Submitted By

Nazeer Manzoor

Submitted to: Dr. Samra Barkaat

Subject: Advanced Chromatographic Techniques
(CHE7019)
Date: 25-11-2022

DEPARTMENT OF M.Phil. Chemistry

Section(A)1stSemester
UNIVERSITY OF RIPHAH INTERNATIONAL FAISALABAD
CAMPUS
Introduction
The bulk physical properties such as strength and toughness of synthetic and
natural polymers, or the activity and efficacy of proteins and biopharmaceuticals
are strongly dependent on their molecular properties. In a large number of
industries, there is a clear and strong desire to control molecular weight and
structure in order to better manipulate and control the bulk properties of the
material. For example, in polymer chemistry, different molecular parameters
can have effects on different bulk properties. For proteins, molecular weight,
and therefore, oligomer state directly relates to their activity either as a
biopharmaceutical, or in an assay. The amount and size of any aggregates
present in a sample will result in a loss of sample activity and in the case of
biopharmaceuticals, can also stimulate an immune response affecting the
efficacy and safety of such drugs. It is therefore necessary to have a reliable
technique for making such measurements. Size Exclusion Chromatography
(SEC), also referred to as Gel Permeation Chromatography (GPC) or Gel Filtration
(GF) is defined as: A separation technique in which separation, mainly according
to the hydrodynamic volume of the molecules or particles, takes place in a
porous non-adsorbing material with pores of approximately the same size as the
effective dimensions in solution of the molecules to be separated.[i] SEC is
mainly used for the characterization of macromolecules including synthetic and
natural polymers (including polysaccharides) as well as proteins or RNA/DNA. As
SEC covers such a broad application range, the main focus areas are slightly
different between applications. For synthetic and natural polymers, the main
purpose of the technique is typically for the determination of molecular mass
averages and molecular mass distribution of the sample. For proteins, the main
focus is typically the determination of monomeric and oligomer states and their
quantification. Further information, such as size, structural and compositional
information, can be derived from multi-detector SEC-systems. In addition to use
at analytical level, SEC can also be applied at a preparative scale for large-scale
separations or purification purposes (whereby eluting molecular weight
fractions can be collected in a suitable container and isolated). SEC can also be
applied to solid matter (nanoparticles) dispersed in a liquid. Despite the
increasing interest in nanoparticle characterization, SEC studies on
nanoparticles still form only a minor area of interest and the main focus of SEC
remains on characterization on a molecular level.
• Size-exclusion chromatography (SEC), also called gelfiltration or gel-
permeation chromatography (GPC), uses porous particles to separate molecules
of different sizes. It is generally used to separate biological molecules, and to
determine molecular weights and molecular weight distributions of polymers It
is usually applied to large molecules or macromolecular complexes such as
proteins and industrial polymers. When an aqueous solution is used to transport
the sample through the column, the technique is known as Gelfiltration
chromatography . When an organic solvent is used as a mobile phase, the
technique is known as Gel-permeation chromatography. The separation of
molecules is called fractionation. Size of pores in beads determines the
exclusion limit (what goes through the beads and what goes around the beads.

PRINCIPLE
A mixture of molecules dissolved in liquid (the mobile phase) is applied to a
chromatography column which contains a solid support in the form of
microscopic spheres, or “beads” (the stationary phase). The mass of beads
within the column is often referred to as the column bed. The beads act as
“traps” or “sieves” and function to filter small molecules which become
temporarily trapped within the pores. Larger molecules pass around or are
“excluded” from the beads. Large sample molecules cannot or can only partially
penetrate the pores, whereas smaller molecules can access most or all pores.
Thus, large molecules elute first, smaller molecules elute later, while molecules
that can access all the pores elute last from the column. Particles of different
sizes will elute (filter) through a stationary phase at different rates.

Separation Mechanism
Prerequisites
SEC is a liquid chromatography (LC) method, a subset of HPLC, and requires the
sample material to be completely dissolved with the individual molecules
dispersed and not interacting. In certain cases, where the aim is to study
assemblies of molecules, e.g. complexes of several proteins, conditions have to
be chosen to keep those complexes intact. Although dissolution is
straightforward for a lot of samples, care has to be taken to make sure samples
do go into solution completely and do not degrade or get modified by the
dissolution process. The separation process The columns used for SEC are filled
with a gel matrix containing highly porous spherical particles. The most common
materials used in these gels include crosslinked polymers like polystyrene,
acrylates, dextran or silica. A constant eluent flow is forced through the column
by an isocratic pump. The sample solution is introduced into the flow by means
of an injection valve and loop. Under ideal conditions, there is no interaction
between the sample and the gel, meaning that the separation process should be
based purely on diffusion of the analyte while the solution travels through the
stationary phase. If there is no adsorption of the analyte to the stationary phase,
the separation process is purely entropic ally driven. The standard free energy
change ∆G° of a chromatographic process is generally described by with ∆H°
being the change in standard enthalpy, ∆S° the change in standard

with ∆H° being the change in standard enthalpy, ∆S° the change in standard
entropy, R the gas constant, T the absolute temperature and k the partition
coefficient. With the separation being free of enthalpy contributions, the above
simplifies for SEC to This shows that the separation process is not expected to
be temperaturedependent. In practice, temperature can be seen to have a small
effect on the result due to its effect on solvent viscosity and the molecules’
diffusion rates. Furthermore, the flow rate dependency of the separation
process is also limited: the flow has to be faster than the re-mixing of
components due to backwards diffusion of previously separated molecules, but
it should not be too fast to allow time for the separation process to happen.
Also, the column packaging material sets an upper limit to the usable flow rates.
Some samples might degrade during the passage through the column if too high
flow rates are applied due to the shear forces within the liquid. Typically, flow
rates for analytical SEC systems are in the range of 0.1 to 1.0 mL/min. To achieve
a purely diffusion driven separation process, a suitable combination of mobile
and stationary phase is required where there is no interaction between the two.
The analyte molecules will then diffuse in and out of the pores of the packaging
material as the mobile phase carries the sample through the column. Whilst a
molecule is inside a pore of the packaging material, its passage through the
column will be delayed compared to the other molecules. Since smaller
molecules will find their way into more of the pores in the packing gel, and also
penetrate more deeply, they will be delayed more than larger molecules. Thus
the result is a separation of the molecules according to their hydrodynamic size
with the larger molecules eluting first. Figure 1 shows the separation process
(schematic) from the time where the sample is introduced into the column until
the sample eluted completely. Figure 1: SEC separation process (schematic)
Clearly there are limits to the separation at the extreme ends of the size
separation. Any molecules that are larger than the largest pores will not
experience any delay in elution and therefore will not be separated from each
other. These will elute together having travelled only through the interstitial
volume of mobile phase between the packaging particles of the column (around
14 mL in the example shown in Figure 2). This is called the “void volume”. Any
molecules which are smaller than the smallest pores will diffuse into all of the
pores in the gel packing matrix and will also not be separated from each other.
These will be the last molecules to elute (around 34 mL in the example shown in
Figure 2). Since almost every injection will include solvent or salt molecules
which are in this size range, a peak in this region will always be visible on the RI
detector and is often called the ‘solvent peak’. The elution of this peak marks
the end of the measurement (called “total permeation volume”, VT) and it is
worth noting that this is therefore independent of the sample. It can be used to
determine the sum of interstitial volume and intra-particle volumes. The
intermediate volume where analyte molecules are able to partially penetrate
the column packaging represents the useful range for SEC separation. Many
columns and pore sizes are available to tailor the separation range to the specific
application.
COMPONENTS OF A SEC
1.. Stationary Phase
2. The Mobile Phase
3. The Columns
4. The Pump
5. Detector

STATIONARY PHASE:
Stationary Phase Semi-permeable, porous beads with well-defined range of
pore sizes. Beads are cross-linked polymers Degree of crosslinking is controlled
carefully to yield different pore sizes. Smaller pore sizes are used for rapid
desalting of proteins or for protein purification. Intermediate pore sizes are
used to separate relatively small proteins. Very large pore sizes are used for
purification of biological complexes. Stationary phase used for gel exclusion
chromatography include dextran (Sephardi™), polyacrylamide and
dextranpolyacrylamide (Sephacryl™). Each is available with a variety of different
ranges of pore size in the beads, permitting separation of macromolecules of
different size A good stationary phase should have following properties:  It
should be chemically inert.  It should be inexpensive.  It should not react with
component to be separated.  It should not react with eluent.  It should be
colorless, uniform in size and shape.  It should be mechanically stable.  Soft
gel e.g.- dextran(Sephardi), Polyacrylamide gels Separation of proteins.  Semi-
rigid gel e.g.- bio beads Separation of non-polar polymers in non-polar solvents.
 Highly rigid gels and glasses Separation of polar systems.

Dextran
A homopolysaccharide of glucose residues. It’s prepared with various degrees
of cross-linking to control pore size. It’s bought as dry beads; the beads swell
when water is added. The trade name is spadix. It’s mainly used for separation
of small peptides and globular proteins with small to average molecular mass.
TYPICAL SEPARATION RANGES THAT CAN BE ACHIEVED USING SEPHADEX ARE
GIVEN BELOW. MOLECULES RANGING FROM 100 TO 600,000 DA CAN BE
SEPARATED DEPENDING ON THE TYPE OF SEPHADEX CHOSEN.
Polyacrylamide
these gels are prepared by cross linking acrylamide with N, N-methylene bis
acrylamide. The pore size is determined by the degree of cross-linking. The
separation properties of polyacrylamide gels are mainly the same as those of
dextran’s. They are sold as bio-gel P. They are available in wide range of pore
sizes.

Agarose
Linear polymers of D-galactose and 3,6 anhydro-1- galactose. It forms a gel
that’s held together with H bonds. It’s dissolved in boiling water and forms a gel
when it’s cold. The concentration of the material in the gel determines the pore
size. The pores of agarose gel are much larger than those of sephadex or bio-
gel p. It’s useful for analysis or separation of large globular proteins or long
linear molecules such as DNA.

Mobile phase
The liquid used to dissolve the biomolecules to make the mobile phase is usually
called a buffer. The mixture of biomolecules dissolved in the buffer is called the
sample. The choice of mobile phase to be used in any separation will depend
on the type of separation to be achieved and component to be separated.  The
most common eluents in for polymers that dissolve at room temperature.e.g.-
Tetrahydrofuran, Chloroform, Dimethyl formamide. MOBILE Phase

SOLVENT SELECTION
The solvents used for mobile phase of SEC are limited to those follows following
criteria: The solvent must dissolve the sample completely. The solvent has
different properties with solute in the eluent: typically, with solvent refractive
index (RI). solvent must not degrade the sample during use Otherwise, the
viscosity of eluent will gradually increase over times. The solvent is not corrosive
to any components of the equipment

Mobile Phase Preparation

high purity of solvent is recommended. Filter mobile phase solvents using 0.5-
micron filter to remove any particular impurities such as dusts, insoluble salts.
Antioxidant is added to dichlorobenzene to keep solvent stable in high
temperature. Other additives eliminate adsorption or interaction of solutes
with column packing materials Size Exclusion Chromatography

Sample preparation
The sample solutions are supposed to be prepared in dilute concentration (less
than 2 mg/mL). A good solvent can dissolve a sample in any proportion in a range
of temperatures. Samples with broad molecular weight distribution may require
higher concentrations. It is recommended to filter the sample solutions before
injecting into columns in order to get rid of clogging and excessively high
pressure problems. Agitation and filtration Generally filtration is required to
remove insoluble impurities. Do not agitate and filter samples that contain very
high MW (>1 million).

COLUMNS
Commercially Available Columns  analytical column- 7.5–8mm diameters. 
Preparative columns-22–25mm for.  Usual column lengths-25, 30, 50, and 60
cm.  Recently, narrow bore columns- 2–3mm diameter have been introduced,
which save time and solve Selecting SEC column Shorter columns save time
and solvent. Small particles (typically 5 mm) provide a better resolution. On
the other hand, 5 mm (or even 3 mm) packing’s are more sensitive towards
contamination by samples containing impurities. Particles as large as 20 mm
have been recommended for very high-molecular-weight polymers. ion.
Small particle size packing’s can sometimes result in shear degradation of large
polymer molecules because the space between particles is very narrow.
Columns with different porosity or mixed-bed columns, provide a better
separation. Handling SEC Columns A column set in SEC should be always run in
the same mobile phase.(isocratic)  SEC columns should never be operated in a
backward direction.  Care should also be taken in connecting columns or in
sample injection.  Replacing a clogged inlet frit is a dangerous operation which
can reduce column performance.  A damaged or dirty check valve of pump,
can also reduce column life.

PUMP
A highly constant flow rate has to be maintained during the entire
chromatogram. This is very important in SEC. A change of the flow rate of only
0.1% can cause an error in molar mass of up to 10%. Most pumps can only
reproduce the flow rate to 0.2–0.3%. In-line filters in the solvent reservoir may
prevent particles from coming into the pump heads, which might damage the
check valves or the pump seals. Types-)Syringe pumps, Reciprocating pumps
DETECTOR Concentration sensitive detectors Bulk Property

Detectors
Refractive Index (RI) Detector Solute Property Detectors- Ultraviolet (UV)
Absorption Detector Evaporative Detectors- Evaporative Light Scattering
Detector (ELSD) Molar mass sensitive detectors  Light Scattering Detectors
Low Angle Light Scattering (LALS) Detectors Multiage Light Scattering (MALS)
detectors Viscosity Detectors- Differential Viscometers Other :- Flame
Ionization Detector (FID), A Mass Spectrometer or A Fourier Transform Infrared
(FTIR).

Advantage:
The advantages of this method include good separation of large
molecules from the small molecules with a minimal volume of
eluate, and that various solutions can be applied without
interfering with the filtration process, all while preserving the
biological activity of the particles to be separated. The technique
is generally combined with others that further separate
molecules by other characteristics, such as acidity, basicity,
charge, and affinity for certain compounds. With size exclusion
chromatography, there are short and well-defined separation
times and narrow bands, which lead to good sensitivity. There is
also no sample loss because solutes do not interact with the
stationary phase. Disadvantages are, for example, that only a
limited number of bands can be accommodated because the time
scale of the chromatogram is short, and, in general, there has
to be a 10% difference in molecular mass to have a good
resolution.

Famous quotes containing the

word advantages:
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women citizens, accustomed to the advantages of our
schools, our churches and the mingling of our social life,
to ask over and over again for so simple a thing as that
“we, the people,” should mean women as well as men;
that our Constitution should mean exactly what it says?”
—Mary F. Eastman, U.S. suffragist. As quoted in History of
Woman Suffrage, vol. 4 Ch. 5, by Susan B. Anthony and
Ida Husted Harper (1902)

“The advantages found in history seem to be of three

kinds, as it amuses the fancy, as it improves the
understanding, and as it strengthens virtue.”
—David Hume (1711–1776)

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shores of Walden Pond, and lived there two years alone, a
life of labor and study. This action was quite native and fit
for him. No one who knew him would tax him with
affectation. He was more unlike his neighbors in his
thought than in his action. As soon as he had exhausted
himself that advantages of his solitude, he abandoned it.”
—Ralph Waldo Emerson (1803–1882).

The disadvantages of size exclusion

chromatography are as follows.

Mobile phase filtering is necessary to prevent the columns and interfere

with detectors.
The major disadvantage of size exclusion chromatography is that the
limited number of peaks can be resolved since the run time is short.

It has poor selectivity compared to other chromatographic techniques.

The load capacity of the sample is another major disadvantage of gel
filtration chromatography.

The disadvantages of size exclusion chromatography are as follows.

Mobile phase filtering is necessary to prevent the columns and interfere
with detectors. The major disadvantage of size exclusion chromatography
is that the limited number of peaks can be resolved since the run time
is short.

APPLICATIONS OF SIZE EXCLUSION

CHROMATOGRAPHY IN PHARMA-CEUTICAL
ANALYSIS

The size-exclusion-chromatography may be used for two specific purposes in the

analysis of pharma-ceutical substances, such as:

(i) Determination of relative component composition, and

(ii) Determination of molecular weight.

1. DETERMINATION OF RELATIVE
COMPONENT COMPOSITION

The assay method along with specific experimental parameters are duly stated in
the official mono-graph. Here, two situations arise, namely:

(a) Equivalent Responses:

In case, all of the components of the sample exhibit equivalent responses
to the detector, then the relative quantity of each component may be
determined conveniently by dividing each peak area by the sum of the peak
areas of the components of interest, and

(b) Non-equivalent Responses:

In case, the responses achieved are not equivalent, calculate the relative
component composition either from the calibration curve obtained with the
calibration standards specified in the official monograph or by any other
method stated in the official monograph.

2. DETERMINATION OF MOLECULAR WEIGHT

The following steps may be followed in a sequential manner to determine the
molecular weight of a pharmaceutical substance:

(i) Follow the method on the sample by employing the specified procedure laid
down in the official monograph,

(ii) Plot a graph of the retention volume of the calibration standards as a function
of the logarithm of the molecular weight,

(iii) The curve, thus obtained, normally approximates to a straight line within the
exclusion and total permeation limits,

(iv) The molecular weight of the component of interest may be determined from
the calibration curve, and

(vi) The calibration is valid only for the particular system employed under the
specified experimental parameters.

3. CORTICOTROPHIN: FOR IMPURITIES OF

HIGHER MOLECULAR WEIGHTS
Materials Required: Corticotrophin: 1 mg; acetic acid (1 M) [prepared by
dissolving 57 ml of

glacial acetic acid in 1000 ml of DW]: 100 ml; sodium dodecyl sulphate (1%
w/v): 10 ml;

Procedure
Dissolve accurately weighed 1 mg of corticotrophin in 1 ml of 1 M acetic acid
containing 1% w/v of sodium dodecyl sulphate. Heat the solution at 100 °C for
10 minutes and allow to cool.

The chromatographic procedure may be performed using (a) a column (about 85

cm × 10 mm) packed with polyacrylamide or cross-linked dextran for
chromatography having a fractionation range for peptides with relative molecular
weights of approximately 1000 to 10,000; (b) 1 M acetic acid as the mobile phase
with a flow rate of 7 ml per hour, and (c) a detection wavelength of 276 nm. Now,
connect the detector, fitted with a flow-cell suitable for liquid chromatography
having a volume of not more than 1 ml, to a strip-chart recorder. Set the detector
and chart recorder at a full-scale sensitivity of 0.5 absorbance unit.
Equilibrate the column with 1 M acetic acid. Apply the cold solution to the top of
the column using 0.4 ml per cm2 of column cross-sectional area. The sum of the
areas of any peaks eluted before the principal peak is not greater than 5.0% of the
sum of the areas of all the peaks in the chromatogram.

4. INSULIN: FOR PROTEINS OF HIGHER

MOLECULAR WEIGHT

Materials Required: Solution (1): Dissolve 10 mg of insulin in 1 ml of

the mobile phase; Solution (2) Dilute 100 μ l of solution (1) to 10 ml with the
mobile phase; and Solution: (3) Dissolve 10 mg of porcine insulin EPCRS* of
bovine insulin EPCRS, as appropriate, in 1 ml of the mobile phase.

Procedure : The chromatographic procedure may be carried out using (a) a

column (60 cm × not less than 7.5 mm) packed with silica gel for chromatography
(10 μ m ; pore size about 13 nm) Water 1-125 ; Toyo Soda TSK 2000 SW ; and
Zorbax GF 250 are suitable ; (b) as the mobile phase with a flow rate of 0.5 ml
per minute a filtered and degassed solution prepared by mixing 20 volumes of
glacial acetic acid and 50 volumes of water, adjusting the pH to 3.0 by the
addition of a 25% v/v solution of ammonia, adding 30 volumes of acetonitrile and
mixing 1 and (c) a detection wavelength of 276 nm.

Inject 50 μ l of each solution. Adjust the sensitivity of the detector so that the
height of the principal peak in the chromatogram obtained with solution (2) is 50-
70% of full-scale deflection. In the chromatogram obtained with solution (1) the
sum of the area of any peak eluting before the principal peak is not greater than
the area of the principal peak in the chromatogram obtained with solution (2)
(1.0%).

5.
HUMAN INSULIN: FOR PROTEINS OF HIGHER
MOLECULAR WEIGHT
Materials Required: Solution (1): Dissolve 10 mg of human insulin in
1 ml of the mobile phase, Solution (2): Dilute 100 μ L of Solution (1) to 10 ml
with the mobile-phase, and Solution (3): Dissolve 10 mg of human insulin
EPCRS in 1 ml of the mobile-phase.
Procedure : The chromatographic procedure may be performed using (a) a
column (60 cm × not less than 7.5 mm) packed with silica gel for chromatography
(10 μ m ; pore size about 13 nm) Water 1-125 ; Toyo Soda TSK 2000 SW and
Zorbax GF 250 are suitable, (b) as the mobile phase with a flow rate of 0.5 ml per
minutes of a filtered and degassed solution prepared by mixing 20 volumes of
glacial acetic acid and 50 volumes of water, adjusting the pH to 3.0 by the
addition of a 25% v/v solution of ammonia, adding 30 volumes of acetonitrile and
mixing, and (c) a detection wavelength of 276 nm.

Inject 50 μ L of each solution. Adjust the sensitivity of the detector so that the
height of the principal peak in the chromatogram obtained with solution (2) is 50
to 70% of full-scale deflection. In the chromatogram obtained with solution (1)
the sum of the areas of any peaks eluting before the principal peak is not greater
than the area of the principal peak in the chromatogram obtained with solution
(2) (1.0%).

6. PLASMA PROTEIN SOLUTION: FOR

POLYMERS AND AGGREGATES
Materials Required: Plasma protein solution: 2.0 ml; mixed phosphate buffer
pH 7.0 with azide [To 1000 ml of a solution containing 1.8% w/v of disodium
hydrogen orthophosphate and 2.3% w/v of sodium chloride and sufficient of a
solution containing 0.78% w/v of sodium dihydrogen orthophosphate and 2.3%
w/v of sodium chloride (about 280 ml) to produce a pH of 7.0 Dissolve sufficient
sodium azide in the resulting solution to give a 0.02% w/v solution]: 1000 ml;

Procedure: The chromatographic procedure may be carried out at room

temperature using (a) a column (1 M × 25 mm) packed with a cross-linked
dextran suitable for fractionation of globular proteins in the range of molecular
weights from 5,000 to 350,000 (Sephadex G-150 is suitable), (b) mixed
phosphate buffer pH 7.0 with azide as the mobile-phase with a flow rate of about
20 ml (4 ml per square centimetre) of column cross-sectional area) per hour, and
(c) a detection wavelength of 280 nm.
Collect the eluate in fractions of about 4 ml and combine the fractions
corresponding to each peak. For each combined fraction carry out the
determination of nitrogen as per BP (1993). Not more than 10% of the total
nitrogen is present in the combined fraction associated with non-retained proteins.
References
http://goldbook.iupac.org/S05705.html as of 5 June 2015
http://www.malvern.com/en/support/resource-center/technical-notes/
TN120103DeterminationTheoreticalPlateNumbers.aspx
http://www.malvern.com/en/support/resource-center/technical-notes/
TN111031WhatIs-dA-dc.aspx
http://www.malvern.com/sls

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