipic acid,
succinic and pthalic acid. Another important characteristic of a gas chromatographic column is the thickness of the
stationary phase. Separation efficiency improves with thinner films. The most common film thickness is 0.25 µm.

Nonvolatile analytes must be chemically converted to a volatile derivative before analysis. For example, amino
acids are not sufficiently volatile to analyze directly by gas chromatography. Reacting an amino acid with 1-
butanol and acetyl chloride produces an esterfied amino acid. Subsequent treatment with trifluoroacetic acid
gives the amino acids volatile N-trifluoroacetyl-n-butyl ester derivative.

The steps involved in GC


1. A vaporized sample is injected onto the chromatographic column. Second, the sample moves through the
column through the flow of inert gas.
2. Separation occurs as a result of unique equilibrium established between the solutes and the stationary phase
(the GC column).
3. The components are recorded as a sequence of peaks as they leave the column.

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Gas chrromatograp
phy columns

1. Packed Column
ns are made upp of glass, nickkel or stainless steel. They arre typically 1.55-10 m long annd 2-4
m in diameter. Contain inerrt support matterials like diaatomaceous eaarth for liquid
mm d stationary ph
hase.
2. Capillary
C (Opeen Tubular) Columns are tyypically 10-50 m long and 0.3-0.5 mm in diameter.
d Cap
pillary
orr open tubular columns aree constructed from fused sillica coated witth a protective polymer. Thhey are
m
more efficient th
han packed collumns. They arre composed of two major paarts: tubing andd stationary phhase. A
thhin film (0.1-10
0.0 micro meteers) of high moolecular weigh
ht, thermally stable
s polymeer is coated on
nto the
in
nner wall of sm
mall diameter tubing.
I. Suppoort coated opeen tubular (S
SCOT) a thin layer
l of a solid support, succh as a diatomaaceous earth, coated
c
with a liquid stationaary phase is atttached to the caapillary’s innerr wall.
II. Porous layer open tubular (PLOT)
( Capilllary GC colum
mns in which thhe stationary phase
p is based on an
ad
dsorbent or a porous polym
mer

III. W
Wall-coated op
pen tubular (WCOT)
( connsist of a capiillary tube whhose walls aree coated with liquid
sttationary phasee

Oven Te
emperature
e

a kept in a heated oven. Coolumn temperaature relies on the boiling pooint of the sam
Columns are mple being anaalyzed,
and shouldd be slightly higher
h than itss boiling pointt. Temperaturee must be stricctly controlledd within fractioons of
degrees.

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Temperature programming

Start at low temperature and gradually ramp to higher temperature.

Detector –There are several types of detectors with each responding to different compounds or properties

1. Flame Ionization Detectors (FID) is only used for organic compounds

Combustion of an organic compound in an H2/air flame results in a flame rich in electrons and ions. If a potential of
approximately 300 V is applied across the flame.
Most carbon atoms, except those in carbonyl and carboxylic groups, generate a signal, making the FID an almost
universal detector for organic compounds. Most inorganic compounds and many gases, such as H2O and CO2,
cannot be detected, making the FID detector ideal for the analysis of atmospheric and aqueous environmental
samples.

2. Electron Capture Detectors (ECD) used for pesticide determination or halogenated compounds

The detector consists of a beta emitter (a beta particle is an electron) such as 63Ni. The emitted electrons ionize
the mobile phase, which is usually N2, resulting in the production of additional electrons that give rise to an electric
current between a pair of electrodes. When a solute capture of electrons elutes from the column, the electric
current decreases. This decrease in electric current serves as the signal. The ECD is highly selective toward solutes
with electronegative functional groups, such as halogens, and nitro groups and is relatively insensitive to amines,
alcohols, and hydrocarbons.

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3. Thermal Conductivity Detectors (TCD) or kathrometer

As the mobile phase exits the column, it passes over a tungsten–rhenium wire filament. The filament’s electrical
resistance depends on its temperature, which, in turn, depends on the thermal conductivity of the mobile phase.
Because of its high thermal conductivity, helium is the mobile phase of choice when using a thermal
conductivity detector (TCD). When a solute elutes from the column, the thermal conductivity of the mobile
phase decreases and the temperature of the wire filament, and thus its resistance, increases. A reference cell,
through which only the mobile phase passes, corrects for any time-dependent variations in flow rate, pressure, or
electrical power, all of which may lead to a change in the filament’s resistance. A TCD detector has the advantage of
universality, since it gives a signal for any solute whose thermal conductivity differs from that of helium.

4. Other detectors

Two common detectors, which also are independent instruments, are Fourier transform infrared spectrophotometers
(GC-FT–IR) and mass spectrometers (GC-MS).
In GC–MS effluent from the column is introduced directly into the mass spectrometer’s ionization chamber in a
manner that eliminates the majority of the carrier gas. In the ionization chamber all molecules (remaining carrier
gas, solvent, and solutes) are ionized, and the ions are separated by their mass-to-charge ratio. Because each
solute undergoes a characteristic fragmentation into smaller ions, its mass spectrum of ion intensity as a function of
mass-to-charge ratio provides qualitative information that can be used to identify the solute.

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Ion-Exchange Chromatography

In ion-exchange chromatography (IEC) the stationary phase is a cross-linked polymer resin, usually
divinylbenzene cross-linked polystyrene, with covalently attached ionic functional groups. The counter ions to
these fixed charges are mobile and can be displaced by ions (present in mobile phase) that compete more favorably
for the exchange sites. Ion-exchange resins are divided into four categories: strong acid cation exchangers; weak
acid cation exchangers; strong base anion exchangers; and weak base anion exchangers.

The ion exchange sites, indicated by R, are mostly in the para position and are not necessarily bound to all
styrene units.

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The ion exxchangers can be


b

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1. Inorganic polymers Aluminosilicates
2. Natural Zeolites
3. Synthetic copolymers of styrene & DVB
OR methacrylic acid

Strong acid cation exchanger Sulfonic acid –SO3-, –CH2CH2SO3-


Weak acid cation exchanger Carboxylic acid –COO–, CH2COO–
Strong base anion exchanger Quaternary amine –CH2N+ (CH3)3
Weak base anion exchanger Amine –N+H3, CH2CH2N+H (CH2CH3)2

The ion-exchange reaction of a monovalent cation, M+, at a strong acid exchange site is

–SO3–– H+(s) + M+(aq) –SO3–– M+(s) + H+(aq)

Strong acid cation exchangers include a sulfonic acid functional group that retains its anionic form (pH 1-14),
and thus its capacity for ion-exchange, in strongly acidic solutions. The functional groups for a weak acid cation
exchanger, however, are fully protonated at pH levels less than 4, thereby losing their exchange capacity. The strong
base anion exchangers are fashioned using a quaternary amine, therefore retaining a positive charge even in strongly
basic solutions. Weak base anion exchangers, however, remain protonated only at pH levels that are moderately
basic. Under more basic conditions, a weak base anion exchanger loses its positive charge and, therefore, its
exchange capacity.

Generally, for weak acids or base which are only ionized at either low pH (weak bases) or high pH (weak
acids) strong anion exchangers are used. And if compounds are strong acid or base then weak anion
exchangers are used.

The equilibrium constant for this ion-exchange reaction, which is also called the selectivity coefficient, is

Highly charged ions bind more strongly than ions of lower charge

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IEX is mostly employed for the purification of the proteins. IEX separates proteins with differences in charge to
give a very high resolution separation with high sample loading capacity. The separation is based on the reversible
interaction between a charged protein and an oppositely charged chromatographic medium. Proteins bind as they are
loaded onto a column. Conditions are then altered so that bound substances are eluted differentially. This elution is
usually performed by increases in salt concentration or changes in pH.
The net surface charge of proteins varies according to the surrounding pH. When above its isoelectric point
(pI) a protein will bind to an anion exchanger, when below its pI a protein will behind to a cation exchanger.
Typically IEX is used to bind the target molecule, but it can also be used to bind impurities if required. IEX can be
repeated at different pH values to separate several proteins which have distinctly different charge properties.

The mobile phase in IEC is usually an aqueous buffer, the pH and ionic composition of which determines a solute’s
retention time. Gradient elutions are possible in which the ionic strength or pH of the mobile phase is changed with
time. For example, an IEC separation of cations might use a dilute solution of HCl as the mobile phase. Increasing
the concentration of HCl speeds the elution rate for more strongly retained cations, since the higher concentration of
H+ allows it to compete more successfully for the ion-exchange sites.
The most common detector measures the conductivity of the mobile phase as it elutes from the column. The
high concentration of electrolyte in the mobile phase is a problem, however, because the mobile-phase ions
dominate the conductivity. For example, if a dilute solution of HCl is used as the mobile phase, the presence of large
concentrations of H3O+ and Cl– produces a background conductivity that may prevent the detection of analytes
eluting from the column. To minimize the mobile phase’s contribution to conductivity, an ion-suppressor
column is placed between the analytical column and the detector. This column selectively removes mobile-phase
electrolyte ions without removing solute ions. For example, in cation ion-exchange chromatography using a dilute
solution of HCl as the mobile phase, the suppressor column contains an anion-exchange resin. The exchange
reaction replaces the ionic HCl with H2O.

H+(aq) + Cl–(aq) + Resin+ –OH– Resin+–Cl– + H2O(l)

Analyte cations elute as hydroxide salts instead of as chloride salts. A similar process is used in anion ion-exchange
chromatography in which a cation ion-exchange resin is placed in the suppressor column. If the mobile phase
contains Na2CO3, the exchange reaction replaces a strong electrolyte, Na2CO3, with a weak electrolyte, H2CO3.

2Na+(aq) + CO3 2–(aq) + 2Resin––H+ 2Resin––Na+ + H2CO3(aq)

Ion suppression is necessary when using a mobile phase containing a high concentration of ions.

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Gel permeattion chrom
matograp
phy or siz
ze exclusiion chrom
matography
Gel-Permeeation Chrom
matography is a mechanicall sorting of molecules
m based on the size of the molecu
ules in
solution. Small molecu
ules are able to permeate more
m pores and
a are, thereefore, retained
d longer than large
molecules..

A sample containing a very


v large mollecule such as thyroglobulin to determine the void volume which doees not
goes into the pores. Th
he elution volum
mes of the staandards are divvided by the ellution volume of the thyroglobulin
(Ve/Vo) annd plotted against the log of the
t standards' molecular
m weigghts.

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KD = (Ve-Vo) / Vi

KD = Partition coefficient where stationary phase is interstitial spaces of the polymer bead
Ve = Elution vol. of solute
Vo = Void vol. the elution of comps that are completely excluded from the gel pores
Vi = Vol. of liquid inside the gel pores available to very smallest molecules

Affinity Chromatography

Affinity chromatography depends upon the specific interactions of biological molecules like
1. Antibody-antigen interaction,
2. Enzyme –inhibitor interaction,
3. DNA-DNA binding,
4. DNA-protein interaction or
5. Receptor agonist / antagonist interactions.

 
 
Affinity Chromatography Surface bound with 
 
Epoxy, aldehyde or aryl ester groups, 
 
Metal Interaction Chromatography surface bound 
 
with Iminodiacetic acid + Ni2+/Zn2+/Co2+  

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Supercritical Fluid Chromatography

Despite their importance, gas chromatography and liquid chromatography cannot be used to separate and analyze all
types of samples. Gas chromatography, particularly when using capillary columns, provides for rapid separations
with excellent resolution. Its application, however, is limited to volatile analytes or those analytes that can be made
volatile by a suitable derivatization. Liquid chromatography can be used to separate a wider array of solutes;
however, the most commonly used detectors (UV, fluorescence, and electrochemical) do not respond as universally
as the flame ionization detector commonly used in gas chromatography.
Supercritical fluid chromatography (SFC) provides a useful alternative to gas chromatography and liquid
chromatography for some samples. The mobile phase in supercritical fluid chromatography is a gas held at a
temperature and pressure exceeding its critical point. Under these conditions i.e. above critical point the mobile
phase is neither a gas nor a liquid. Instead, the mobile phase is a supercritical fluid whose properties are
intermediate between those of a gas and a liquid. Specifically, supercritical fluids have viscosities that are
similar to those of gases, which means that they can move through either capillary or packed columns without
the need for the high pressures encountered in HPLC. Analysis time and resolution, although not as good as in
GC, are usually better than that obtainable with conventional HPLC. The density of a supercritical fluid, however, is
much closer to that of a liquid, accounting for its ability to function as a solvent. The mobile phase in SFC,
therefore, behaves more like the liquid mobile phase in HPLC than the gaseous mobile phase in GC. The most
common mobile phase for supercritical fluid chromatography is CO2. Its low critical temperature, 31 °C, and
critical pressure, 72.9 atm, are relatively easy to achieve and maintain. Although supercritical CO2 is a good
solvent for nonpolar organics, it is less useful for polar solutes. The addition of an organic modifier, such as
methanol, improves the mobile phase’s elution strength.

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SPECTROSCOPY

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The electromagnetic spectrum

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List of important
i ttables and d
diagrams

                         

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Proton NM
MR differs fro
om 13C NMR in a number of
o ways.
• 1H is the major isotope of hydrogen (999.985% naturaal abundance),, while 13C is only
o a minor issotope (1.1%)
• 1H NMR is quantitativee: the area undeer the peak tellls us the numbeer of hydrogenn nuclei, while 13C NMR maay give
strong or weak
w m the same nuumber of 13C nuuclei
peaks from
13
• Protons interact magn
netically (‘coupple’) to reveall the connectivvity of the strructure, while C is too raare for
13
coupling between C nucclei to be seen

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Some loss in mass spectra

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Solvent cutoff in UV/ VIS spectroscopy

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INSTRUMENTATION

1. UV/Vis spectroscopy: Electronic transitions


Source: Tungsten lamp: for visible region i.e. 400-800nm
Deuterium lamp: For ultraviolet region i.e. 190-400 nm
• Sample cell is made up of Quartz
• Solvent generally used are 95 % Ethanol, 1,4-Dioxane
• Lambert-beer’s law: A= εbc
• Graph is plotted between concentration vs optical density (Absorbance) o
Detector: Photomultiplier tubes
2. IR spectroscopy: Viberational changes for compounds having fluctuating dipole
moment
Source: Globar source (silicon carbide rod)
Nerst glower (Ytterinium etc )
Incandescent lamp
Detectors: Bolometers, Thermocouples, Golay cells (xenon gas), Pyroelectric
(Triglycerine sulphate), Photo cells
• Cells are constructed of metal halides like NaCl or KBr

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• For examination of solid samples either by KBr pellet or mull techniques uses
Nujol (high boiling point petroleum oil or mineral oil) is used but when
hydrocarbon in the sample interfere with it than hexachlorobutadiene is used.
• Most common solvent in IR is CCL4 and CS2
• Hook’s law:

3. NMR: Spin changes


Radio waves oscillator and magnetic field (1Tesla = 104 Gauss)
Chemical shift is measured in ppm and it is independent on magnetic field.
Coupling constant is measured in Hz.
4. ESR: Rotational changes
Microwaves (Klystron oscillator) and electromagnets
Internal standard: DPPH
5. Mass spectroscopy: No electromagnetic radiations are used
Source: Electron impact
Chemical ionization (carrier gas Methane and ammonia)
Fast atom bombardment (carrier gas Argon)
Laser desorption (Matrix Assisted Laser Desorption Ionization) is Soft technique
used for protein samples
Electron spray Ionization
Mass analyzers: Quadra pole
Magnetic sector
Electronic sector
Detector: Electron multiplier tubes

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Faraday Cup

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