ABSOLUTE MOLECULAR WEIGHT OSMOMETRY

OSMOMETRY

LIGHT SCATTERING METHODS

ULTRACENTRIFUGATION

Nill PATRICK U. DULCE

BSChE 5
10:30-11:30 TTHS
ABSOLUTE MOLECULAR WEIGHT OSMOMETRY
Absolute molecular weight osmometry or absolute molar mass is a process used to
determine the characteristics of molecules.
The first absolute measurements of molecular weights (i.e. made without reference to
standards) were based on fundamental physical characteristics and their relation to the molar
mass. The most useful of these were membrane osmometry and sedimentation.
Another absolute instrumental approach was also possible with the development of light
scattering theory by Einstein, Raman, Debye, Zimm, and others. The problem with measurements
made using membrane osmometry and sedimentation was that they only characterized the bulk
properties of the polymer sample. Moreover, the measurements were excessively time consuming
and prone to operator error. This was achieved by the advent of size exclusion
chromatography (SEC). SEC is based on the fact that the pores in the packing material of
chromatography columns could be made small enough for molecules to become temporarily lodged
in their interstitial spaces. As the sample makes its way through a column the smaller molecules
spend more time traveling in these void spaces than the larger ones, which have fewer places to
"wander". The result is that a sample is separated according to its hydrodynamic volume. As a
consequence, the big molecules come out first, and then the small ones follow in the elutent. By
choosing a suitable column packing material it is possible to define the resolution of the system.
Columns can also be combined in series to increase resolution or the range of sizes studied.
The next step is to convert the time at which the samples eluted into a measurement of
molar mass. This is possible because if the molar mass of a standard were known, the time at which
this standard eluted should be equal to a specific molar mass. This is significant for polymer
analysis because a single polymer could be shown to have many different components, and the
complexity and distribution of which would also affect the physical properties. However this
technique has shortcomings. For example, unknown samples are always measured in relation to
known standards, and these standards may or may not have similarities to the sample of interest.
The measurements made by SEC are then mathematically converted into data similar to that found
by the existing techniques.
The problem was that the system was calibrated according to the Vh characteristics of
polymer standards that are not directly related to the molar mass. If the relationship between the
molar mass and Vh of the standard is not the same as that of the unknown sample, then the
calibration is invalid. Thus, to be accurate, the calibration must use the same polymer, of the same
conformation, in the same eluent and have the same interaction with the solvent as the hydration
layer changes Vh.
Benoit showed that taking into account the hydrodynamic volume would solve the problem. In his
publication, Benoit showed that all synthetic polymers elutes on the same curve when the log of the
intrinsic viscosity multiplied by the molar mass was plotted against the elution volume. This is the
basis of universal calibration which requires a viscometer to measure the intrinsic viscosity of the
polymers. Universal calibration was shown to work for branched polymers, copolymers as well as
starburst polymers.
For good chromatography, there must be no interaction with the column other than that
produced by size. As the demands on polymer properties increased, the necessity of getting
absolute information on the molar mass and size also increased. This was especially important in
pharmaceutical applications where slight changes in molar mass or shape may result in
different biological activity. These changes can actually have a harmful effect instead of a beneficial
one.
 To obtain molar mass, light scattering instruments need to measure the intensity of light
scattered at zero angle. This is impractical as the laser source would outshine the light scattering
intensity at zero angle. The 2 alternatives are to measure very close to zero angle or to measure at
many angle and extrapolate using a model (Rayleigh, Rayleigh-Gans-Debye, Berry, Mie, etc.) to zero
degree angle.
Traditional light scattering instruments worked by taking readings from multiple angles,
each being measured in series. A low angle light scattering system was developed in the early 1970s
that allowed a single measurement to be used to calculate the molar mass. Although measurements
at low angles are better for fundamental physical reasons (molecules tend to scatter more light in
lower angle directions than in higher angles), low angle scattering events caused by dust and
contamination of the mobile phase easily overwhelm the scattering from the molecules of interest.
When the low-angle laser light scattering (LALLS) became popular in the 1970s and mid-1980s,
good quality disposable filters were not readily available and hence multi-angle measurements
gained favour.
Multi-angle light scattering was invented in the mid-1980s and instruments like that were
able to make measurements at the different angles simultaneously but it was not until the later
1980s (10-12) that the connection of multi-angle laser light scattering (MALS) detectors to SEC
systems was a practical proposition enabling both molar mass and size to be determined from each
slice of the polymer fraction.

Light scattering measurements can be applied to synthetic

polymers, proteins, pharmaceuticals and particles such as liposomes, micelles, and encapsulated
proteins. Measurements can be made in one of two modes which are un-fractionated (batch mode)
or in continuous flow mode (with SEC, HPLC or any other flow fractionation method). Batch mode
experiments can be performed either by injecting a sample into a flow cell with a syringe or with
the use of discrete vials. These measurements are most often used to measure timed events
like antibody-antigen reactions or protein assembly. Batch mode measurements can also be used to
determine the second virial coefficient (A2), a value that gives a measure of the likelihood of
crystallization or aggregation in a given solvent. Continuous flow experiments can be used to study
material eluting from virtually any source. More conventionally, the detectors are coupled to a
variety of different chromatographic separation systems. The ability to determine the mass and size
of the materials eluting then combines the advantage of the separation system with an absolute
measurement of the mass and size of the species eluting.
 The addition of an SLS detector coupled downstream to a chromatographic system allows
the utility of SEC or similar separation combined with the advantage of an absolute detection
method. The light scattering data is purely dependent on the light scattering signal times the
concentration; the elution time is irrelevant and the separation can be changed for different
samples without recalibration. In addition, a non-size separation method such as HPLC or IC can
also be used. As the light scattering detector is mass dependent, it becomes more sensitive as the
molar mass increases. Thus it is an excellent tool for detecting aggregation. The higher the
aggregation number, the more sensitive the detector becomes.

WHAT IS OSMOMETRY?

VAPOR PRESSURE OSMOMETRY

Vapor pressure osmometry is a technique to measure the number average molecular

weight of a polymer. It is based upon Raoult's law that governs change in vapor pressure of a
solution based on the mole fraction of the solute.

The vapor pressure osmometer is composed of two chambers: one for pure solvent and the
other to contain solution, where the solute is the polymer whose molecular weight is
unknown. Thermistors in each chamber provide an electrical signal (the actual measurement) of
differential heating to achieve a vapor equilibrium in each chamber.

By measuring solutions of different concentrations of solute with a known molecular weight

standard, a plot of concentration versus electrical differential can be prepared. Similarly, the
unknown is then prepared and measured with the number average molecular weight derived from
the standard plot.

MEMBRANE OSMOMETRY

The membrane osmometry is a technique for the determination of molecular masses of

polymers by means of osmosis. The phenomenon of osmosis describes the attempt of solvent
molecules to go through a semipermeable membrane into a solution. The detection of the so
originated osmotic pressure can be determined into the number average molecular weight
Manganese of the solved polymer.

The membrane osmometer consists of

two chambers, whereupon one contains pure
solvent and the other the polymer solution. The
chambers are separated with a semipermeable
membrane. Beside the polymer concentration of
the solution the detected low-pressure of the
solvent chamber is the measuring value. The
pressure sensors of the osmometer are very
sensitive and are able to detect the pressure,
which is indicated of a 1.1 mm high water head.
Theoretically, molecular masses up to 1,000
kg/mol are determinable. In practice molecular
masses up to 100 kg/mol are reliable
determinable.
MEMBRANE OSMOMETRY

The measurement of the freezing point of a solution is related to the osmotic concentration of that
solution. Osmotic concentration can be thought of as the concentration of particles of solute per
unit amount of solvent. Sometimes this is called an apparent concentration since the manner in
which the solute reacts with the solvent affects the number of particles that are formed. The
manner in which the solute and solvent interact is called activity. This refers not only to the
degree of dissociation of the solute (if the solute is an ionic salt), but also to a number of other
factors including the degree to which the water molecules are free to enter the solution. If freezing
point is measured over a number of known time intervals, then the rate of reaction can be
measured. Some physical chemists prefer to interpret the freezing point of a solution as the
concentration of water in that solution. Others relate freezing point to the water potential of the
solution. For the purposes of this discussion, it is most convenient to describe freezing point as the
concentration of particles in solution. Freezing point wont tell you how big these particles are, or
what shape they have, or if they are charged. It only tells you how many. At relatively low
concentrations, freezing point is linear with the number of dissolved particles. Twice as many
particles will depress the freezing point twice as much, three times as many, three times as much,
and so forth.

LIGHT SCATTERING
Light scattering is a form of scattering in which light in the form of propagating energy is
scattered. Light scattering can be thought of as the deflection of a ray from a straight path, for
example by irregularities in the propagation medium, particles, or in the interface between two
media. Deviations from the law of reflection due to irregularities on a surface are also usually
considered to be a form of scattering. When these irregularities are considered to be random and
dense enough that their individual effects average out, this kind of scattered reflection is commonly
referred to as diffuse reflection.

Most objects that one sees are visible due to light scattering from their surfaces. Indeed, this
is our primary mechanism of physical observation Scattering of light depends on
the wavelength or frequency of the light being scattered. Since visible light has wavelengths on the
order of hundreds of nanometers, objects much smaller than this cannot be seen, even with the aid
of a microscope. Colloidal particles as small as 1 m have been observed directly in aqueous
suspension.

Mechanisms of diffuse
reflection include surface scattering from
roughness and subsurface scattering from
internal irregularities such as grain
boundaries inpolycrystalline solids.

The transmission of various

frequencies of light is essential for applications
ranging from window glass to fiber optic
transmission cables and infrared (IR) heat-
seeking missile detection systems. Light
propagating through an optical system can
be attenuated by absorption, reflection and
scattering.
 The interaction of light with matter can reveal important information about the structure
and dynamics of the material being examined. If the scattering centers are in motion, then the
scattered radiation is Doppler shifted. An analysis of the spectrum of scattered light can thus yield
information regarding the motion of the scattering center. Periodicity or structural repetition in the
scattering medium will cause interference in the spectrum of scattered light. Thus, a study of the
scattered light intensity as a function of scattering angle gives information about the structure,
spatial configuration, or morphology of the scattering medium. With regard to light scattering
in liquids and solids, primary material considerations include.

Crystalline structure: How close-packed its atoms or molecules are, and whether or not the
atoms or molecules exhibit the long-range order evidenced in crystalline solids.
Glassy structure: Scattering centers include fluctuations in density and/or composition.
Microstructure: Scattering centers include internal surfaces in liquids due largely to density
fluctuations, and microstructural defects in solids such as grains, grain boundaries, and
microscopic pores.

In the process of light scattering, the most critical factor is the length scale of any or all of these
structural features relative to the wavelength of the light being scattered.

An extensive review of light scattering in fluids has covered most of the mechanisms which
contribute to the spectrum of scattered light in liquids, including density, anisotropy, and
concentration fluctuations. Thus, the study of light scattering by thermally driven density
fluctuations (or Brillouin scattering) has been utilized successfully for the measurement of
structural relaxation and viscoelasticity in liquids, as well as phase
separation, vitrification, and compressibility in glasses.

In addition, the introduction of dynamic light scattering and photon correlation

spectroscopy has made possible the measurement of the time dependence of
spatial correlations in liquids and gases in the relaxation time gap between 106 and 102 s in
addition to even shorter time scales or faster relaxation events. It has therefore become quite
clear that light scattering is an extremely useful tool for monitoring the dynamics of structural
relaxation in glasses on various temporal and spatial scales and therefore provides an ideal tool for
quantifying the capacity of various glass compositions for guided light wave transmission well into
the far infrared portions of the electromagnetic spectrum.

Note: Light scattering in an ideal defect-free crystalline (non-metallic) solid which provides no
scattering centers for incoming lightwaves will be due primarily to any effects of anharmonicity
within the ordered lattice. Lightwave transmission will be highly directional due to the
typical anisotropy of crystalline substances, which includes their symmetry group and Bravais
lattice. For example, the seven different crystalline forms of quartz silica (silicon dioxide, SiO2) are
all clear, transparent materials.
LIGHT SCATTERING METHODS

RAYLEIGH SCATTERING

Rayleigh scattering is the elastic scattering of light by molecules and particulate

matter much smaller than the wavelength of the incident light. It occurs when light penetrates
gaseous, liquid, or solid phases of matter. Rayleigh scattering intensity has a very strong
dependence on the size of the particles (it is proportional to the third power of their diameter). It is
inversely proportional to the fourth power of the wavelength of light, which means that the shorter
wavelengths in visible white light (violet and blue) are scattered stronger than the longer
wavelengths toward the red end of the visible spectrum. This type of scattering is therefore
responsible for the blue color of the sky during the day and the orange colors during sunrise and
sunset. Rayleigh scattering is the main cause of signal loss in optical fibers.
MIE SCATTERING

Mie scattering is a broad class of scattering of light by spherical particles of any diameter.
The scattering intensity is generally not strongly dependent on the wavelength, but is sensitive to
the particle size. Mie scattering coincides with Rayleigh scattering in the special case where the
diameter of the particles is much smaller than the wavelength of the light; in this limit, however, the
shape of the particles no longer matters. Mie scattering intensity for large particles is proportional
to the square of the particle diameter.

TYNDALL SCATTERING

Tyndall scattering is similar to Mie scattering without the restriction to spherical geometry
of the particles. It is particularly applicable to colloidal mixtures and suspensions.

BRILLOUIN SCATTERING

Brillouin scattering occurs from the interaction of photons with acoustic phonons in solids,
which are vibrational quanta of lattice vibrations, or with elastic waves in liquids. The scattering
is inelastic, meaning it is shifted in energy from the Rayleigh line frequency by an amount that
corresponds to the energy of the elastic wave or phonon, and it occurs on the higher and lower
energy side of the Rayleigh line, which may be associated with the creation and annihilation of a
phonon. The light wave is considered to be scattered by the density maximum or amplitude of the
acoustic phonon, in the same manner that X-rays are scattered by the crystal planes in a solid. In
solids, the role of the crystal planes in this process is analogous to the planes of the sound waves or
density fluctuations. Brillouin scattering measurements require the use of a high-contrast Fabry
Prot interferometer to resolve the Brillouin lines from the elastic scattering, because the energy
shifts are very small (< 100 cm1) and very weak in intensity. Brillouin scattering measurements
yield the sound velocities in a material, which may be used to calculate the elastic constants of the
sample.
RAMMAN SCATTERING

Raman scattering is another form of inelastic light scattering, but instead of scattering from
acoustic phonons, as in Brillouin scattering, the light interacts with optical phonons, which are
predominantly intra-molecular vibrations and rotations with energies larger than acoustic
phonons. Raman scattering may therefore be used to determine chemical composition and
molecular structure. Since most Raman lines are stronger than Brillouin lines, and have higher
energies, standard spectrometers using scanningmonochromators may be used to measure them.
Raman spectrometers are standard equipment in many chemical laboratories.

DYNAMIC LIGHT SCATTERING

Dynamic light scattering (DLS) is the most common used light scattering method, and
is a technique in physics that can be used to determine the size distribution profile of
small particles in suspension or polymers in solution. In the scope of DLS, temporal fluctuations are
usually analyzed by means of the intensity or photon auto-correlation function (also known
as photon correlation spectroscopy or quasi-elastic light scattering). In the time domain analysis,
the autocorrelation function (ACF) usually decays starting from zero delay time, and faster
dynamics due to smaller particles lead to faster decorrelation of scattered intensity trace. It has
been shown that the intensity ACF is the Fourier transformation of the power spectrum, and
therefore the DLS measurements can be equally well performed in the spectral domain. DLS can
also be used to probe the behavior of complex fluids such as concentrated polymer solutions.

A monochromatic light source, usually a laser, is shot through a polarizer and into a sample.
The scattered light then goes through a second polarizer where it is collected by a photomultiplier
and the resulting image is projected onto a screen. This is known as a speckle pattern.

All of the molecules in the solution are being hit with the light and all of the molecules
diffract the light in all directions. The diffracted light from all of the molecules can either interfere
constructively (light regions) or destructively (dark regions). This process is repeated at short time
intervals and the resulting set of speckle patterns are analyzed by an autocorrelator that compares
the intensity of light at each spot over time. The polarizers can be set up in two geometrical
configurations. One is a vertical/vertical (VV) geometry, where the second polarizer allows light
through that is in the same direction as the primary polarizer. In vertical/horizontal (VH) geometry
the second polarizer allows light not in same direction as the incident light.
ULTRACENTRIFUGATION

ULTRACENTRIFUGE

The ultracentrifuge is a centrifuge optimized for spinning a rotor at very high speeds,
capable of generating acceleration as high as 1000000 g (approx. 9800 km/s).There are two
kinds of ultracentrifuges, the preparative and the analytical ultracentrifuge. Both classes of
instruments find important uses in molecular biology, biochemistry, and polymer science.

Theodor Svedberg invented the analytical ultracentrifuge in 1925, and won the Nobel Prize
in Chemistry in 1926 for his research on colloids and proteins using the ultracentrifuge.
The vacuum ultracentrifuge was invented by Edward Greydon Pickels in the Physics Department at
the University of Virginia. It was his contribution of the vacuum which allowed a reduction
in friction generated at high speeds. Vacuum systems also enabled the maintenance of
constant temperature across the sample, eliminating convection currents that interfered with the
interpretation of sedimentation results.

In 1946, Pickels cofounded Spinco (Specialized Instruments Corp.) to market analytical and
preparative ultracentrifuges based on his design. Pickels considered his design to be too
complicated for commercial use and developed a more easily operated, foolproof version. But
even with the enhanced design, sales of analytical centrifuges remained low, and Spinco almost
went bankrupt. The company survived by concentrating on sales of preparative ultracentrifuge
models, which were becoming popular as workhorses in biomedical laboratories. In 1949, Spinco
introduced the Model L, the first preparative ultracentrifuge to reach a maximum speed of
40,000 rpm. In 1954, Beckman Instruments, now Beckman Coulter, purchased the company,
forming the basis of its Spinco centrifuge division.

ANALYTICAL ULTRACENTRIFUGE

In an analytical ultracentrifuge, a sample being spun can be monitored in real time through
an optical detection system, using ultraviolet light absorption and/or interference optical refractive
index sensitive system. This allows the operator to observe the evolution of the sample
concentration versus the axis of rotation profile as a result of the applied centrifugal field. With
modern instrumentation, these observations are electronically digitized and stored for further
mathematical analysis. Two kinds of experiments are commonly performed on these instruments:
sedimentation velocity experiments and sedimentation equilibrium experiments.

Sedimentation velocity experiments aim to interpret the entire time-course of

sedimentation, and report on the shape and molar mass of the dissolved macromolecules, as well as
their size-distribution. The size resolution of this method scales approximately with the square of
the particle radii, and by adjusting the rotor speed of the experiment size-ranges from 100 Da to
10 GDa can be covered. Sedimentation velocity experiments can also be used to study reversible
chemical equilibria between macromolecular species, by either monitoring the number and molar
mass of macromolecular complexes, by gaining information about the complex composition from
multi-signal analysis exploiting differences in each components spectroscopic signal, or by
following the composition dependence of the sedimentation rates of the macromolecular system, as
described in Gilbert-Jenkins theory.

Sedimentation equilibrium experiments are concerned only with the final steady-state of
the experiment, where sedimentation is balanced by diffusion opposing the concentration
gradients, resulting in a time-independent concentration profile. Sedimentation equilibrium
distributions in the centrifugal field are characterized by Boltzmann distributions. This experiment
is insensitive to the shape of the macromolecule, and directly reports on the molar mass of the
macromolecules and, for chemically reacting mixtures, on chemical equilibrium constants.

The kinds of information that can be obtained from an analytical ultracentrifuge include the
gross shape of macromolecules, the conformational changes in macromolecules, and size
distributions of macromolecular samples. For macromolecules, such as proteins, that exist
in chemical equilibrium with different non-covalent complexes, the number and
subunit stoichiometry of the complexes and equilibrium constant constants can be studied.

Analytical ultracentrifugation has recently seen a rise in use because of increased ease of
analysis with modern computers and the development of software, including a National Institutes
of Health supported software package, SedFit.

ANALYTICAL ULTRACENTRIFUGATION

An analytical ultracentrifuge spins a rotor at an accurately controlled speed and

temperature. The concentration distribution of the sample is determined at known times using
absorbance measurements. The concentration, c, is determined for solutes obeying the Beer-
Lambert law.

This figure displays a schematic diagram of the Beckman Optima XL-A absorbance
system. A high intensity xenon flask lamp allows the use of wavelengths between 190 and
800nm. The lamp is fired briefly as a selected sector passes the detector.

An example of an analytical ultracentrifuge (Beckman Optima XL-A).

PREPARATIVE ULTRACENTRIFUGE

Preparative ultracentrifuges are available with a wide variety of rotors suitable for a great
range of experiments. Most rotors are designed to hold tubes that contain the samples. Swinging
bucket rotors allow the tubes to hang on hinges so the tubes reorient to the horizontal as the rotor
initially accelerates. Fixed angle rotors are made of a single block of material and hold the tubes in
cavities bored at a predetermined angle. Zonal rotors are designed to contain a large volume of
sample in a single central cavity rather than in tubes. Some zonal rotors are capable of dynamic
loading and unloading of samples while the rotor is spinning at high speed.

Preparative rotors are used in biology for pelleting of fine particulate fractions, such as
cellular organelles (mitochondria, microsomes, ribosomes) and viruses. They can also be used
for gradient separations, in which the tubes are filled from top to bottom with an increasing
concentration of a dense substance in solution. Sucrose gradients are typically used for separation
of cellular organelles. Gradients of caesium salts are used for separation of nucleic acids. After the
sample has spun at high speed for sufficient time to produce the separation, the rotor is allowed to
come to a smooth stop and the gradient is gently pumped out of each tube to isolate the separated
components.

REFERENCES
http://osmolality.com/pdf/Osmometry.pdf
https://en.wikipedia.org/wiki/Vapor_pressure_osmometry
https://en.wikipedia.org/wiki/Dynamic_light_scattering
https://en.wikipedia.org/wiki/Ultracentrifuge
http://www.bioc.rice.edu/bios576/AU/AU_Page.html
http://www.wee-solve.de/en/osmometry.html
http://osmolality.com/pdf/Freezing%20Point%20Osmo.pdf
https://en.wikipedia.org/wiki/Absolute_molar_mass