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1Chromatography CourseDr Ehab Aboueladab-Lecturer of Biochemistry-Mansoura University-Branch Damietta
Chapter One
Aim Separation Techniques
1-Biological fluids are extremely complex in composition.
2-Chemical analysis would be impossibleif it were necessary to completelyisolate each substance prior to its measurement.
3- An optimal method tests for a specific substance in the presence of all
others, requiring no isolation of the substance under analysis.
4- A test is specific when none of the other substances present interfere.
However, virtually all chemical tests are subject to at least some
interference.
5-This is one of the most important problems in clinical chemistry. Therefore
some type of separation procedure is required.
7-Separation in clinical chemistry usually is based on differences in the
size, solubility or chargeof the substances involved.
Introduction
Chromatography is a separation method in which the analyte is
contained in a mobile phase and pumped through a stationary phase.
Sample components interact differently with these two phases and elute
from the column at different retention times tR. Since the first description
of chromatography by Russian botanical scientist Mikhail Semenovich
Tswett is discovery of chromatography. He used a column of powdered
calcium carbonate to separate green leaf pigments into a series ofcolored bands by allowing a solvent to percolate through the column bed.
Since these experiments by Tswett many scientists have made substantial
contributions to the theory and practice of chromatography. Not least among
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these is A. J. P. Martin who received the Nobel Prize in 1952 for the
invention of partition chromatography (with R. L. K. Synge) and in the
same year with A. T. James he introduced the technique of gas-liquid
chromatography. Chromatography is now an important tool used in allbranches of the chemical and life sciences.
Chromatographic separations can be described quantitatively with a number
of parameters including the capacity factor k , the selectivity factor , the
plate number N or height equivalent of a theoretical plate H and the
resolution RS. The optimum flow rate of a chromatographic separation can
be determined with the van Deemter equation. In bioanalytical chemistry,
chromatography is mainly employed for the isolation and purification of
proteins. Reversed p hase chromatography can separate biomolecules
according to their interaction with the hydro phob ic stat ionary phase and
the hydro phi l ic mobl i le phase. This separation method can be coupled to
an ESI mass spectrometer. Ion exchange chromatography separates
molecules depending on their net charge. Aff in i ty chrom atographymakes
use of molecular recognition between biomolecules; and size exclusion
chromatography allows for the separation of molecules depending on
their size.
1-Definition of Chromatography
Chromatography is essentially a physical method of separation in
which the components to be separated are distributed between two phases
one of which is stationary (stationary phase)while the other (the mobilephase)through it in a definite direction.
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chromatography (GSC), liquidliquid chromatography (LLC), and liquidsolid
chromatography (LSC),
4- Main Type of Chromatography
In general, there are four main types which can be classified asfollows:
4.1-Liquid-Solid chromatography
Classical adsorption chromatography(Tswett column)
Ion-exchange chromatography
4.2. Gas-Solidchromatography
4.3. Liquid-Liquidchromatography
Classicalpartition chromatography
Paper chromatography
4.4Gas-Liquidchromatography
5-Separation techniques
Technique Property Description
Precipitation Solubility Some of the substances
precipitate while the others
remain dissolved
Ultra-filtration or Dialysis Molecular size Some of the substances
pass through a layer or
sheet of porous material
while the other substances
are retainedExtraction Solubility Some of the substances
dissolve (partition) more in
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water. While other
substances dissolve more
organic solvent in contact
with the waterThin layer
Chromatography
or
Column Chromatography
Solubility Some of the substances
dissolve (partition) more in
the immobile file of water on
a solid supporting medium
(or stick more to the
exposed areas of the solid
supporting medium) while
the other substances
dissolve more in the
surrounding film of flowing
organic solvent
Gas liquid
Chromatography
Solubility Some of the substances
dissolve more in the
immobile film of wax or oil-
like material on a solid
supporting medium. While
the others dissolve more in
surrounding stream of
flowing gas.Gel filtration
Chromatography
Molecular Size Some of the substances
diffuse into the pores in a
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porous, solid material while
others remain outside in the
surrounding stream of
flowing waterIon-exchange
Chromatography
Electrical
charge
Some of the substances are
bound by immobile charges
on the solid supporting
medium while others are not
bound
Electrophoresis
Chromatography
Electrical
charge
The substances with more
charge move faster and,
therefore, further.
Substances with opposite
charges move in opposite
directions.
6-Adsorption chromatography
Adsorption column chromatography is the oldest form of
chromatography. Whether two or more substances of a mixture can be
separated by adsorption chromatography depends on a number of factors.
Most important is the strength with which each component of mixture is
adsorbed and its solubility in the solvent used for elution.The degree to
which a particular substance is adsorbed depends on the type of bondswhich can be formed between the solute molecules and the surface of the
adsorbent.
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5-Purpose of Chromatography
Analytical- determine chemical composition of a samplePreparative- purify and collect one or more components of a sample
Other classification of Chromatographic Methods
Chromatography is classified according to three ways:
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1. According to the physical state of the mobile phase:
Liquid chromatography: This subdivided according to the stationary
phase into liquid-liquid or liquid-solid chromatography.Gas chromatography: This subdivided according to the stationary
phase into Gas-liquid or Gas-solid chromatography.
2. According to the method of contact between the mobile phase and
stationary phase:
Column chromatography: the stationary phase is placed in a 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 columns walls.
Planar chromatography: the stationary phase coats a flat glass, metal, or
plastic plate and is placed in a reservoir containing the mobile phase
which moves by capillary action carrying with it the sample components
3. According to the chemical or physical mechanism responsible for
separating the samples constituents.(attractive forces)
Adsorption chromatography: for polar non-ionic compounds
Ion Exchange chromatography: for ionic compounds
Anion: analyte is anion; bonded phase has positive charge
Cation: analyte is cation; bonded phase has negative charge
Partition chromatography: based on the relative solubility of analyte in
mobile and stationary phases Normal: analyte is non-polar organic; stationary phase MORE polar
than the mobile phase
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Reverse: analyte is polar organic; stationary phase LESS polarthan
the mobile phase
Size Exclusion chromatography: stationary phase is a porous matrix.
6-The Principle of ChromatographyChromatography is a separation method where the analyte is contained
within a liquid or gaseous mobile phase, which is pumped through a
stationary phase.
Usually, one phase is hydrophilic and the other lipophilic. The components of
the analyte interact differently with these two phases. Depending on their
polarity, they spend more or less time interacting with the stationary phase
and are thus retarded to a greater or lesser extend. This leads to the
separation of the different components present in the sample. Each sample
component elutes from the stationary phase at a specific time, its retention
time tR(Fig. 1.1). As the components pass through the detector, their signal
is recorded and plotted in the form of a chromatogram.
Chromatographic methods can be classified into
Gas chromatography (GC) and liquid chromatography (LC) depending on
the nature of the mobile phase involved.
Gas chromatographycan be applied only to gaseous or volatile substances
that are heat-stable. The mobile phase, an inert carrier gas such as
nitrogen, hydrogen or helium, is pumped through a heated
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Figure1-1
column. This column can be packed with a silicon oxide based material or
is coated with a polymeric wax. The sample is vaporised, pumped throughthe column and the analytes are detected in the gas stream as they exit the
column. Analyte detection can be achieved by either flame ionisation or
thermal conductivity. GC is not commonly used for the analysis of
biomolecules since large molecular weight compounds such as peptides and
proteins are thermally destroyed before evaporation. Smaller molecules such
as amino acids, fatty acids, peptides and certain carbohydrates can be
analysed if they are modified chemically to increase their volatility. Some cell
cultures produce volatile metabolites such as aldehydes, alcohols or
ketones. These can be analysed readily via GC.
In liquid chromatography, the sample is dissolved and pumped through a
column containing the stationary phase. LC is more versatile than GC as it is
not restricted to volatile and heat-stable samples; the sample only has to
dissolve completely in the mobile phase. Common detection methods are
UV spectroscopy, measurement of refractive index, fluorescence, electrical
conductivity and mass spectrometry.
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Modes of operation can be classified as normal and reversed phase
chromatography.
In normal phase chromatography, the stationary p hase consists of a
hydrophilic material such as silica particles and the mobile phase is ahydrophobic organic solvent such as hexane.
In reversed phase chromatography, on the other hand, the stationary
phase is hydrophobic and the mobile phase is a mixture of polar solvents, for
example water and acetonitrile. Biomolecules are generally soluble in polar
solvents; hence, reversed phase chromatography is the method of choice for
amino acids, peptides, proteins, nucleic acids and carbohydrates.
7-Comparison of classical and bioanalytical chemistry
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The optimisation of chromatography is aimed towards completely
separating all of the components of a sample in the shortest possible time.
This can, for example be achieved by modifying the composition of the
mobile phase, choosing a different stationary phase or by changing the flowrate. A typical chromatogram is depicted in (Fig. 1.2).
Fig. 1.2.Definition of retention time, tR, and peak width, w.
The sample is injected into the chromatographic column at t = 0 s.
Substances that are not retarded by the stationary phase leave the column
at zero retent ion t im e, t0, corresponding to the flow rate of the mobile
phase. Compounds A and B are retarded by the stationary phase and leavethe column at their retention times tR(A) and tR(B), respectively. The peak
width, w, is defined as the intersection of the tangents on each side of the
peak with the baseline. These basic parameters, retention time and peak
width, can be used to derive a number of other parameters that express the
quality of the achieved chromatographic separation. In the following
paragraphs, a brief summary of the most important parameters of
chromatographic theory are discussed.
The capacity factor k' (equation1. 1)describes the velocity of the analyte
relative to the velocity of the mobile phase. Each compound spends a
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(equations 1.3)
(equations 1.4)
The parameters that influence band broadening can be approximated by the
van Deemter equation (equation 1.5) which is valid for gas and liquid
chromatography as well as capillary electrophoresis
(equations 1.5)
In this simplified equation, the height of theoretical plates, H, is given as a
sum of three terms. The first term, A,describes the influence of the column
packing on band broadening. This so-called Eddy diffusion is constantfor
a given column and independent of the flow rate. The second term, B/u,
describes the diffusion in or opposed to the direction of flow. This
longitudinal diffusion is inversely proportional to the flowrate u. The third
term, Cu, describes the resistance to mass transfer between the stationary
and mobile phase which is directly proportional to the flow rate. By plotting H
as a function of u, the optimum flow ratefor a chromatographic separation
can be determined (Fig. 1.3).
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Fig. 1.3. A van Deemter plot for the determination of the optimum flow rate.
The ultimate goal of a separation is to achieve a high resolution, Rs,
(equations 1.6 and 1.7). If Rs = 1.5, then peaks of identical area overlap by
only 0.3 %, an Rs = 1equals a peak overlap of 4 %. Peak resolution can be
optimized by increasing the selectivity and minimizing band broadening.
Resolution
(equations 1.6)
valid for < 1.2
(equations 1.7)
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As can be seen from equation 1.7, the capacity factor k' has a great
influence on the resolution. Usually the components in the sample have a
wide variety of k values. If conditions are optimised such that the firstcompounds to elute have k' values between the optimum of 1 and 5, then the
other compounds with higher k valueselute much later and show excessive
band broadening. If, on the other hand, conditions are optimised for the later
eluting compounds, then the resolution will be poor for the compounds that
elute first. This general elutionproblem can be overcome by decreasing k'
during the separation. In LC, the composition of the mobile phase can be
changed during the separation. This is called a gradient elut ion as
opposed to an isocratic elution, where the composition of the mobi le phase
remains unchanged during the separation process. In GC, a temperature
gradient can be applied during separation rather than operating under
isothermic conditions. Generally, the first step in trying to achieve a good
separation of the sample mixture is to choose a stationary phase with which
the analyte can interact. Then, the composition and gradient of the mobile
phase can be chosen to optimise the capacity factor and resolution.
Chromatographic theory as outlined in the above paragraphs can be
applied to the analysis of smaller molecules such as amino acids,
peptides and short biopolymers. Care has to be taken for larger
biomolecules such as high molecular weight proteins. These often show
different behavior and the theory can only be applied to a limited extent.
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hydroxyl groups of the silica particle with silanes containing non-polar
hydrocarbon chains. Any chain length from ethyl silane (C2) to n-octadecyl
silane (ODS) (C18) is used, although octyl silane (C8) and ODS are the most
commonly employed chain lengths. For analytical separations, the particlesize is typically 5 m or smaller. In preparative liquid chromatography, where
the goal is to isolate a compound of interest for further analysis or
investigation, larger particles with a higher capacity and larger column
diameters are used. The pore size of the silica particles is usually about 10
nm, resulting in a very large surface area, as much as 100 to 400 m2/g.
This gives the analytes ample opportunity to interact with the stationary
phase whilst flowing through the separation column.
The mobile phase is based on a polar solvent system consisting of an
aqueous buffer and acetonitrile or methanol. Gradient elution is often
employed to increase resolution and shorten separation times. This is
achieved by increasing the organic solvent and thus decreasing the mobile
phase polarity and the retention of less polar analytes during the separation
process. Solvents can be classified according to their elution strength and
polarity (Fig. 1.5).
Buffer systems based on ammonium acetate, phosphate or hydrogen
carbonate are usually added at concentrations of about 20 mMto adjust the
pH of the mobile phase to values between 2 and 8. Ion pairing reagents can
be used at low concentrations, typically 0.1%, to increase the hydrophobicity
of charged analytes. They
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Fig. 1.5. Solvents ordered according to polarity and elution speed of the
analytes.
Fig. 1.6.Instrumental setup of an HPLC gradient system.
form ion-pair complexes with the analyte. Anionic ion pairing reagents such
as trifluoroacetic acid (TFA) bind to positively charged analytes,
whereas cationic ion pairing reagents such as tetra alkyl ammonium salts
can be used to bind to negatively charged analytes. These complexes are
retarded more by the stationary phase and are thus easier to separate than
the largely unretained charged analytes alone.
In modern chromatography, the separation columns are tightly packed with
small particles of about 15m in diameter. To achieve ambient flow rates in
these columns, high pressures of up to 300400 bars must be generated. A
typical instrumental setup for this high pressure or high performance liquid
chromatography (HPLC) is shown in Fig. 1.6.
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Computer controlled pumps move the mobile phase through the system.
Aqueous solvent A and organic solvent B are mixed to the desired
composition. In the case of gradient elution, the composition is gradually
altered during the separation.Sample volumes are injected with either a manual loop and valve system or
automatically via an auto sampler. Depending on the column dimensions
sample volumes can be as low as several nL and as high as a mL. Often the
column is situated inside an oven which is thermostatically regulated to
maintain a constant temperature. After eluting from the column, the analytes
pass through the detector.
UV detection using a fixed wavelength could be performed at = 210 nm
for peptides and = 254 nm or = 280 nm for proteins. More expensive
instruments have diode array detectors (DAD) which can take several
whole spectra per second and allow for more unambiguous identification.
High sensitivity can be achieved via fluorescence detection of derivatised
amino acids and peptides. A more recent developm ent is to couple
liquid chromatography systems to an elect rospray ion isation m ass
spectrometer, ESI-MS.
Mass spectrometry allows universal detection at very high sensitivity and
also gives structural information about the analyte. However, not all buffers
commonly employed for liquid chromatography are compatible with mass
spectrometers.
In recent years, there has been a trend to develop ever smaller liquidchromatography systems. LC systems on micro and even nanoscales have
been demonstrated. Shorter and smaller columns with smaller particles offer
faster analysis times, decreased solvent consumption and require less
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sample. The differences between preparative, analytical, micro and nano
LC are summarized in Table 1.2.
Table 1.2. Differences between preparative, analytical, micro and nano
liquid chromatography.
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Chapter Two
ADSORPTION CHROMATOGRAPHYIn adsorption chromatography the compounds to be separated are
adsorbed onto the surface of a solid material. The compounds are desorbed
from the solid adsorbent by eluting solvent.
1-Separation of the compounds depends on
1-The relativebalance between the aff in i ties of the comp ound s
for the adsorbent and their solubi l i ty in the solvent.
2-The chemical nature of the subs tances.
3-The nature of the solvent.
4-The nature of the adsorbent.
Solid adsorbents commonly used are alumina, si l ica gel , charcoal
(act ive carbo n), cel lulose, starch, and suc ros e.
Solvents commonly usedare hexane, benzene, petroleum ether, diethyl
ether, chlo rofo rm, methylene chlo r ide, var ious alco hols (ethyl , pro py l ,
n-bury l and t-buty l alcohols), and various aqueous b uf fers and sal ts,
some in combination with organic solvents
Adsorption chromatography is a column that is packed with the adsorbents.
The adsorbent is prepared and poured into the column with an inert support
at the bottom. Suitable supports include plast ic disc s, or sheetsof ny lon
or Tef lon fabr ics.The adsorbent bed must be homogeneous and f ree of bubb les, cracks,
or sp aces between the adsorbents and the wal ls of the column .
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Figure 2-1:Collection of fractions from a column by an automatic fraction
a device that accumulates from an elution column the same predetermined
volume in each of a series of tubes that automatically change position when
the proper volume has been collected .This may be accomplished in various
ways. For example, set volume, with a timer, or by counting drops with adrop counter. The latter is frequently used and is usually the most reliable
and flexible. The fraction collector may be Equipped with a detection cell
that automatically measures some parameter of the solution going into the
tubes and may correlated with fraction number and automatically recorded.
The detection cell is frequently a small spectrophotometer that can measure
absorbances at a fixed wavelength or at variable wavelengths. Other
detecting cell use index of refraction, optical rotation, and other properties.
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Figure 2-2: Adsorption chrornatography
A = adsorbent, S=Sample, ES= eluting solvent
(A) Application of sample to the column.
(B) Adsorption of sample onto adsorbent.
(C)Addition of elution solvent.
(D) and (E) Partial fraction of sample components.
(F) Complete fractionation of sample. (G) and (H) Separation of all three
components at various stages on the adsorbents.
(I) Elution of the first component from the column.
The substances adsorbed on the column support can be eluted in threeways
In the simp lest method, a single solvent continuously flows through
the column until the compounds have been separated and eluted from
the column
Stepwise elution, in which twoor mo re di f ferent solvents of fixed
volume are added in sequence to elute the desired compounds.
Gradient elut ion, in which the composition of the solvent is
continuously changing. The latter method is used to effect separations
that are difficult because of a tendency of component to be eluted in
broad. Trailing bands when a single solvent is used. Gradient elution
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frequently provides a means of sharpening the bands, a simple linear
gradient has two solvents, A and B, in which A is the starting solvent
and B is the final solvent. Solvent B is allowed to flow into solvent A as
solvent A flows into the column. The composition of solvent A is, thus,constantly changing as solvent B is constantly being added to A (Fig.
2-3).
Gradients other than linear gradients (e.g., exponent ial , concave. or
convex) may be obtained by introducing a third vessel and varying the
composition of the solvents in the vessels. These eluting methods are also
used with other column chromatographic methods.
2-Activation of adsorbent
Many adsorbents such as alumin a, si l ica gel , and act ive carbon and
Mg si l icatecan obtain commercially, but they require activation before use.
Activation is achieved by heating and there is usually an optimum
temperature for activation, for e.g. alumina is about 400oC. For reduced
activityby the controlled addi t ion of w ater, and the subsequent activity is
Figure 2-3: Gradient elution. Flow of solvent B into solventAWith mixing,
continuously changing the composition of solvent Aas it flows into column
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related to the amount of water added. Brookman and Schodder
established five grades of aluminaGrade I is the most active and the is
simply alumina heated at about 3500
C for several hours. Grade IIhas about
2-3% water, Grade III5-7%, Grade IV9-11 %, Grade Vfilm. (Least active)
about 15%.
3-Retention
The retention is a measure of the speed at which a substance moves in a
chromatographic system. In continuous development systems like HPLC or
GC, where the compounds are eluted with the eluent, the retention is usually
measured as the retention time Rt or tR, the time between injection anddetection. In interrupted development systems like TLC the retention is
measured as the retention factor Rf, the run length of the compound divided
by the run length of the eluent front:
The retention of a compound often differs considerably between
experiments and laboratories due to variations of the eluent, the stationary
phase, temperature, and the setup. It is therefore important to compare the
retention of the test compound to that of one or more standard compounds
under absolutely identical Conditions.
4-Plate theory
The plate theory of chromatography was developed by Martin and
Synge.The plate theory describes the chromatography system, the mobile
and stationary phases, as being in equilibrium. The partition coefficient K
is based on this equilibrium, and is defined by the following equation:
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K is assumed to be independent of concentration, and can change ifexperimental conditions are changed, for example temperature is increased
or decreased. As Kincreases, it takes longer for solutes to separate. For a
column of fixed length and flow, the retention time (tR) and retention
volume (Vr) can be measured and used to calculate K
5- Column chromatography
1. Small plug of wool (or cotton)
2. Sandto cover "dead volume"
3. Silica gel, length = 5.5 - 6 inch (Note 1inch=2.54cm).
4. Tap column on bech (carefully) to remove air bubbles inside
the column
5. Add solvent system
6. Add sandon top of silica
7. The top of the silica gel should not be allowed to run dry.
8. Sample is diluted (20-25% solution)
9. The sample is applied by pipette
10. Solvent used to pack the column is reused
11. Walls of column are washed with a few milliliters of eluant
12. Column is filled with eluant
13. Flow controller is secured to column and adjusted 2.0 in / min.
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Figure 2-5
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Table 2-1: Common adsorbents and the type of compounds
Solid Suitable for separation of
Alumina Steriods, vitamins, ester, and alkaloids
Silica gel Steriods, amino acids, alkaloids
Carbon Peptides, carbohydrates, amino acid
Magnesium carbonate Porphyrins
Magnesium silicate Steriods, ester, glycerides, alkaloids
Magnesia Similar to alumina.
Ca(OH)2 Carotenoids.
CaCO3 Carotenoids and xanthophylls.
Ca Phosphate Enzymes, protein, and polynucleotide
Starch Enzymes.
Sugar Chlorophyll.
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33Chromatography CourseDr Ehab Aboueladab-Lecturer of Biochemistry-Mansoura University-Branch Damietta
Chapter Three
PAPER CHROMATOGRAPHY
Paper chromatography is a type of liquid-liquid partitionchromatography that may be performed by ascending or descending
solvent f low. Each mode has its advantages and disadvantages.
Ascending c hromatography involves relatively simple and inexpensive
equipment compared with descending chromatography and usually gives
more uniform migration with less diffusion of the sample "spots."
Descending chrom atography, on the other hand is usually faster because
gravity aids the solvent flow and with substances of relatively low mobility.
The solvent can run off the paper. Giving a longer path for migration. To
resolve compounds with low mobility. Ascending chromatography may be
performed more than once utilizing a multiple-ascent technique.
For descending chromatography, papers 22 cm wide and 56 cm
long can be used. To facilitate the flow of solvent from the paper, the bottom
of the paper is serrated with a pair of pinking shears. Three pencil lines are
drawn 25 mm apart at the top of the sheet, and small aliquot of the sample
(10-50 ml) is placed at a marked spot on the third line. The spot is kept as
small as possible by adding the aliquot in small increments. With drying in
between. This may be expedited with a hair dryer. Several samples,
including standards, are placed 15-25 mm apart.
The paper is then folded along the other two lines and placed in thesolvent trough of the descending tank (Fig. 3-1).This has been equilibrated
with solvent beforehand to ensure a saturated atmosphere. The paper is
irrigated with solvent until the solvent reaches the bottom or for a longer
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period, allowing the solvent to flow off the end of the paper, if necessary. The
chromatogram is then removed dried and developed to reveal the locations
of the compounds. (Part II gives methods of locating carbohydrates, amino
acids. proteins. nucleotides and nucleic acids and lipids.)In ascending chromatography, a paper approximately 25 cm x 25
cm is used. A pencil line 20-25 mm from the bottom is drawn across the
paper
Fig. 3-1 Steps in descending paper chromatography
and aliquots (10-50l) of the samples and standards are spotted
approximately 15-25 mm apart along the line. The spots are dried and the
paper is rolled into a cylinder and stapled so that the ends of the paper are
not touching (Fig. 3-2). Solvent is poured into the bottom of a
chromatographic chamber, and the cylinder is placed inside. The chamber is
closed and solvent is allowed to flow up
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Fig.3-2 Steps in ascending paper chromatography
The paper by capillary action. The chamber may be a simple wide-mouth,
screw top, gallon jar or a cylinder with a ground-glass edge and a glass plate
top. As with descending chromatography, the chamber should be
equilibrated with solvent beforehand. Contrary to a popular misconception, if
the chamber has been sealed and is airtight, the paper does not have to be
removed as soon as the solvent reaches the top. When multiple ascents are
performed, the paper is removed, thoroughly dried, and returned to the
chamber for another ascent of solvent.
The resolved compounds on a paper chromatogram may be detected by
their color if they are colored, by their fluorescence if they are fluorescent, by
a color that is produced from a chemical reaction on the paper after sprayingor dipping the chromatogram with various reagents, or by autoradiography if
the compounds are radioactive. Identification of compounds on a
chromatogram is usually based on a comparison with authentic compounds
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(standards). A quantitative comparison may be made by measuring the Rf,
which is the ratio of the distance the compound migrates to the distance the
solvent migrates. A better comparison is the ratio of the distance a particular
compound migrates to the distance a particular standard migrates. Forexample, in the separation of carbohydrates, the standard might be glucose
and the ratio would be RGlcor for amino acids, the standard might be glycine
and the ratio would be RGly
A useful modification is two-dimensional paper chromatography, in
which the sample is spotted in the lower left-hand corner and irrigated in the
first dimension with solvent A. The chromatogram is removed from the
solvent dried, turned 90, and irrigated in the second dimension with solvent
B, giving a two-
Fig. 3-3 Two-dimensional paper or thin-layer chromatography
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dimensional separation (Fig. 3-3). An application of this procedure has
been developed for the study of enzyme specificity in which a solution of the
enzyme is sprayed onto the chromatogram between the first irrigation andthe second to see what products are formed by the action of the enzyme on
the compounds separated in the first dimension.
Fig.3-4. Elution of compounds from paper chromatograms for preparative
chromatography or quantitative determination
eluted with water. To accomplish the elution, tabs of chromatographic paper
are stapled to the narrow ends of each strip. As shown in Figure 3.4, one
end is fitted with two pieces of glass (cut microscope slides), which arc held
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together with rubber bands, and the bottom end is cut tapered, like a pipet
tip. This assembly is played so that one end lies in a chromatographic trough
containing water, and the elution of the strip occurs by capillary flow of the
water down the paper strip into a baker.Usually less than 1 mL of water is sufficient to effect quantitative
elution, the samples are quantitatively diluted to a specific volume, and a
chemical analysis is performed for the specific compound separated. This
technique also may be used as a preparative procedure to obtain small
quantities of pure compound from a mixture of compounds.
In an alternate quantitative procedure, the compounds in the sample are
radioactively labeled and separated in the usual way, and an autoradiogram
is prepared. The labeled compounds are located on the chromatogram by
comparing their positions on the autoradiogram. The radioactive compounds
are cut out and placed into a liquid scintillation cocktail, and the radioactivity
is determined by heterogeneous liquid scintillation counting
In paper chromatography, the mobile phase (solvent) carries the
components of the sample on the stationary phase (filter paper) separating
them according to the differences in the migration rate (depends on the
molecular weight , polarity and adsorption ability)
Components
For one-dimensional paper chromatography, either ascending or
descending development can be carried out in simple units. Descendingdevelopment is more often used because it is faster and more suitable for
long paper sheets.
The stationary phase (filter paper)
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The mobile phase (solvent may be in a reservoir)
Procedures
1. Make the initial line on the paper.
2. Apply the solvent alone on the initial line.3. Wait till the solvent migration is stopped, then make the final line.
4. Spot the sample, and then apply the solvent either in ascending or
descending or concentric manner.
5. In case of colored sample: Calculate the rate of flow (Rf) directly then
compare it with stander in order to know the unknown sample (qualitatively).
6. In case of the colorless sample: use UV-lamb to detect the spot
position then determine the (Rf). Rf depends on the temp., solvent, type of
paper Rf = distance of sample migration / distance of solvent migration
Appl icat ions
1. Separation of amino acids
2. Separation of the plant pigments
Advantages
1. Simple
2. Cheap
Disadvantages
1. Time consuming.
2. Need high quantity of sample.
3. With weak solvent power.
4. limited use5. Difficulty detection of spots
6. Difficulty isolation of separated substances.
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Chapter Four
Thin layer chromatography
This technique is particularly useful for the separation of very smallamounts of material. The general pr inc ip le involved is similar to that
involved in column chromatography, i.e. it is primarily adsorption
chromatography, although other partition effects may also be involved. A
glass sheet is covered by a uniform thin layer of an adsorbent. Adsorbents
used in TLC, di f fer f rom colum n adsorb ents. It contains a binding agent
such as calcium sulphate, which facilitates the adsorbent sticking to the
glass plate. The plates are prepared by spreading slurry of adsorbent in
water over them, starting at one end, and moving progressively to the other
and then dry ing them in an oven at 100-120C. Dryingserves to remove
the water and to leave a coating of adsorbent on the plate. Equipment is
available which will ensure the production of an even coating of adsorbent
over a series of glass plates. The normal thickness of slurry layer used is
0.25 mm for qualitative analysis, but layers up to 5-10 mm thick may be
made for preparative work.
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The sample is applied to the plate by micropipette or syringes, as spot2.5
cm from one endand at least an equal distance from the edge. The solvent
is removed from the sample by the use of an air blower. All spots should be
placed on equal distance from the end of the plate.Separation takes place in glass tank which contains the developing solvent
(mo bi le phase)to a depth of 1.5 cm, this is allowed to stand for at least 1
hour with a glass plate over the top of the tank to ensure that the
atmosphere within the tank becomes saturated with solvent vapor.
Then, the thin layer plate is placed vert ical lyin the tank so that, it stands in
the solvent with the end bearing the sample in the solvent.
The cover plate is replaced and separation of the compounds then occurs as
the solvent travels up the plate. After the solvent had reached the wanted
level , the run is s topp ed. The chromatographic separation is completed the
spots of the component substances can be detected by different
methods:
1-Many commercially available TLC adsorbents contain a fluorescent
dye, the plate is examined under UV light, the separated components
show up as blu e, gr een, blackarea.
2. Spraying the plate with 50% sulphur ic acid and heat ing so, the
compounds become charred and show spots
3. Spraying the plates with speci f ic color reagents will stain up certain
compounds e.g. ninh yd r in for am ino acid (aa) , ani l ine for aldoses.
SolventsUniversal TLC System:
Petroleum ether - ethyl acetate
Very polar solvent addi t ives:
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Methanol > ethanol > isopropanol
Moderately po lar addi t ives:
Acetonitrile > ethyl acetate > chloroform, dichloromethane > diethyl ether >toluene
Non-polar so lvents:
Cyclohexane, petroleum ether, hexane, pentane
TLC Visualization (Detecting the spots)
Non-dest ruct ive techniqu es:
1.Ultraviolet lamp. Shows any UV-active spots
2.Plate can be stained with iodine.
Bottle containing silica and a few crystals of iodine (especially good for
unsaturated compounds)
Destruct ive techniq ues
Staining Solutions immerse the plate as completely as possible in the stain
and remove it quickly. Heat carefully with a heating
Stains Use/Comments
Anisaldehyde Good general reagent, gives a range of colors
PMA Good general reagent, gives blue/greenspots
Vanillin Good general reagent, gives a range of colors
Ceric sulfate Fairly general reagent, gives a range of colors
DNP Mainly for aldehydes and ketones, gives orange
spots
Permangante Mainly for unsaturated compounds and alcohols,
gives yellow spots
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Thin-Layer Chromatography of Amino acidsAmino acids may be separated by two-dimensional TLC using either
si l ica gel or cel lulos eas the separating medium. Two different solvents are
used for each type of TLC plate and a different type of separation is
achieved for each type. The amino acids are visualized with two types of
ninhydrin sprayfor the silica gel and the cellulose gel media.
Ninhydr in Sprays for amino acid detect ion
For silica gel TLC: The plate is sprayed with a solution of 300 mg of
ninhydrin + 3 ml of glacial acetic acid + 100 ml of butyl alcohol and heated
for 10 minutes at 110C.
For cellulose TLC:
The plate is sprayed with a solution of 500 mg of ninhydrin + 350 ml of
absolute ethanol + 100 ml of glacial acetic acid + 15 ml of 2,4,6-
trimethylpyridine and heated for 10 minutes at 110C.
Two-dimensional TLC separation of amino acids.
On s i l ica gel Gwith
Solvent I, chlorolorm-17% methanol (v/v)-ammonia (2:2:1, v/v/v) and
Solvent II, phenol-water (75:25, v/v).
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on cel lulose MN 300with
Solvent III, 1-butanol-acetone-diethylamine-water (10:10:2:5,v/v/v/v, pH
12.0) and
Solvent IV, 2-propanol-formic acid (99%)-water (40:2:10, v/v/v, pH 2.5)
Thin-Layer Chromatography of Carbohydrates
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Carbohydrates may be separated on commercial silica gel plates using
a variety of solvents to achieve specific separations. The results of the
separation depend on the particular plate used. Whatman K5 silica gel and
Merck silica gel 60 plates give good results.Solvent for TLC separations of carbohydrates
Solvent: Acetonitrile-water (35:15, v/v)with four ascents (45 minutes each
for a 20-cm plate) will separate mono-, di, and trisaccharides
The visualization of carbohydrates on thin layer si l ica gel plates is
obtained by spraying with sul fu r ic acid -methanol (1: 3, v/v) followed by
heat ing for 10 m in utes at 110-120C. Most carbohydrates give black to
brown spots on a white background.
Examples of some TLC separation systems
Compounds Adsorbent Solvent system (v/v)
Amino acids Silica Gel G 96% Ethanol/water (70/30)
Butan-1-ol/acetic acids/
water (80/20/20)
Mono and di
saccharides
Kieselguhr G (sodium
acetate)
Kieselguhr G
(sodium phosphate
pH5)
Ethyl acetate/propan-1-ol
(65/35). Butan-1-ol /
acetone/phosphate buffer
pH5 (40/50/10)
Neutral lipids Silica Gel G Petroleum ether/diethyl
ether/acetone (90/10/1)
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Cholesterol
Esters
Silica Gel G Carbon tetrachloride/
chloroform (95/5)
Carotenoids Kieselguhr G Petroleum ether/propan-1-
ol (99/1)
Phospholipids Silica Gel G Chloroform/methanol/water
(65/25/4)
Advantages of TLC.
The speedat which separation is achieved. With a volatile solventas
the mobi le phasethe time involved may be as low as 30 minu tes, but even
with non-volatile solvents the time involved is rarely longer than 90
minutes.
Summ ary for TLC
Principle
As in paper chromatography
Components glass or plastic plate: as a support to the stationary phase
stationary phase (silica gel, alumina or agar)
mobile phase solvent system
Procedures
(a) Preparing the plate
1- Prepare a glass plate. for example (20X20)Cm
2- Dissolve suitable amount of the silica gel in water path.
3- Spread it on glass plate homogeneously. Then wait till solidification.
(b) Running the sample
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2- Limited to non-volatile compounds
3- Less accurate and less sensitive
Advantages1- Need small quantity of sample.
2- With greater solvent power.
3- easy detection of spots
4- easy isolation of separated substances
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The procedure of two-dimensional thin-layer chromatography
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Developing solvent mixtures that have been recommended for two dimensional TLC separation of
underivatised amino-acids
Organic component of the solvent continues migrating, thus forming the mobile
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phase. Therefore, compounds soluble to organic component move faster than
compounds soluble to aqueous component. -Thus, molecules are separated
according to their polarities.
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Chapter FiveGel filtration
Biomolecules are purified using chromatography techniques that separate
them according to differences in their specific properties, as shown in Figure
5.1.and Table 5.1.
Property Technique
Size Gel filtration (GF), also called size
exclusion
Charge Ion exchange chromatography (IEX)
Hydrophobicity Hydrophobic interaction
chromatography (HIC)
Reversed phase chromatography
(RPC)
Biorecognition (ligand
specificity)
Affinity chromatography (AC)
Table 5.1.
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Fig. 5.1Separation principles in chromatography purification.
Gel filtration has played a key role in the puri f icat ion of enzymes,
polysacchar ides, nucle ic acids, prote ins and other bio logical
macromolecules. Gel filtration is the simplest and mildest of all the
chromatography techniques and separates molecules on the basis of
differences in size. The technique can be applied in two distinct ways:
1. Group separations:
The components of a sample are separated into two major groups
according to size range. A group separation can be used to remove high orlow molecular weight contaminants(such as phenol red from culture fluids)
or to desalt and exchange buffers.
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2. High resolution fractionation of biomolecules:
The components of a sample are separated according to di f ferences
in their mo lecular size.High resolution fractionation can be used to isolateone or more components, to separate monomers from aggregates, to
determine m olecular w eightor to perform a molecular weight distribution
analysis.
Gel filtration can also be used to facilitate the refolding of denatured proteins
by careful control of changing buffer conditions.
Gel filtration is a robust technique that is well suited to handling biomolecules
that are sensitive to changes in pH, concentration of metal ions or co-
factors and harsh environmental conditions. Separations can beperformed
in the presence of essential ions or cofactors, detergents, urea,
guanidine hydrochloride, at high or low ionic strength, at 37 C or in the
cold roomaccording to the requirements of the experiment
Gel filtration in practice
Gel filtration separates molecules according to differences in size as
they pass through a gel filtration medium packed in a column. Unlike ion
exchange or affinity chromatography, molecules do not bind to the
chromatography medium so buffer composition does not directly affect
resolution (the degree of separation between peaks).
Separation by gel filtrationGel filtration medium is packed into a column to form a packed bed. The
medium is a porous matr ixin the form of sphericalparticles that have been
chosen for their chemical and physical stabi l i ty, and inertness (lack of
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reactivity and adsorptive properties). The packed bed is equilibrated with
buffer which fills the pores of the matrix and the space in between the
particles. The liquid inside the pores is sometimes referred to as the
stat ionary phaseand this liquid is in equilibrium with the liquid outside theparticles, referred to as the mobi le phaseas shown in Figure 2.
Gel filtration is used in group separation mode to remove small
molecules from a group of larger moleculesand as a fast, simple solution for
buffer exchange. Small molecules such as excess salt (desalting) or free
labels are easily separated. Samples can be prepared for storage or for
other chromatography techniques and assays. Gel filtration in group
separation mode is often used in protein pur i f icat ion schemes for
desalting and buffer exchange
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Fig. 5. 2.Common terms in gel filtration
Sephadex G-10, G-25 and G-50 are used for group separations. Large
sample volumes up to 30% of the total column volume (packed bed) can be
applied at high flow rates using broad, short columns. Figure 3 shows the
elution profile (chromatogram) of a typical group separation. Large
mo lecules are eluted in or just af ter the void volum e, Voas they pass
through the column at the same speed as the flow of buffer. For a well
packed column the void v olum e is equivalent to approximately 30% of
the tota l colum n volum e. Small molecules such as salts that have fullaccess to the pores move down the column, but do not separate from each
other. These molecules usually elute just before one total column volume,
Vt, of buffer has passed through the column. In this case the proteins are
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detectedby monitoring theirUV absorbance, usually atA280 nm, and the
sal ts are detectedby monitoring the cond uct iv i ty of the buf fer.
Fig. 5.3. Typical chromatogram of a group separation. The UV (protein) and
conductivity (salt) traces enable pooling of the desalted fractions and
facilitate optimization of the separation.
The theoretical elution profile (chromatogram) of a high resolution
fractionation. Molecules that do not enter the matrix are eluted in the void
volume, Voas they pass directly through the column at the same speed as
the flow of buffer. For a well packed column the vo id vo lumeis equivalent to
approximately 30% of the total column volume (packed bed). Molecules with
partial access to the pores of the matrix elute from
the column in order of decreasing size. Small molecules such as salts that
have full access to the pores move down the column, but do not separate
from each other. These molecules usually elute just before one total column
Sample:(His)6 protein eluted from HiTrap
Chelating HP with
sodium phosphate 20 mM,
sodium chloride 0.5 M,
Imidazole 0.5 M, pH 7.
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volume, Vt, of buffer has passed through the column, Fig. 5.4.
Fig. 5.4.Theoretical chromatogram of a high resolution fractionation (UV
absorbance).
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Separation examples
Fig. 5.5 Cytochrome C, Aprotinin, Gastrin I, Substance P,
(Gly)6, (Gly)3and Gly
Media Selection
Chromatography media for gel filtration are made from p orous
matr iceschosen for their inertness and chemical and physical stability. The
size of the pores within a particle and the particle size distribution are
carefully controlled to produce a variety of media with different selectivities.
Today'sgel filtration media cover a molecular weightrange from 100 to80 000 000, from peptides to very large proteins and protein complexes.
Figure.5.7.
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Sephacryl is suitable for fast, high recovery separations at laboratory and
industrial scale
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Sephadexis ideal for rapid group separations such as desalting and buffer
exchange.
Sephadexis used at laboratory and production scale, before, between or
after other chromatography purification steps.Determination molecular weight
VeV0
Kav= --------------
VtV0
WhereVe= elution volume for the proteinVo= column void volume
Vt= total bed volume
On semi logarithmic graph paper, plot the Kav value for each protein
standard (on the l inear scale) against the corresponding molecular
weight (on the logar i thmic scale). Draw the straight line which best fits the
points on the graph. Then, calculate the corresponding Kav for the
component of interest and determine its molecular weight from the
calibration curve.
Sephadex:
Rapid group separation of high and low molecular weight substances,
such as desalting, buffer exchange and sample clean up
Sephadex is prepared by cross-linking dextran with epichlorohydrin.
Variations in the degree of cross linking create the different Sephadex media
and influence their degree of swelling and their selectivity for specific
molecular sizes (Table.5.2).
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Product Fractionation
range, Mr
(globularproteins)
pH stability Bed
volume
ml/gdry
Sephad
ex
Particle size,
wet
Sephadex G-
10
700)from smaller molecules (Mr 30000 frommolecules Mr
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Therefore, The technique chosen must discriminate between the target
protein and any remaining contaminants
Gel Filtration = Gel Permeation Chromatography =Size Exclusion Chromatography
Size exclusion chromatography (SEC), also called gel permeation
Chromatography (GPC) or gel filtration chromatography (GFC) is a
technique for separates molecules according to their molecular size. Gel
particles form the stationary phase of this type of chromatography; the
mobile phase is the solution of moleculesto be separated and the eluting
solvent, which most frequently is water or a dilute buffer. The sample is
applied to the gel, if the molecules are too large for the pores; they never
enter the gel and move outside the gel bed with the eluting solvent. Thus,
the very large molecules in a mixture move the fastest through the gel bed
and the smaller molecules, which can enter the gel pores, are retarded and
move more slowly through the gel bed. In gel chromatography, molecules
are, therefore, eluted in order of decreasing molecular size
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Fig.5.11Gel permeation chromatography. Open circles represent porous gel
molecules: large solid Circles represent molecules too large to enter the gel
through the pores, and smaller solid circles represent molecules capable of
entering the gel pores
Three types of polymers are principally used-dextran, polyacrylamide,
and agarose
Dextranis a polysaccharide composed of (-1--->6)-linked glucose residues
with (-1, 3) branch linkages. It is synthesized from sucroseby an enzyme
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produced by the bacter ium Leuconostoc mesenteroides B -512F. The
dextran is cross-linked to various extents by reaction with epichlorohydr in
to give gel beads with different pore sizes Fig 5.12. Cross-linked dextrans
are commercially produced by Pharmacia Fine Chemicals, lnc ., (Uppsala,
Sweden),and sold under the trade name Sephadex. Sephadex gels in th e
so -cal led G-ser ies, where the G-numbers refer to the amou nt of w ater
gained wh en the beads are sw el led in water (Table 1) have different
degrees of cross-linking, hence different pore sizes. This gives gels that
have capabilities of separating different ranges of molecular weights and
have different molecular exclusion limits. The exclusion limit is the molecular
weight of the smallest peptide or globular protein that will not enter the gel
pore. Sephadex G-10, the highest cross-linked dextran, has a water regain
of about 1mL/g of dry gel and Sephadex G-200, the lowest cross-linked
dextran, has a water regain of about 20 mL/g of dry gel. In the swelling
process, the gels become filled with water.
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Fig.5.12. Structure ofepichlorohydr in c ross l inked Dextran
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Table 5.1: Properties of gels used in gel permeation (filtration)
chromatography
Gel
Waterregain
(mL/g)
Exclusionlimit
Maximumhydrostatic
pressure
cm H2O
Maximumflow rate (ml,
min)
Sephadex G-10 1.0 700 200 100
Sephadex G-15 1.5 1500 200 100
Sephadex G-25 2.5 5000 200 50
Sephadex G-50 5.0 30000 200 25
Sephadex G-75 7.5 70000 160 6.4
Sephadex G-100 10.0 150000 96 4.2
Sephadex G-150 15.0 300000 36 1.9
Sephadex G-200 20.0 600000 16 1.0
Sepharose 6B NA 4 x 10 200 1.2
Sepharose CL 6B NA 4 x 10 >200 2.5
Sepharose 4B NA 20 x 106 80 0.96
Sepharose CL 4B NA 20 x 10 120 2.17
Sepharose 2B NA 40 x 10 40 0.83
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Sepharose CL 2B NA 40 x 10 50 1.25
Bio-Gel P-2 1.5 1800 >100 110
Bio-Gel P-4 2.4 4000 >100 95
Bio-Gel P-6 3.7 6000 >100 75
Bio-Gel P-10 4.5 20000 >100 75
Bio-Gel P-30 5.7 40000 >100 65
Bio-Gel P-60 7.2 60000 100 30
Bio-Gel P-100 7.5 100000 100 30
Bio-Gel P-150 9.2 150000 100 25
Bio-Gel P-200 14.7 200000 75 11
Bio-Gel P-300 18.0 400000 60 6
Bio-Gel A-0.5m NA 500000 >100 3
Bio-Gel A-1.5m NA 1.5 x 10 >100 2.5
Bio-Gel A-5m NA 5 x 106 >100 1.5
Bio-Gel A-15m NA 15 x 106 90 1.5
Bio-Gel A-50m NA 50 x 10 50 1.0
Bio-Gel A-150m NA 150 x 10
6
30 0.5
Bio-Gel is a trade name of Bio-Rad Laboratories, Sephadex and
Sepharose are trade name of Pharmacia Fine Chemical
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Polyacrylamide gels are long polymers of acrylamide cross-linked withN.N'methylene-bisacrylamide (Fig. 5.13).
Fig.5.13. Structureof cross-linked polyacrylamide
The gels are commercially produced by BioRad Laboratories, Richmond.
California, as the Bio-Gel P series. Like the Sephadex G series. the Bio-
Gels differ in degree of cross-linking and in pore size; the Bio-Gels,
however. have a wider range of pore sizes than is available in the Sephadex
G seriesfor the exclusion limits and properties of the different Bio-Gels.
Agaroseis a gel material with pore sizes larger than cross-linked dextran or
polyacrylamide. Agarose is the neutral polysaccharide fraction of agar. It is
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composed of a linear polymer of D-galactopyranose linked ( 1->4) 3,6
anhydro-L-galactopyranose, which is linked (1-> 3)(Fig.5. 14).
D-galactose (-1->4) 3, 6-Anhydro-L-galactoseFig.5.14. Structure of the repeating unit of agarose, D-galactopyranose
linked (-1->4) to 3, 6-anhydro-L-galactopyranose, which is linked (-1-3) tothe next D-galactopyranose residue
When the polysaccharide is dissolved in boiling water and cooled, it forms a
gel by forming inter-and intramolecular hydrogen bonds. The pore sizes are
controlled by the concentration of the agarose. High molecular weight
materials such as protein aggregates, chromosomal DNA, ribosomes,
viruses, and cells have been fractionated on agarose gels. Bio-Rad markets
the agarose Bio-Gel A serieswith different molecular exclusion limits, and
Pharmacia markets agarose as Sepharose and Sepharose CL. The latter
is Sepharose cross-linked by reacting with alkaline 2, 3-dibromopropanol
to give an agarose gelwith increased thermal and chemical stability. Table
5.1gives the properties of the different Sephadex, Bio-Gel, and Sepharose
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gels. The separations that may be achieved by gel permeation
chromatography are based on differences in the molecular sizes of the
molecules. The method is used for both preparative and analytical purposes.
The latter has been especially useful in determining the molecular weights of
proteins. The proteins are chromatographed on a gel column and the elution
volume of the protein determined. Proteins with known molecular weights
are also chromatographed and the elution volumes determined. Then, from a
plot of log molecular weight versus elution volume, the molecular weight of
an unknown protein may be determined (Fig. 5.15).
Fig.5.15.Molecular weight determination of proteins by gel permeation chromatography
using Sephadex G-100 as the gel bed: log molecular weight is plotted versus elution
volume.
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Gel chromatography provides a rapid and mild method of removing salts and
other small molecules from high molecular weight biomolecules. The sample
containing the biomolecules and the salt is passed over a gel column whose
exclusion limit is below the molecular weight of the biomolecules. The
biomolecules which do not enter the gel emerge in the void volume of the
column, while the salts enter the gel and are retarded, and therefore are
removed from the biomolecules.
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Summary Gel filtration chromatography
(Size-exclusion chromatography)
Principle
This technique separate proteins according to their size and shape, as theypass through a stationary phase (cross-linked polymer =sephadex) by the
help of mobile phase (without binding). Larger proteins or molecules, which
can not penetrate the sephadex pores, move around the sephadex in space
between them faster than the smaller molecules which may penetrate the
sephadex pores taking long time to elute from the column.
Components
1. Column: as a support to the stationary phase
2. Stationary phase (pours matrix in the form of spherical particles,
stable, inert e.g. sephadex or agarose)
3. Mobile phase (buffer system)
Procedures
1. (Loading step): spherical particles of the sephadex are packed into the
column
2. (Sampling step): sample is applied to the column
3. Buffer (mobile phase) and sample move through the column. The
sample components diffuse in and out of the pores of the matrix (sephadex)
according to their size.
4. Larger proteins or molecules move faster than the smaller molecules
and leave the column first5. Separation completed as the entire buffer volume is passed.
Applications
1. Separation of neutral proteins and larger molecules including polymers
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and biomolecules according to size.
2. The determination of formula weights.
Disadvantages1. Limited applications
2. Low purification
Advantages
1. Provides a rapid means for separating larger molecules
2. Use only one buffer (coast effective)
3. Do not need elution step because there are no bonds formed.
Note: Gel Filtration
Separation based on size
Molecular sieve chromatography
Size exclusion chromatography
Media composed of crosslinkedpolymers
Pore size of matrix determines degree of interaction
Larger moleculesare excluded and migrate faster
Smaller moleculesare included and are retained longer
Dextran (=Sephadex)
Agarose (=Sepharose)
Polyacrylamide choose matrix with desired characteristics
Size range
does not interact with solute include 0.15-1 M NaCl in buffer
Load sample in smallest possible volume
elute in one column volume
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Practical Considerations
Sephadex
Code Range (kDa)
G-25 1-5, G-50 2-30, G-100 4-150, G-150 5-300, G-200 5-600Applications:
Purification
Desalting
Size determination
Calculating Size
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Chapter Six
Ion-exchange chromatography
Ion-exchange chromatography is a variation of adsorption
chromatography in which the solid adsorbent has charged groups chemically
linked to an inert solid. Ions are electrostatically bound to the charged
groups; these ions may be exchanged for ions in an aqueous solution. Ion
exchangers are most frequently used in columns to separate molecules
according to charge. Because charged molecules bind to ion exchangers
reversibly. Molecules can be bound or eluted by changing the ionic strength
or pH of the eluting solvent.
Two types of ion exchanger are available: those with chemically
bound negat ive charges are cal led cat ion exchangers and those with
chemically bound posi t ive charges are cal led anion exc hangers. The
charges on the exchangers are balanced by counterions such as chloride
ions for the anion exchangers and metal ions for the cation exchangers.
Sometimes buffer ions are the counterions. The molecules in solution which
are to be adsorbed on the exchangers also have net charges which are
balanced by counterions. As an example of an ion-exchange process, let us
say that the molecules to he adsorbed from solution have a negative charge
(X-), which is counterbalanced by sodium ions (Na+). Such negatively
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Cellulose and cross-linked dextran (Sephadex) are used as the
solid supports and charged groups such as diethylaminoethyl (DEAE) or
carboxymethyl (CM) are chemically linked to them to give anion and
cat ion and the exchangers respect ively. The preparation and commercial
availability of these materials beginning in the 1960 provided the biochemist
with powerful tools for separat ion of p roteins and nucle ic acid Figure 2
presents partial structures of DEAE-cellulose and CMcellulose
Figure 6.2. Partial structures of diethylaminoethyl-cellulose and carboxymethyl-
cellulose.
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The DEAE and CMgroups are shown attached to the C6-hydroxyl group of
glucose. The DEAE and CM groups are also found attached to the hydroxyl
groups of C2and C3. The total degree of substitution of the DEAE and CM
groupsmust be less than one group per five glucose residues to maintain a
water-insoluble product.
Table 6.1. Pretreatment steps for DEAE-cellulose and CM -cellulose ion exchangers
Cellulose First treatment Intermediate
pH
Second treatment
DEAE 0.5 M HCl 4 0.5 M NaOH
CM 0.5 M NaOH 8 0.5 M HCl
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The dry ion-exchange celluloses are pretreated with acid and base to swell
the exchangers so that they become fully accessible to the charged
macromolecules in solution. The weighed exch anger is sus pended in 15
volumes (w/v) of the "f i rs t treatment," acid o r alkali depending on th e
exchanger (Table. 1),and is al low ed to stand at least 30 m inutes but no t
mo re than 2 hours. The supernatant is decanted and the exchanger is
washed until the effluent is at the "intermediate pH" The exchanger is stirred
into 15 volumes of the "second treatment" and allowed to stand for an
additional 30 minutes. The second treatment is repeated and the exchanger
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is washed with distilled water until the effluent is close to neutral pH. The
treated exchanger is placed into the acid component of the buf fer (the pH
shou ld be less than 4.5) and degassed under vacuum 10 cm Hg
pressure) with stirring, until bubbling stops The exchanger is then titrated
with the basic component of the buffer to the desired pH, filtered, and
suspended in fresh buffer to complete the pretreatment. The exchanger is
ter) above the
settled exchanger are removed by decantation. Buffer is added to the
exchanger so that the final volume of the slurry is l50% of the settled wet
volume of the exchanger. The column is then packed with the slurry of the
exchanger, the sample is applied, and elution is performed as described for
adsorption chromatography.
Three general methods are used for eluting molecules from the
exchanger:
(a)Changing the pH o f the buf fer to a value at which binding is weakened
(i.e., the pH is lowered for an anion exchanger and raised for a cation
exchanger),
(b)Increasing the ionic st rength by increasing the concentration of salt in
the elution solvent, thereby weakening the electrostatic interactions between
the adsorbed molecule and the exchanger, and
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(c)Performin g aff ini ty elut ion. In affinity elution the adsorbed molecule is
usually a macromolecule that is desorbed from the affinity ligand by adding
a molecule that is charged and of opp osi te signs to the net chargeon
the macromolecule and has a specific affinity for the macromolecule. Thus,
the reduction of the net charge on the macromolecule weakens its
electrostatic interaction with the exchanger sufficiently to permit the elution of
the macromolecule from the affinity ligand.
The stages of anion exchange chromatography.
An example of the use of ions exchange resins
Is the purification of Cytochrome C:
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Cytoch rom e C has an isoelectr ic p oint (pI) of 10.05; that is at pH
10.05 the number of positive charges will equal the number of negative
charges. A columncontaining a cation exchangerbuffered, at pH 8.5, is
prepared. This columnhas a full negative charge. Cytochrome Cat pH
8.5 has a full positive charge. An Impure solution of Cytochrome C at
pH 8.5 placed on the column, and water is passed through the
column (the pI of proteins is usually 7.0 or less) but Cytochrome C is
held firmly by electrostatic attraction to the resin heads. If the eluting
solvent pH is raised to about 10, the Cytochrome C will now has a
net zero charge and will pass rapidly through as a pure component
Summary Ion-exchange chromatography
Principle
Ion exchange chromatography separates molecules (proteins) according to
their differences between the overall charges. The proteins to be separated
must have a charge opposite to that of stationary phase in order to bind. Ion
exchange has two types according to the stationary phase charge:
1. Cation-exchanger: in which the stationary phase is charged
negatively in order to binds with positive molecules (cations)
2. Anion-exchanger: in which the stationary phase is charged positively
in order to binds with negative molecules (anions)
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A-Cation-exchange chromatography
Cation-exchange chromatography can be classified as: either strong or
weak. A strong cation exchanger contains strong acid which stable along
pH1-14. Whereas, weak cation exchanger contains weak acid which loss its
charge as the pH decrease below 4-5
The sample must be charged positive in order to bind with the negative
matrix (strong or weak acid). H+
B-Anion-exchange chromatography
Anion-exchange chromatography can be classified as: either strong or
weak. A strong anion exchanger contains strong base which stable along
pH1-14. Whereas, weak anion exchanger contains weak base which loss its
charge as the pH increase over 9
The sample must be charged negative in order to bind with the positive
matrix (strong or weak base).OH-
Components
1. The column containing the stationary phase (anion or cation
exchanger) on suitable matrix
2. Washing and eluting buffer
3. pump to withdrew the buffer
4. Detector
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Procedures
Before carry out the process, you must answer two important questions:
a) What is the sample charge? If +Ve, use cation exchanger. ifVe, use
anion exchanger
b) What is the suspected strength of the charge? If weak +Ve, use weak
cation exchanger, if strong +Ve, use strong cation exchanger, if weak Ve,
use weak anion exchanger, if strongVe use strong anion exchanger.
e.g.the sample is weak negative proteins. So we will use anion exchanger
contain weak base.
1. (Loading step): the column is packed with the matrix that charged with
weak positive charge by adding weak base e.g. DEAE- cellulose (stationary
phase)
2. (Sampling step): apply the sample in the column: the negatively
charged proteins bind to positively charged matrix whereas; the positively
charged proteins flow down to the exterior. Some negative charged
contaminants can bind to matrix.
3. (Washing step): apply washing buffer (Tris-HCL) to remove the
contaminants remaining the target proteins.
4. (Elution step): now, we need to separate the target proteins from the
matrix, so we apply an eluting buffer that has the same charge of protein in
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order to substitutes it (ion exchange). Separation can be done also by ion
exclusion and ion pairing.
5. (Gradient step): make gradient elution with different buffer till you
obtain 100% correct proteins. i.e. repeat washing and eluting steps with
different buffer
6. (Detection step): after separation carry out detection by electrophoresis
Applications
Recommended