Definition and Classification of Chromatography Chromatography course
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Aim Separation Techniques
1-Biological fluids are extremely complex in composition.
2-Chemical analysis would be impossible if it were necessary to
completely isolate 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 charge of the substances
involved.
Definition and Classification of Chromatography Chromatography course
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INTRODUCTION
The Russian botanist M. S. Tswett is discovery of
chromatography. He used a column of powdered calcium
carbonate to separate green leaf pigments into a series of
colored 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 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 all branches of the chemical and life sciences.
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 mobile phase) through
it in a definite direction.
2- Classification of chromatographic methods
The common feature of all chromatographic methods is
two phases, one stationary and the other mobile
A classification can be made depending upon whether the
stationary phase is solid or liquid. If it is solid, the method is
Definition and Classification of Chromatography Chromatography course
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termed adsorption chromatography; if it is liquid the method
is partition chromatography.
One of the two phases is a moving phase (the mobile phase),
while the other does not move (the stationary phase). The
mobile phase can be either a gas or a liquid, while the
stationary phase can be either a liquid or solid.
3- Classification scheme
One classification scheme is based on the nature of the two
phases. All techniques which utilize a gas for the mobile phase
come under the heading of gas chromatography (GC). All
techniques that utilize a liquid mobile phase come under the
heading of liquid chromatography (LC). Additionally, we
have gas–liquid chromatography (GLC), gas–solid
chromatography (GSC), liquid–liquid chromatography (LLC),
and liquid–solid chromatography (LSC),
4- Main Type of Chromatography
In general, there are four main types which can be
classified as follows:
4.1-Liquid-Solid chromatography
Classical adsorption chromatography (Tswett column)
Ion-exchange chromatography
4.2. Gas-Solid chromatography
4.3. Liquid-Liquid chromatography
Classical partition chromatography
Paper chromatography
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4.4 Gas-Liquid chromatography
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 retained
Extraction Solubility Some of the substances
dissolve (partition) more
in water. While other
substances dissolve
more organic solvent in
contact with the water
Thin 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
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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 porous, solid material
while others remain
outside in the
surrounding stream of
flowing water
Ion-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.
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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 bonds
which can be formed between the solute molecules and the
surface of the adsorbent.
Chromatography
Adsorption Chromatography
Solid stationary phase Partition Chromatography
Liquid Stationary Phase
Liquid
mobile phase Gas mobile
phase
Gas mobile
phase Liquid
mobile phase
Adsorption Chromatography Chromatography course
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All chromatographic separations are carried out using a
mobile and a stationary phase, the primary classification of
chromatography is based on the physical nature of the mobile
phase. The mobile phase can be a gas or a liquid which gives
rise to the two basic forms of chromatography, namely, gas
chromatography (GC) and liquid chromatography (LC). The
stationary phase can also take two forms, solid and liquid,
which provides two subgroups of GC and LC, namely; gas–
solid chromatography (GSC) and gas–liquid chromatography
(GLC), together with liquid solid chromatography (LSC) and
liquid chromatography (LLC). The different forms of
chromatography are summarized in Table.1
Most thin layer chromatography techniques are considered
liquid-solid systems although the solute normally interacts with
a liquid-like surface coating on the adsorbent or support or, in
some cases an actual liquid coating.
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Table 1: The Classification of Chromatography
1-ADSORPTION CHROMATOGRAPHY
In 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.
2-Separation of the compounds depends on
1-The relative balance between the affinities of the compounds
for the adsorbent and their solubility in the solvent.
2-The chemical nature of the substances.
3-The nature of the solvent.
4-The nature of the adsorbent.
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Solid adsorbents commonly used are alumina, silica gel,
charcoal (active carbon), cellulose, starch, calcium phosphate
gels, calcium hydroxylapatite, and sucrose.
Solvents commonly used are hexane, benzene, petroleum
ether, diethyl ether, chloroform, methylene chloride, various
alcohols (ethyl, propyl, n-buryl and t-butyl alcohols), and
various aqueous buffers and salts, 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 plastic discs, or sheets of nylon or Teflon fabrics.
The adsorbent bed must be homogeneous and free of bubbles,
cracks, or spaces between the adsorbents and the walls of the
column.
The choice of the eluting solvent, although very important,
depends on the nature of the substances to be separated and
the adsorbent, and hence affords considerable latitude. The
process of eluting the sample components from the adsorbent by
the solvent is termed development. As illustrated in Figure 1,
the compounds in the mixture that are more soluble in the
solvent and have less affinity for the adsorbent move more
quickly down the column.
If the substances are colored, as they were in Tswett's
experiment, they are readily visible as they separate, However,
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many substances are not colored, and in these instances, as the
development proceeds, fractions are collected at the bottom of
the column, and the different fractions are analyzed for
compounds of the types that are being separated, For example, if
proteins are being separated,
the fractions would be analyzed for protein by measurement of
the UV absorbance at 280 nm. If carbohydrates or nucleic
acids are being separated analytical measurements for
carbohydrates or nucleic acids. The collection of fractions by an
automatic fraction collector,
Figure 1: Collection of fractions from a column by an automatic fraction
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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 a
drop 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: Adsorption chrornatography
A = adsorbent, S=Sample, ES = eluting solvent
(A) Application of sample to the of 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.
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The substances adsorbed on the column support can be
eluted in three ways
(a) in the simplest method, a single solvent continuously
flows through the column until the compounds have
been separated and eluted from the column
(b) Stepwise elution, in which two or more different
solvents of fixed volume are added in sequence to
elute the desired compounds.
(c) Gradient elution, 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
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. 3).
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Figure 3: Gradient elution. Flow of solvent B into solvent A With
mixing, continuously changing the composition of solvent A as it flows
into column
Figure 4: Elution of chromatography column with a gradient of
increasing salt concentration.
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Gradients other than linear gradients (e.g., exponential,
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.
3-Activation of adsorbent
Many adsorbents such as alumina, silica gel, and active
carbon and Mg silicate can 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 activity by the
controlled addition of water, and the subsequent activity is
related to the amount of water added. Brookman and Schodder
established five grades of alumina Grade I is the most active
and the is simply alumina heated at about 350 0
C for several
hours. Grade II has about 2-3% water, Grade III 5-7%, Grade
IV 9-11 %, Grade V film. (Least active) about 15%.
4-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 and detection. In
interrupted development systems like TLC the retention is
measured as the retention factor Rf, the run length of the
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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.
5-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:
K is assumed to be independent of concentration, and can
change if experimental conditions are changed, for example
temperature is increased or decreased. As K increases, 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
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6- Column chromatography
1. Small plug of wool (or cotton)
2. Sand to 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 sand on 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.
Adsorption Chromatography Chromatography course
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Figure 5
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Column as that illustrated in Fig.5 may be used:
Typical chromatographic column.
Mixture sorbed on top of column.
Partial separation
Complete separation
Table 2: 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.
Thin layer chromatography Chromatography course
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Thin layer chromatography
This technique is particularly useful for the separation of very
small amounts of material. The general principle 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, differ from column adsorbents. It contain 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 drying them in an oven at 100-
120°C. Drying serves 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 spot
2.5 cm from one end and 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 (mobile 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 vertically in 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 stopped. 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 blue, green, black area.
2. Spraying the plate with 50% sulphuric acid and heating so,
the compounds become charred and show spots
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3. Spraying the plates with specific color reagents will stain up certain
compounds e.g. ninhydrin for amino acid (aa) , aniline for aldoses.
Solvents Universal TLC System:
petroleum ether - ethyl acetate
Very polar solvent additives:
methanol > ethanol > isopropanol
Moderately polar additives:
acetonitrile > ethyl acetate > chloroform, dichloromethane >
diethyl ether > toluene
Non-polar solvents:
cyclohexane, petroleum ether, hexane, pentane
TLC Visualization (Detecting the spots)
Non-destructive techniques:
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)
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Destructive techniques
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/green spots
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
Thin-Layer Chromatography of Amino acids
Amino acids may be separated by two-dimensional TLC using either
silica gel or cellulose as 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 spray for the silica gel and the cellulose gel media.
Ninhydrin Sprays for amino acid detection
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 110°C.
Thin layer chromatography Chromatography course
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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 110°C.
Two-dimensional TLC separation of amino acids.
On silica gel G with
solvent I, chlorolorm-17% methanol (v/v)-ammonia (2:2:1, v/v/v) and
solvent II, phenol-water (75:25, v/v).
on cellulose MN 300 with
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)
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Thin-Layer Chromatography of Carbohydrates
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 silica gel plates is
obtained by spraying with sulfuric acid-methanol (1: 3, v/v) followed by
heating for 10 minutes at 110-120°C. Most carbohydrates give black to
brown spots on a white background.
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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)
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 speed at which separation is achieved. With a volatile solvent
as the mobile phase the time involved may be as low as 30 minutes, but
even with non-volatile solvents the time involved is rarely longer than
90 minutes.
PAPER CHROMATOGRAPHY
Paper chromatography is a type of liquid-liquid partition
chromatography that may be performed by ascending or descending
solvent flow. Each mode has its advantages and disadvantages.
Ascending chromatography involves relatively simple and inexpensive
equipment compared with descending chromatography and usually gives
more uniform migration with less diffusion of the sample "spots."
Descending chromatography, 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 the
solvent trough of the descending tank (Fig. 1). Which 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 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. 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. 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
Fig. 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
docs 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 spraying or 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 (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. For example, in the separation
of carbohydrates, the standard might be glucose and the ratio would be
RGlc or 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 Two-dimensional paper or thin-layer chromatography
dimensional separation (Fig. 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
and the second to see what products are formed by the action of the
enzyme on the compounds separated in the first dimension.
Paper chromatography has been used to establish the structural
homology of a series of oligomers obtained by enzymic synthesis, by acid
or enzymic hydrolysis, or by isolation from a natural source. The RF of
each separated homologue is determined and a French-Wild plot is made
by plotting log [RF / (1-RF)]
against the number of monomers in the oligomer. If the isolated
compounds fall on a straight line of this plot, they belong to a
homologous series, differing from each other by one monomer residue
(Fig. 4). Compounds separated by paper chromatography may be
quantitatively determined. Aliquots (50-200,l) of the solution
containing the substances to be separated and quantitatively determined
are streaked along the separation line. Aliquots of the solution (5-10,l)
are also spotted on the two outside edges of the streak and are used as
location standards. The chromatogram is irrigated in the usual way, and
vertical sections of the location standards are cut out and developed to
reveal the positions of the compounds. After drying, these standards are
placed alongside the streaked sections and the undeveloped compounds
are located; horizontal strips containing the individual compounds are cut
out and
Fig. 4. French-Wild plots (log RF / 1-RF), versus number of monomer units per
molecule) correlating paper chromatographic mobility with the number of
homologous monomer residues in oligosaccharide molecules.
Fig. 5. 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 5,
one end is fitted with two pieces of glass (cut microscope slides), which
arc held 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
Paper Chromatography
What is Chromatography?
Chromatography is a technique for separating mixtures into their
components in order to analyze, identify, purify, and/or quantify the
mixture or components.
Uses for Chromatography
Chromatography is used by scientists to:
Analyze – examine a mixture, its components, and their relations
to one another
Identify – determine the identity of a mixture or components
based on known components
Purify – separate components in order to isolate one of interest for
further study
Quantify – determine the amount of the a mixture and/or the
components present in the sample
Separate • Analyze
• Identify
• Purify
• Quantify
Components Mixture
Real-life examples of uses for chromatography:
• Pharmaceutical Company – determine amount of each chemical
found in new product
• Hospital – detect blood or alcohol levels in a patient’s blood
stream
• Law Enforcement – to compare a sample found at a crime scene
to samples from suspects
• Environmental Agency – determine the level of pollutants in the
water supply
• Manufacturing Plant – to purify a chemical needed to make a
product
Definition of Chromatography
Detailed Definition:
Chromatography is a laboratory technique that separates
components within a mixture by using the differential affinities of the
components for a mobile medium and for a stationary adsorbing medium
through which they pass.
Terminology:
• Differential – showing a difference, distinctive
• Affinity – natural attraction or force between things
• Mobile Medium – gas or liquid that carries the components
(mobile phase)
• Stationary Medium – the part of the apparatus that does not
move with the sample (stationary phase)
Simplified Definition:
Chromatography separates the components of a mixture by
their distinctive attraction to the mobile phase and the stationary phase.
Explanation:
• Compound is placed on stationary phase
• Mobile phase passes through the stationary phase
• Mobile phase solubilizes the components
• Mobile phase carries the individual components a certain
distance through the stationary phase, depending on their
attraction to both of the phases
Illustration of Chromatography
Components Affinity to Stationary Phase Affinity to Mobile Phase
Blue ---------------- Insoluble in Mobile Phase
Black
Red
Yellow
Mixture Components
Separation
Stationary Phase
Mobile Phase
Principles of Paper Chromatography
Capillary Action – the movement of liquid within the spaces of a
porous material due to the forces of adhesion, cohesion, and surface
tension. The liquid is able to move up the filter paper because its
attraction to itself is stronger than the force of gravity.
Solubility – the degree to which a material (solute) dissolves into a
solvent. Solutes dissolve into solvents that have similar properties.
(Like dissolves like) This allows different solutes to be separated by
different combinations of solvents.
Separation of components depends on both their solubility in
the mobile phase and their differential affinity to the mobile phase
and the stationary phase.
Paper Chromatography Experiment
What Color is that Sharpie?
Overview of the Experiment
Purpose:
To introduce students to the principles and terminology of
chromatography and demonstrate separation of the dyes in Sharpie Pens
with paper chromatography.
Time Required:
Prep. time: 10 minutes
Experiment time: 45 minutes
Costs:
Less than $10
Materials List
• 6 beakers or jars
• 6 covers or lids
• Distilled H2O
• Isopropanol
• Graduated cylinder
• 6 strips of filter paper
• Different colors of Sharpie pens
• Pencil
• Ruler
• Scissors
• Tape
Preparing the Isopropanol Solutions
Prepare 15 ml of the following isopropanol solutions in appropriately
labeled beakers:
- 0%, 5%, 10%, 20%, 50%, and 100%
Preparing the Chromatography Strips
Cut 6 strips of filter paper
Draw a line 1 cm above the bottom edge of the strip with the pencil
Label each strip with its corresponding solution
Place a spot from each pen on your starting line
Developing the Chromatograms
Place the strips in the beakers
Make sure the solution does not come above your start line
Keep the beakers covered
Let strips develop until the ascending solution front is about 2 cm
from the top of the strip
Remove the strips and let them dry
Developing the Chromatograms
Developing the Chromatograms
Observing the Chromatograms
Concentration of Isopropanol
0
% 20
% 50
% 70
% 100
%
Alternative Experiments
Protein purification Chromatography
Dr.Ehab Aboueladab (Assistance. Prof.of Biochemistry, Mansoura University) 1صفحة
1. Ammonium Sulfate Fraction of Protein Mixtures
Increasing the salt concentration to a very high level will
cause proteins to precipitate from solution without denaturation if
done in a gentle manner. First, we want to understand why the
protein precipitates. A protein in a buffer solution is very highly
hydrated, in other words, the ionic groups on the surface of the
protein attract and bind many water molecules very tightly:
This graphic illustrates how proteins in solution are hydrated
by water molecules. When a lot of salt, such as ammonium sulfate,
is added to the protein solution, the salt ions attract the water
molecules away from the protein. This is partly since the salt ions
have a much greater charge density than the proteins. So as the salt
is added and these small ions bind water molecules, the protein
molecules are forced to interact with themselves and begin to
Protein purification Chromatography
Dr.Ehab Aboueladab (Assistance. Prof.of Biochemistry, Mansoura University) 2صفحة
aggregate:
So when enough salt has been added, the proteins will be
begin to precipitate. If this is carried out at a cold temperature like
in ice, the proteins will precipitate without denaturation. Thus, the
proteins can be collected by centrifugation and then redissolved in
solution using a buffer with low salt content.
This process is called "Salting Out" and works best with divalent
anions like sulfate, especially ammonium sulfate which is highly
soluble at ice temperatures.
Salting out or ammonium sulfate precipitation is useful for
concentrating dilute solutions of proteins. It is also useful for
fractionating a mixture of proteins. Since large proteins tend to
precipitate first, smaller ones will stay in solution. Thus, by
analyzing various salt fractions, one can find conditions where the
Protein purification Chromatography
Dr.Ehab Aboueladab (Assistance. Prof.of Biochemistry, Mansoura University) 3صفحة
protein you are studying precipitates and many of the other
proteins in the mixture stay in solution. The end result is that you
are also able to increase the purity of your protein of interest while
you concentrate it using an ammonium sulfate fractionation
procedure.
2. Dialysis of Proteins
After a protein has been ammonium sulfate precipitate and
taken back up in buffer at a much greater protein concentration
than before precipitation, the solution will contain a lot of residual
ammonium sulfate which was bound to the protein. One way to
remove this excess salt is to dialyze the protein against a buffer
low in salt concentration.
This graphic illustrates the dialysis process. First, the
Protein purification Chromatography
Dr.Ehab Aboueladab (Assistance. Prof.of Biochemistry, Mansoura University) 4صفحة
concentrated protein solution is placed in dialysis bag with small
holes which allow water and salt to pass out of the bag while
protein is retained. Next the dialysis bag is placed in a large
volume of buffer and stirred for many hours (16 to 24 hours),
which allow the solution inside the bag to equilibrate with the
solution outside the bag with respect to salt concentration. When
this process of equilibration is repeated several times (replacing the
external solution with low salt solution each time), the protein
solution in the bag will reach a low salt concentration:
The graphic illustrates the complete dialysis process, except
for it suggests you do this with distilled water. Really you want to
do this process with buffer to prevent the protein from denaturing
due to the fact that distilled or deionized water is too low in salt
and may have an undesirable pH for your protein, which may
Protein purification Chromatography
Dr.Ehab Aboueladab (Assistance. Prof.of Biochemistry, Mansoura University) 5صفحة
cause it to denature.
In fact, dialysis is a good way to exchange the buffer the protein is
in at the same time you get rid of excess salt. For example, the
GOT after ammonium sulfate precipitation contains a mixture of
buffers as well as excess salt. So we use the buffer we want for the
next step in the purification, which is ion-exchange
chromatography, as the external solution during dialysis. After the
dialysis process, the protein solution is dialyzed against the starting
buffer for the ion-exchange chromatography step, not only will the
salt be removed but the protein will now be in the buffer needed
for the next step and ready to go. Sometimes, proteins will
precipitate during the dialysis process and you will need to
centrifuge the solution after dialysis to remove any particles which
would interfere with the next step – such as ion-exchange
chromatography where particles would clog the column and
prevent the chromatography step from working. In addition, you
may lose enzyme activity during dialysis. So it is a good idea to
keep some of your protein solution as a sample before it is put in
the dialysis bag so that it can be assayed for enzyme activity
before and after dialysis.
3. Alternative Methods for Desalting and Concentration of Proteins
There are several ways to get rid of excess salt in a protein
solution. One rapid method is to use a small gel filtration column
Protein purification Chromatography
Dr.Ehab Aboueladab (Assistance. Prof.of Biochemistry, Mansoura University) 6صفحة
which contains a gel with very small pores which will only allow
water and salt inside the gel particles and will exclude the protein.
This method works very well and can be done at 4°C so that little
or no enzyme activity is lost during processing. A small amount of
dilution of the protein solution will take place during processing,
but it is possible by this method to exchange the buffer and prepare
the protein solution.
Another way to both concentrate a protein and exchange the
buffer, which completely avoids precipitation, is called
ultrafiltration:
Ultrafiltration is done a device which can withstand high
pressure. First, the ultrafiltration device is fitted with an ultrafilter
membrane of the desired molecular weight cut off such that you
protein of interest will be retain in the cell. Next, the pressure cell
Protein purification Chromatography
Dr.Ehab Aboueladab (Assistance. Prof.of Biochemistry, Mansoura University) 7صفحة
is filled with the protein solution and nitrogen gas at about 50 psi is
applied while the cell is stirred gently at 4°C. After about 1 hour,
the solution will be decreased in volume usually without loss of
activity. To exchange the buffer the cell is filled with the desired
buffer and the concentration process are repeated.
Desalting
Before an ion-exchange chromatographic step or after an
ammonium sulfate fractionation, it is usually necessary to remove the salt
from the solution of protein. Desalting is accomplished in one of two
ways: dialysis or gel filtration.
Dialysis
Dialysis is performed by filling a section of dialysis tubing (a semi
permeable membrane) having a sufficiently small molecular weight "Cut-
off", with the protein solution, and placing the filled tubing in a large
volume of buffer. The decrease in salt concentration can be calculated
easily from the ratio of the volumes inside and outside of the bag.
Dialysis requires a few hours, after which the bag may be
transferred to fresh buffer if the reduction in salt concentration effected
by one cycle is deemed to be insufficient. In dialysis, all small molecules,
including salt ions, metal ions and cofactors, pass through the membrane,
which retains only macromolecules. Neither tightly bound metal ions and
cofactors, nor counterions to the macromolecule are effectively removed.
Since the initial solution in the bag is of much greater osmotic
strength than the surrounding buffer, the bag generally increases in
volume. The volume of the contents of the bag must be measured after
dialysis if either total protein or total enzyme units are to be calculated.
Ion Exchange Chromatography
Since proteins have different net charge and charge distribution,
ion exchange chromatography can be an effective purification tool. For
bench-top preparations, usually gravity-flow columns are employed, but
HPLC and automated HPLC-like systems have grown in popularity. For
gravity flow or for use with low pressure peristaltic pumps, ion exchange
media are usually carbohydrate based. Charged groups are attached to
solid supports (“inert phase”) such as Sepharose, Sephadex and cellulose.
Since these carbohydrates are compressible, they are not used in higher-
pressure systems, and more rigid inert phases such as TSK (a polyether-
coated gel) are used. For higher pressures, reinforced Polysaccharides,
and organically coated silica (e.g., TSK) are used. The resins, especially
poly (styrenediviny1benzene) described by HIRS for use with enzymes
were used by MOORE and STEIN in their famous amino acid analyzer.
They are commonly employed for ion exchange chromatography of small
molecules, but have given way to the ion exchange polysaccharides for
preparative applications in enzymology. The charged groups used with
the solid supports depend to some extent on the chemistry of the support
material itself, but are remarkably similar. Groups containing charged
nitrogen atoms are almost universally used for anion exchange media.
These include, from strong to weak, quaternary amino methyl or ethyl
(QAE), tertiary amino (diethylaminoethyl, DEAE, or diethylaminomethyl)
and secondary plus tertiary nitrogens (polyethylenimine, PEI). The
quaternary amino compounds are positively charged at any pH, but the
others must be used at a pH below the pK, of the protonated form (- 10,
for DEAE). The conjugate base of the strongly acidic sulfonic acid (i.e.,
alkyl or aryl sulfonate) and the weakly acidic carboxylic acids (e.g.,
carboxymethyl, CM) are the most common charged groups employed in
cation exchangers. The carboxymethyl packing must be used at a pH
above their pK4. Methods for determining the optimal pH for separation
of proteins depends, of course, on the proteins. Since most proteins are
acidic, they are negatively charged at pH 7-8. They therefore adsorb to a
positively charged stationary phase to which they act as counterions,
providing that other anions are not available to play the role of
counterion. The cationic stationary phase is known as an anion
exchanger because it functions by exchanging one anionic counterion for
another. Anionic proteins may bind more tightly to anion- exchange
stationary phases than simple salts because they possess more negative
charges than a simple anion. However, it is not the total charge on a
protein, but the charge density that determines the affinity. More
precisely, it is the charge distribution. Since a protein may interact with a
stationary phase on one side at a time, proteins with densely charged
patches may be bound more tightly. At pH values below the isoelectric
point of a protein, the net charge is positive, so negatively charged
stationary phases (cation exchange phases) are used. If a protein has an
isoelectric point near neutrality, either a cation exchange or an anion
exchange system can be used, depending on the pH employed. The
important considerations in choosing an optimal pH for separation of
enzymes by ion exchange chromatography have been reviewed. Protein
solutions are generally desalted, then loaded onto a column packed with a
stationary phase having the appropriate charge. Loading can often be
done as rapidly as the columns will flow without undue pressure; proteins
that adsorb are retained at the top of the column. As long as the capacity
of the column is not exceeded, liters of a (desalted, buffered) crude
extract can be loaded onto a column of modest size, so that a pre-
chromatography concentration step is not needed. After loading, the
column is washed with the loading buffer to remove unabsorbed and
weakly adsorbed proteins. The adsorbed proteins are then eluted by
washing the column with buffers of increasing salt concentration (e.g.,
NaCl), which corresponds to increasing solvent strength. This method of
elution using a series of isocratic (constant strength) elutions of
progressively increasing strength is known as batch elution. The ion
having a charge of the same sign as the protein can act as a displacing ion
by competing for charged sites on the stationary phase. At some
concentration, the eluting ion competes effectively with the protein,
which accordingly, spends a larger fraction of its time in the mobile
phase, leading to elution. This concentration would be ideal to purify the
protein of interest providing that more loosely bound proteins were
removed first, because it affords the maximum discrimination among
the charge densities of the proteins still on the column. However, the
protein might elute as a broad, dilute band. A simple and common
solution to elution is to employ a linear concentration gradient of salt,
Such a gradient can cover a range from 0 to 1 M NaCl over the volume of
a few hundred ml to a few liters, depending on the dimensions of the
column and the steepness of the gradient desired.
A major advantage of gradient elution is that proteins having a
wide range of affinities for the column can be eluted in a single run. The
information obtained from a gradient elution may be used to determine an
optimum salt concentration to be used in isocratic elution, but the
procedure is not straightforward. The theory of gradient elution is messy,
even in the simplest case. One egregious misstatement appears in
numerous papers on enzyme purification “the enzyme elutes at such and
such a concentration of sodium chloride”. Because the gradient travels
much more rapidly in the column than the protein (the protein is retained
to some extent), the concentration of sodium chloride in which the
enzyme actually appears at the bottom of the column is much higher than
the concentration at which it began to elute appreciably. Thus, the
concentration in which it appears to elute (concentration of sodium
chloride in the fraction in which the activity appears) is much too strong
for use as an isocratic eluent. In addition, the concentration in which the
enzyme appears varies with the dimensions of the column; longer
columns cause the enzyme to appear to elute in a higher salt
concentration, simply because the gradient progresses as the enzyme
moves down the column. To exercise maximum control over the system,
it is useful to separate the effects of pH from those of ionic strength
during ion exchange chromatography. One of the ions involved in the
buffering system bears the same charge as the protein and can therefore
act as a displacing ion. The concentration of this ion should not change
with pH, so it should not be the one involved in the equilibrium with
solvent protons. Buffering ions selected for use in ion exchange
chromatography should have the same charge as the column, i.e., cations
for an anion exchange column, anions for cation exchange. Hence,
phosphate buffers are used for cation exchange chromatography, and
Tris (for instance) buffers are used for anion exchange. It is necessary
for the column to be completely equilibrated with the starting solvent.
Equilibration can be checked by measurement of both pH and ionic
strength (e.g., by conductivity) prior to loading the column. Elution from
an ion-exchange column could also be accomplished using a change in
pH. Stepwise pH changes are sometimes employed, but do not generally
produce high resolution of complex mixtures. Reproducible continuous
pH gradients are difficult to obtain because so many of the components in
the system engage in acid-base equilibria. A workable system along these
lines has been devised using a proprietary mixed-bed packing and a
multi-component buffer system to elute proteins at their isoelectric pH.
The process is called chromatofocusing because of a loose analogy to
isoelectric focusing gel electrophoresis.
Gel filtration
Biomolecules are purified using chromatography techniques that separate
them according to differences in their specific properties, as shown in
Figure 1. and Table 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 1.
Fig. 1. Separation principles in chromatography purification.
Gel filtration has played a key role in the purification of enzymes,
polysaccharides, nucleic acids, proteins and other biological
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
or low molecular weight contaminants (such as phenol red from culture
fluids) or to desalt and exchange buffers.
2. High resolution fractionation of biomolecules:
The components of a sample are separated according to differences
in their molecular size. High resolution fractionation can be used to
isolate one or more components, to separate monomers from aggregates,
to determine molecular weight or 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
be performed 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 room according 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 filtration
Gel filtration medium is packed into a column to form a packed bed. The
medium is a porous matrix in the form of spherical particles that have
been chosen for their chemical and physical stability, and inertness (lack
of 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
stationary phase and this liquid is in equilibrium with the liquid outside
the particles, referred to as the mobile phase as shown in Figure 2.
Gel filtration is used in group separation mode to remove small
molecules from a group of larger molecules and 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 purification schemes for desalting and
buffer exchange
.
Fig. 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 molecules are eluted in or just after the void volume, Vo as they
pass through the column at the same speed as the flow of buffer. For a
well packed column the void volume is equivalent to approximately 30%
of the total column volume. 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 volume,
Vt, of buffer has passed through the column. In this case the proteins are
detected by monitoring their UV absorbance, usually at A280 nm, and
the salts are detected by monitoring the conductivity of the buffer.
Fig. 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, Vo as they pass directly through the column at the
same speed as the flow of buffer. For a well packed column the void
volume is equivalent to approximately 30% of the total column volume
(packed bed). Molecules with partial access to the pores of the matrix
elute from
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.
Column: HiTrap Desalting 5 ml
Buffer: Sodium phosphate 20 mM,
Sodium chloride 0.15 M, pH 7.0
Void volume :Vo,
Total column volume :Vt
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 volume, Vt, of buffer has passed through the column, Fig. 4.
Fig. 4.Theoretical chromatogram of a high resolution fractionation (UV
absorbance).
Separation examples
Fig. 5. Cytochrome C, Aprotinin, Gastrin I, Substance P,
(Gly)6, (Gly)3 and Gly
Comparison of the selectivity of Superdex 75 prep grade and Superdex
200 prep grade for model proteins Figure.6
Superdex 75 prep grade (a)
gives excellent resolution of the three proteins in the Mr range 17 000 to
67 000 while the two largest proteins elute together in the void volume.
Superdex 200 prep grade (b) resolves the two largest proteins completely. The three smaller proteins
are not resolved quite as well as the larger ones or as in (a). The void
volume (Vo) peak at 28 minutes in (b) is caused by protein aggregates.
Fig. 6. Columns : a) HiLoad 16/60 Superdex 75 prep grade
b) HiLoad 16/60 Superdex 200 prep grade
Sample : 1. Myoglobin 1.5 mg/ml, Mr 17 000
2. Ovalbumin 4 mg/ml, Mr 43 000
3. Albumin 5 mg/ml, Mr 67 000
4. IgG 0.2 mg/ml, Mr 158 000
5. Ferritin 0.24 mg/ml, Mr 440 000
Sample volume : 0.5 ml
Buffer : 0.05 M phosphate buffer,
0.15 M NaCl,
0.01% sodium azide, pH 7.0
Flow : 1.5 ml/min (45 cm/h)
Media Selection Chromatography media for gel filtration are made from porous
matrices chosen 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's gel filtration media cover a molecular weight
range from 100 to 80 000 000, from peptides to very large proteins and
protein complexes. Figure.7
Superdex is the first choice for high resolution, short run times and high recovery.
Sephacryl is suitable for fast, high recovery separations at laboratory and industrial
scale
Superose offers a broad fractionation range, but is not suitable for large scale or
industrial scale separations.
Sephadex is ideal for rapid group separations such as desalting and buffer exchange.
Sephadex is used at laboratory and production scale, before, between or after other
chromatography purification steps.
The selectivity of a gel filtration medium depends solely on its pore
size distribution and is described by a selectivity curve. Gel filtration
media are supplied with information on their selectivity, as shown for
Superdex in Figure 8. The curve has been obtained by plotting a
partition coefficient Kav against the log of the molecular weight for a
set of standard proteins
Fig. 8. Selectivity curves for Superdex
Fig. 9. Defining fractionation range and exclusion limit from a selectivity curve.
Selectivity curves are usually quite linear over the range Kav = 0.1
to Kav = 0.7 and it is this part of the curve that is used to determine the
fractionation range of a gel filtration medium Figure 9.
Determination molecular weight
Ve – V0
Kav =--------------
Vt – V0
where Ve = elution volume for the protein
Vo = column void volume
Vt = total bed volume
On semilogarithmic graph paper, plot the Kav value for each protein
standard (on the linear scale) against the corresponding molecular
weight (on the logarithmic 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. 2 ).
Product Fractionation
range, Mr
(globular
proteins)
pH stability Bed volume
ml/g dry
Sephadex
Particle size,
wet
Sephadex G-10 <7×102
Long term: 2–13
Short term: 2–13 2-3 55–165 μm
Sephadex G-25
Coarse
1×103–5×10
3 Long term: 2–13
Short term: 2–13 4-6 170–520 μm
Sephadex G-25
Medium
1×103–5×10
3 Long term: 2–13
Short term: 2–13 4-6 85–260 μm
Sephadex G-25
Fine
1×103–5×10
3 Long term: 2–13
Short term: 2–13 4-6 35–140 μm
Sephadex G-25
Superfine
1×103–5×10
3 Long term: 2–13
Short term: 2–13 4-6 17–70 μm
Sephadex G-50
Fine
1×103–3×10
4 Long term: 2–10
Short term: 2–13 9-11 40–160 μm
• Sephadex G-10 is well suited for the separation of biomolecules such
as peptides (Mr >700) from smaller molecules (Mr <100).
• Sephadex G-50 is suitable for the separation of molecules Mr >30000
from molecules Mr<1500 such as labeled protein or DNA from
unconjugated dyes. The medium is often used to remove small
nucleotides from longer chain nucleic acids.
• Sephadex G-25 is recommended for the majority of group separations
involving globular proteins. These media are excellent for removing
salt and other small contaminants away from molecules that are greater
than Mr 5000. Using different particle sizes enables columns to be packed
according to application requirements
Sephadex is prepared by cross-linking dextran with
epichlorohydrin, illustrated in Figure 10 The different types of
Sephadex vary in their degree of cross-linking and hence in their degree
of swelling and selectivity for specific molecular sizes, as shown
Fig. 10. Partial structure of Sephadex.
Why use different techniques at each stage
In order to final removal of trace contaminants. Adjustment of pH,
salts or additives for storage. Then, end product of required high level
purity Therefore, The technique chosen must discriminate between the
target protein and any remaining contaminants
Gel Filtration (or)
Gel Permeation Chromatography (or)
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 molecules to 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
Fig.1 Gel 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
Dextran is a polysaccharide composed of (-1--->6)-linked glucose
residues with (-1,3) branch linkages. It is synthesized from sucrose by
an enzyme produced by the bacterium Leuconostoc mesenteroides B-
512F. The dextran is cross-linked to various extents by reaction with
epichlorohydrin to give gel beads with different pore sizes Fig.2. Cross-
linked dextrans are commercially produced by Pharrnacia Fine
Chemicals, lnc., (Uppsala, Sweden), and sold under the trade name
Sephadex. Sephadex gels in the so-called G-series, where the G-
numbers refer to the amount of water gained when the beads are
swelled 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.
Fig.2. Structure of epichlorohydrin cross linked Dextran
Table 1: Properties of gels used in gel permeation (filtration) chromatography
Gel
Water
regain
(mL/g)
Exclusion
limit
Maximum
hydrostatic
pressure cm
H2O
Maximum
flow 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 106 200 1.2
Sepharose CL 6B NA 4 x 106 >200 2.5
Sepharose 4B NA 20 x 106 80 0.96
Sepharose CL 4B NA 20 x 106 120 2.17
Sepharose 2B NA 40 x 106 40 0.83
Sepharose CL 2B NA 40 x 106 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 106 >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 106 50 1.0
Bio-Gel A-150m NA 150 x 106 30 0.5
Bio-Gel is a trade name of Bio-Rad Laboratories
Sephadex and Sepharose are trade name of Pharmacia Fine Chemical
Polyacrylamide gels are long polymers of acrylamide cross-linked with
N.N'methylene-bisacrylamide (Fig. 3).
Fig.3. Structure of 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 series See Table. 1 for the exclusion limits and properties of
the different Bio-Gels.
Agarose is a gel material with pore sizes larger than cross-linked dextran
or polyacrylamide. Agarose is the neutral polysaccharide fraction of agar.
It is composed of a linear polymer of D-galactopyranose linked ( 1->4)
3,6 anhydro-L-galactopyranose, which is linked (1-> 3) (Fig. 4).
D-galactose (-1->4) 3,6-Anhydro-L-galactose
Fig.4. Structure of the repeating unit of agarose, D-galactopyranose linked (-1->4)
to 3,6-anhydro-L-galactopyranose, which is linked (-1-3) to the 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 series with 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 gel with increased
thermal and chemical stability. Table 1 gives the properties of the
different Sephadex, Bio-Gel, and Sepharose 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).
Fig.5. 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.
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.
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 negative charges are called cation exchangers and those with
chemically bound positive charges are called anion exchangers. 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 charged molecules can be chromatographed on an anion
exchanger (A+), which has chloride ions as the counterion to give A
+Cl
-.
When (Na+ X
-) molecules in solution interact with the ion exchanger, the
X- displaces the chloride ion from the exchanger and becomes
electrostatically bound to give A+X
-, simultaneously releasing sodium
ions. This process of ion exchange is illustrated in Figure 1. A similar
but opposite process would take place for positively charged molecules
(Y+ Cl
-) which would be chromatographed on cation exchangers (C
-Na
+).
Thus the cation exchangers will bind positively charged molecules from
solution and the anion exchangers will bind negatively charged molecules
from solution.
One of the inert materials used in this type of chromatography is
cross-linked polystyrene, to which the charged groups are chemically
bound. In the separation of biologically important macromolecules,
such as enzymes and proteins.
Figure 1. The process of anion-exchange chromatography
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 cation and the exchangers respectively. The preparation and
commercial availability of these materials beginning in the 1960 provided
the biochemist with powerful tools for separation of proteins and
nucleic acid Figure 2 presents partial structures of DEAE-cellulose and
CM –cellulose
Figure 2. Partial structures of diethylaminoethyl-cellulose and carboxymethyl-
cellulose. The DEAE and CM groups are shown attached to the C6-hydroxyl group
of glucose. The DEAE and CM groups are also found attached to the hydroxyl groups
of C2 and C3. The total degree of substitution of the DEAE and CM groups must be
less than one group per five glucose residues to maintain a water-insoluble product.
Table 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
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 exchanger is suspended in 15
volumes (w/v) of the "first treatment," acid or alkali depending on the
exchanger (Table. 1), and is allowed to stand at least 30 minutes but not
more 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 is washed with distilled water until the effluent is close to
neutral pH. The treated exchanger is placed into the acid component of
the buffer (the pH should 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 allowed to settle and the "fines" (fragments < 10 m in
diameter) 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 of the buffer 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 strength by increasing the concentration of salt
in the elution solvent, thereby weakening the electrostatic interactions
between the adsorbed molecule and the exchanger, and
(c) Performing affinity elution. 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 opposite signs to the net
charge on 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:
Cytochrome C has an isoelectric point (pI) of 10.05; that is at pH 10.05
the number of positive charges will equal the number of negative
charges. A cloumn containing a cation exchanger buffered, at pH 8.5,
is prepared. This column has a full negative charge. Cytochrome C at
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.
Gel Filtration
Ion exchange chromatography
Affinity chromatography
Histadine ,Aspartic,glycine,tyrosine
In anion exchange chromatography,which seprate first and why
AFFINITY CHROMATOGRAPHY
Affinity chromatography is a specialized type of adsorption
chromatography in which a specific type of molecule is covalently linked
to an inert solid support. This specific molecule called a ligand, has a
high binding affinity for one of the compounds in a mixture of
substances. The process uses the unique biological property of the
substance to bind to the ligand specifically and reversibly and provides a
high degree of selectivity in the isolation and purification of biological
molecules
Fig. 1. The steps of affinity chromatography
A solution containing the substance to be purified. Usually a
macromolecule such as a protein (enzyme, antibody, hormone. etc.).
Polysaccharide or nucleic acid is passed through a column containing an
insoluble inert polymer to which the ligand has been covalently attached.
The ligand may be specific competitive inhibitors, substrate analogues,
product analogues, coenzymes and so on. Molecules in the mixture not
having affinity for the ligand pass through the column. Wide molecules
that have specific affinity for the ligand are bound and retained on the
column. The specifically adsorbed molecules) can be eluted by changing
the ionic strength the pH or by the addition of a competing ligand. In one
chromatographic step. The method is capable of isolating a single
substance in a pure form. It has thus become a powerful tool in the
isolation and purification of enzymes, antibodies, antigens, nucleic acids.
Polysaccharides, coenzyme or vitamin binding proteins, repressor
proteins, transport proteins, drug or hormone receptor structures and
other biochemical materials.
The Inert Support and the Ligand
The inert solid supports are the same materials discussed in the
preceding sections: cross-linked dextran cross-linked polyacrylamide,
agarose and cellulose. The macromolecules to be separated should not be
retarded by a gel filtration process but should be retarded only by the
specific interaction with the ligand. The ligand must be a molecule that
display, special and unique affinity for the macromolecule to be purified
it also must have a chemical group that can be modified for covalent
linkage to the solid support without destroying or seriously decreasing its
interaction with the macromolecule to be purified. Also for successful
affinity chromatography, the chemical groups of the ligand that arc
critical for the binding of the macromolecule to be purified must be
sufficiently distant from the solid support to minimize steric interference
with the binding process. This steric problem has been solved by adding a
long, hydrocarbon chain spacer arm to the solid support and coupling the
ligand to the end of the arm. Alternatively the hydrocarbon arm may be
attached to the ligand and the arm attached to the solid support.
Attachment of the Ligand to the Solid Support
The polysaccharide solid supports-cross-linked dextran, agarose,
and cellulose can be activated by reaction with alkaline cyanogen
bromide. The products that arc formed upon coupling of the activated
polysaccharides with amino compounds are derivatives of amino carbonic
acid. The reactions are the following:
If the ligand contains an amino group, it can be coupled directly to
the activated polysaccharide. A spacer arm can be introduced by
sequential reaction with a diaminoalkane and glutaraldehyde. The amino
group on the ligand can then be coupled to the free aldehyde group.
If the ligand contains an aldehyde group instead of an amino group,
it can be coupled directly to the free amino group of the diaminoalkane.
Ligands may be coupled to polyacrylamide by displacing the amide group
of the polyacrylamide by heating with a diaminoalkane (c), followed by
reaction with glutaraldehyde (d).
The Schiff base that results from the reaction of glutaraldehyde
with an amino group may be stabilized by reduction with sodium
cyanoborohydride without affecting the aldehyde group. The ligand can
then be coupled to the aldehyde group.
Another method of activating polyacrylamide is to form the hydrazide
derivative by reaction with hydrazine hydrate. When an amino, aldehyde,
or hydrazide group is incorporated onto the solid support, the support
becomes activated so that ligands may be attached through amino,
carboxyl, phenolic, or imidazole groups.
Gel electrophoresis The movement of a charged presented by Equation 1.0 subjected to an
electric field:
(Equation 1.0)
where
E = the electric field in volts/cm
q = the net charge on the molecule
f = frictional coefficient, which depends on the mass and shape of the
molecule
V = the velocity of the molecule
The charged particle moves at a velocity that depends directly on the
electrical field (E) and charge (q) but inversely on a counteracting force
generated by the viscous drag (f ) The applied voltage represented by E in
Equation 1.0 is usually held constant during electrophoresis, although
some experiments are run under conditions of constant current (where the
voltage changes with resistance) or constant power (the product of
voltage and current). Under constant-voltage conditions, Equation 1.0
shows that the movement of a charged molecule depends only on the
ratio q/f. For molecules of similar conformation (for example, a
collection of linear DNA fragments or spherical proteins), varies with
size but not shape; therefore, the only remaining variables in Equation
1.0 are the charge (q) and mass dependence of (f ) meaning that under
such conditions molecules migrate in an electric field at a rate
proportional to their charge-to-mass ratio. The movement of a charged
particle in an electric field is often defined in terms of mobility, , the
velocity per unit of electric field (Equation 2.0).
(Equation 2.0)
This equation can be modified using Equation 1.0.
(Equation 3.0)
In theory, if the net charge, (q), on a molecule is known, it should be
possible to measure (f) and obtain information about the hydrodynamic
size and shape of that molecule by investigating its mobility in an electric
field. Attempts to define (f) by electrophoresis have not been successful,
primarily because Equation 3.0 does not adequately describe the
electrophoretic process. Important factors that are not accounted for in
the equation are interaction of migrating molecules with the support
medium and shielding of the molecules by buffer ions. This means that
electrophoresis is not useful for describing specific details about the
shape of a molecule. Instead, it has been applied to the analysis of purity
and size of macromolecules. Each molecule in a mixture is expected to
have a unique charge and size, and its mobility in an electric field will
therefore be unique. This expectation forms the basis for analysis and
separation by all electrophoretic methods The technique is especially
useful for the analysis of ammo acids, peptides, proteins, nucleotides,
nucleic acids, and other charged molecules.
Method of Electrophoresis
All types of electrophoresis are based on the principles just
outlined. The major difference between methods is the type of support
medium, which can be either cellulose or thin gels. Cellulose is used as a
support medium for low-molecular-weight biochemical such as ammo
acids and carbohydrates, and polyacrylamide and agarose gels are widely
used as support media for larger molecules. Geometries (vertical and
horizontal), buffers, and electrophoretic conditions for these two types of
gels provide several different experimental arrangements, as described
below.
Polyacrylamide Gel Electrophoresis (PAGE)
Gels formed by polymerization of acrylamide have several positive
features in electrophoresis:
A) High resolving power for small and moderately sized proteins
and nucleic acids (up to approximately 1 X 106 daltons),
B) Acceptance of relatively large sample sizes,
C) Minimal interactions of the migrating molecules with the
matrix, and
D) Physical stability of the matrix that gels can be prepared with
different pore sizes by changing the concentration of cross-linking
agents. Electrophoresis through polyacrylamide gels leads to enhanced
resolution of sample components because the separation is based on both
molecular sieving and electrophoretic mobility The order of molecular
movement in gel filtration and PAGE is very different, however in gel
filtration, large molecules migrate through the matrix faster than small
molecules The opposite is the case for gel electrophoresis, where there
is no void volume in the matrix, only a continuous network of pores
throughout the gel. The electrophoresis gel is comparable to a single bead
in gel filtration. Therefore, large molecules do not move easily through
the medium, and the rate of movement is small molecules followed by
large molecules.
Polyacrylamide gels are prepared by the free radical polymerization
of acrylamide and the cross-linking agent N,N'- methylene-bis-
acrylamide. Chemical polymerization is controlled by an initiator-catalyst
system, ammonium persulfate-N,N,N,َN َ tetramethylethylenediamine
(TEMED). Photochemical polymerization may be initiated by riboflavin
in the presence of ultraviolet (UV) radiation. A standard gel for protein
separation is 7.5% polyacrylamide. It can be used over the molecular size
range of 10,000 to 1,000,000 daltons; however, the best resolution is
obtained in the range of 30,000 to 300,000 daltons. The resolving power
and molecular size range of a gel depend on the concentrations of
acrylamide and bis-acrylamide Lower concentrations give gels with
larger pores, allowing analysis of higher-molecular-weight biomolecules
In contrast, higher concentrations of acrylamide give gels with smaller
pores, allowing analysis of lower-molecular-weight biomolecules
(Table 1.0) Effective Range of Separation of DNA by PAGE
Acylamide
(% W/V)
Range of Separation
(bp)
Bromophenol
Blue
Xylene Cyanol
35 1000-2000 100 450
50 80-500 65 250
80 60-400 50 150
120 40-200 20 75
200 5-100 10 50
Polyacrylamide electrophoresis can be done using either of two
arrangements, column or slab. Figure 1.0 shows the typical arrangement
for a column gel. Glass tubes (10 cm X 6 mm l.d.) are filled with a
mixture of acrylamide, N,N'-methylene-bis-acrylamide, buffer, and free
radical initiator catalyst. Polymerization occurs in 30 to 40 minutes. The
gel column is inserted between two separate buffer reservoirs. The upper
reservoir usually contains the cathode and the lower the anode. Gel
electrophoresis is usually carried out at basic pH, where most biological
polymers are anionic; hence, they move down toward the anode. The
sample to be analyzed is layered on top of the gel and voltage is applied
to the system. A "tracking dye" is also applied, which moves more
rapidly through the gel than the sample components. When the dye band
has moved to the opposite end of the column, the voltage is turned off
and the gel is removed from the column and stained with a dye.
Chambers or column gel electrophoresis is commercially available or can
be constructed from inexpensive materials.
Slab gels are now more widely used than column gels. A slab gel on
which several samples may be analyzed is more convenient to make and
use than several individual column gels. Slab gels also offer the
advantage that all samples are analyzed m a matrix environment that is
identical in composition. A typical vertical slab gel apparatus is shown in
Figure 2.0.
The polyacrylamide slab is prepared between two glass plates that are
separated by spacers Figure 3.0.
The spacers allow a uniform slab thickness of 0.5 to 2.0 mm, which is
appropriate for analytical procedures. Slab gels are usually 8 X 10 cm or
10 X 10 cm, but for nucleotide sequencing, slab gels as large as 20 X 40
cm are often required. A plastic "comb" inserted into the top of the slab
gel during polymerization forms indentations in the gel that serve as
sample wells. Up to 20 sample wells may be formed. After
polymerization, the comb is carefully removed and the wells are rinsed
thoroughly with buffer to remove salts and any unpolymerized
acrylamide. The gel plate is clamped into place between two buffer
reservoirs, a sample is loaded into each well, and voltage is applied. For
visualization, the slab is removed and stained with an appropriate dye.
Perhaps the most difficult and inconvenient aspect of
polyacrylamide gel electrophoresis is the preparation of gels. The
monomer, acrylamide, is a neurotoxin and a cancer suspect agent; hence,
special handling is required. Other necessary reagents including catalysts
and initiators also require special handling and are unstable- In addition,
it is difficult to make gels that have reproducible thicknesses and
compositions. Many researchers are now turning to the use of precast
polyacrylamide gels. Several manufacturers now offer gels precast in
glass or plastic cassettes. Gels for all experimental operations are
available including single percentage (between 3 and 27%) or gradient
gel concentrations and a variety or sample well configurations and buffer
chemistries. Several modifications of PAGE have greatly increased its
versatility and usefulness as an analytical tool.
Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophorosis
(SDS-PAGE)
The electrophoretic techniques previously discussed are not applicable
to the measurement of the molecular weights of biological molecules
because mobility is influenced by both charge and size. If protein samples
are treated so that they have a uniform charge, electrophoretic mobility
then depends primarily on size (see Equation 2.0). The molecular
weights of proteins may be estimated if they are subjected to
electrophoresis in the presence of a detergent, sodium dodecyl sulfate
(SDS), and a disulfide bond reducing agent, mercaptoethanol. This
method is often called "denaturing electrophoresis." When protein
molecules are treated with SDS, the detergent disrupts the secondary,
tertiary, and quaternary structure to produce linear polypeptide chains
coated with negatively charged SDS molecules. The presence of
mercaptoethanol assists in protein denaturation by reducing all disulfide
bonds. The detergent binds to hydrophobic regions of the denatured
protein chain in a constant ratio of about 14 g of SDS per gram of protein.
The bound detergent molecules carrying negative charges mask the native
charge of the protein In essence, polypeptide chains of a constant
charge/mass ratio and uniform shape are produced The electrophoretic
mobility of the SDS-protein complexes is influenced primarily by
molecular size the larger molecules are retarded by the molecular sieving
effect of the gel, and the smaller molecules have greater mobility
Empirical measurements have shown a linear relationship between the
log molecular weight and the electrophoretic mobility Figure 4.0
In practice, a protein of unknown molecular weight and subunit
structure is treated with 1% SDS and 0.1 M mercaptoethanol in
electrophoresis buffer. A standard mixture of proteins with known
molecular weights must also be subjected to electrophoresis under the
same conditions. Two sets on standards are commercially available, one
for low-molecular-weight proteins (molecular weight range 14,000 to
100,000) and one for high-molecular weight proteins D5,000 to 200,000)
Figure 5.0
a stained gel after electrophoresis of a standard protein mixture
After electrophoresis and dye staining, mobilities are measured and
molecular weights determined graphically SDS-PAGE is valuable for
estimating the molecular weight of protein subunits This modification of
gel electrophoresis finds its greatest use in characterizing the sizes and
different types of subunits in oligomeric proteins. SDS-PAGE is limited
to a molecular weight range of 10,000 to 200,000. Gels of less than
2.5% acrylamide must be used for determining molecular weights above
200,000, but these gels do not set well and are very fragile because of
minimal cross-linking. A modification using gels of agarose-acrylamide
mixtures allows the measurement of molecular weights above 200,000.
Agarose Gel Electrophoresis
The electrophoretic techniques discussed up to this point are useful for
analyzing proteins and small fragments of nucleic acids up to 350,000
daltons (500 bp) in molecular size; however, the small pore sizes in the
gel are not appropriate for analysis of large nucleic acid fragments or
intact DNA molecules. The standard method used to characterize RNA
and DNA in the range 200 to 50,000 base pairs 50 kilobases) is
electrophoresis with agarose as the support medium.
Agarose, a product extracted from seaweed, is a linear polymer of
galactopyranose derivatives. Gels are prepared by dissolving agarose in
warm electrophoresis buffer. After cooling the gel mixture to 50°C, the
agarose solution is poured between glass plates as described for
polyacrylamide. Gels with less than 0.5% agarose are rather fragile and
must be used in a horizontal arrangement (Figure 4.8). The sample to be
separated is placed in a sample well made with a comb, and voltage is
applied until separation is complete. Precast agarose gels of all shapes,
sizes, and percent composition are commercially available. Nucleic acids
can be visualized on the slab gel after separation by soaking in a solution
of ethidium bromide, a dye that displays enhanced fluorescence when
intercalated between stacked nucleic acid bases. Ethidium bromide may
be added directly to the agarose solution before gel formation. This
method allows monitoring of nucleic acids during electrophoresis.
Irradiation of ethidium bromide treated gels by UV light results in
orange-red bands where nucleic acids are present. The mobility of nucleic
acids in agarose gels is influenced by the agarose concentration and the
molecular size and molecular conformation of the nucleic acid. Agarose
concentrations of 0.3 to 2.0% are most effective for nucleic acid
separation Table 2.0
Figure 6.0
The separation of DNA fragments on agarose gels. Like
proteins, nucleic acids migrate at a rate that is inversely proportional
to the logarithm of their molecular weights; hence, molecular weights
can be estimated from electrophoresis results using standard nucleic
acids or DNA fragments of known molecular weight. The DNA
conformations most frequently encountered are superhelical circular
(form I), nicked circular (form II), and linear (form III). The small,
compact, supercoiled form I molecules usually have the greatest mobility,
followed by the rodlike, linear form III molecules. The extended, circular
form II molecules migrate more slowly. The relative electrophoretic
mobility of the three forms of DNA, however, depends on experimental
conditions such as agarose concentration and ionic strength.
Isoelectric Focusing of Proteins
Another important and effective use of electrophoresis for the analysis of
Proteins are isoelectric focusing (IEF), which examines electrophoretic
mobility as a function of pH. The net charge on a protein is pH
dependent. Proteins below their isoelectric pH (pHI or the pH at which
they have zero net charge) are positively charged and migrate to a
medium of fixed pH toward the negatively charged cathode at a pH
above its isoelectric point, a protein is deprotonated and negatively
charged and migrates toward the anode If the pH of the electrophoretic
medium is identical to the pHI of a protein, the protein has a net charge
of zero and does not migrate toward either electrode. Theoretically, it
should be possible to separate protein molecules and to estimate the pH:
of a protein by investigating the electrophoretic mobility in a series of
separate experiments in which the pH of the medium is changed. The pH
at which there is no protein migration should coincide with the pHI of the
protein. Because such a repetitive series determine the pHI, IEF has
evolved as an alternative method for performing a single electrophoresis
run in a medium of gradually changing pH.
Figure 7.0
illustrates the construction and operation of an IEF pH gradient. An acid,
usually phosphoric, is placed at the cathode; a base, such as
triethanolamine, is placed at the anode. Between the electrodes is a
medium in which the pH gradually increases from 2 to 10. The pH
gradient can be formed before electrophoresis is conducted or formed
during the course of electrophoresis. The pH gradient can be either broad
(pH 2-10) for separating several proteins of widely ranging pHI values or
narrow (pH 7-8) for precise determination of the pHI of a single protein. P
in Figure 7.0 represents different molecules of the same protein in two
different regions of the pH gradient. Assuming that the pH in region 1 is
less than the pHI of the protein and the pH in region 2 is greater than the
pHI of the protein, molecules of P in region 1 will be positively charged
and will migrate m an applied electric field toward the cathode. As P
migrates, it will encounter an increasing pH, which will influence its net
charge. As it migrates up the pH gradient, P will become increasingly
deprotonated and its net charge will decrease toward zero. When P
reaches a region where it's net charge is zero (region 3), it will stop
migrating. The pH in this region of the electrophoretic medium will
coincide with the pHI of the protein and can be measured with
Illustration of isoelectric a surface microelectrode, or the position of the
protein can be compared to that of a calibration set of proteins of bown
pHI values. P molecules in region 2 will be negatively charged and will
migrate toward the anode. In this case, the net charge on P molecules will
gradually decrease to zero as P moves down the pH gradient, and P
molecules originally in region 2 will approach region 3 and come to rest.
The P molecules move in opposite directions, but the final outcome of
IEF is that P molecules located anywhere m the gradient will migrate
toward the region corresponding to their isoelectric point and will
eventually come to rest in a sharp band; that is, they will "focus" at a
point corresponding to their pHI. Since different protein molecules in
mixtures have different pHI values, it is possible to use IEF to separate
proteins In addition; the pHI of each protein in the mixture can be
determined by measuring the pH of the region where the protein is
focused. The pH gradient is prepared in a horizontal glass tube or slab.
Special precautions must be taken so that the pH gradient remains stable
and is not disrupted by diffusion or convective mixing during the
electrophoresis experiment. The most common stabilizing technique is to
form the gradient in a polyacrylamide, agarose, or dextran gel. The pH
gradient is formed in the gel by electrophoresis of synthetic
polyelectrolyte, called ampholytes, which migrate to the region of their
pHI values just as proteins do and establish a pH gradient that is stable for
the duration of the IEF run. Ampholytes are low-molecular-weight
polymers that have a wide range of isoelectric points because of their
numerous ammo and carboxyl or sulfonic acid groups. The polymer
mixtures are available in specific pH ranges (pH 5-7, 6-8, 3.5-10, etc.). It
is critical to select the appropriate pH range for the ampholyte so that the
proteins to be studied have pHI values in that range. The best resolution
is, of course, achieved with an ampholyte mixture over a small pH range
(about two units) encompassing the pHI of the sample proteins. If the pHI
values for the proteins under study are unknown, an ampholyte of wide
pH range (pH 3-10) should be used first and then a narrower pH range
selected for use. The gel medium is prepared as previously described
except that the appropriate ampholyte is mixed prior to polymerization.
The gel mixture is poured into the desired form (column tubes, horizontal
slabs, etc.) and allowed to set. Immediately after casting of the gel, the
pH is constant throughout the medium, but application of voltage will
induce migration of ampholyte molecules to form the pH gradient. The
standard gel for proteins with molecular sizes up to 100,000 daltons is
7.5% polyacrylamide; however, if larger proteins are of interest, gels with
larger pore sizes must be prepared. Such gels can be prepared with a
lower concentration of acrylamide (about 2%) and 0.5 to 1% agarose to
add strength. Precast gels for isoelectric focusing are also commercially
available. The protein sample can be loaded on the gel in either of two
ways. A concentrated, salt-free sample can be layered on top of the gel as
previously described for ordinary gel electrophoresis. Alternatively, the
protein can be added directly to the gel preparation, resulting in an even
distribution of protein throughout the medium. The protein molecules
move more slowly than the low-molecular-weight ampholyte molecules,
so the pH gradient is established before significant migration of the
proteins occurs. Very small protein samples can be separated by IER. For
analytical purposes, 10 to 50 g is a typical sample size. Larger sample
sizes (up to 20 mg) can be used for preparative purposes.
Common abbreviations in chromatography
GF: gel filtration (sometimes referred to as SEC: size exclusion
chromatography)
IEX: ion exchange chromatography (also seen as IEC)
AC: affinity chromatography
RPC: reverse phase chromatography
HIC: hydrophobic interaction chromatography
CIPP: Capture, Intermediate Purification and Polishing
MPa: megapascals
psi: pounds per square inch
SDS: sodium dodecyl sulphate
CIP: cleaning in place
A280nm, A214nm:
UV absorbance at specified wavelength
Mr: relative molecular weight
N: column efficiency expressed as theoretical plates per meter
Ve: elution volume is measured from the chromatogram and
relates to the molecular size of the molecule.
Vo: void volume is the elution volume of molecules that are
excluded from the gel filtration medium because they are
larger than the largest pores in the matrix and pass straight
through the packed bed
Vt: total column volume is equivalent to the volume of the packed
bed
Rs: resolution, the degree of separation between peaks
Kav and
logMr:
partition coefficient and log molecular weight, terms used
when defining the selectivity of a gel filtration medium
In product names
HMW: high molecular weight
LMW: low molecular weight
HR: high resolution
pg: prep grade
PC: precision column
SR: solvent resistant
Protein
------------Negative--------------------------o-----------------------Positive-----------------------
(pH>pI) (pH=pI) (pH<pI)
anion exchange resin cation exchange resin
(resin is positive A+) (resin is negative C-)
At pH > pI, protein net charge is negative At pH < pI, protein net charge is positive At pH = pI, protein net charge is zero
Isoelectric point (pI)
At pH > pI, use an anion exchange resin (positive resin) At pH < pI, use a cation exchange resin(negative resin)
nonpolar molecules lack charge
polar, uncharged molecules carry one or more partial charges
-Organic component of the solvent
continues migrating, thus forming the
mobile phase.
-Therefore, compounds soluble to organic
component move faster than compounds
soluble to aqueous component.
-Thus, molecules are separated according
to their polarities.
Differential precipitation Salt (or other solute) is added to a solution that contains a
mixture of proteins
Ammonium sulfate is most popular for “salting out”
-Effective, highly soluble, & does not tend to denature protein
Individual proteins precipitate at specific [(NH4)2SO4]
Depends on properties of specific solute (salt), not ionic strength
per se
Precipitated proteins are:
- isolated in centrifuge
- resuspended in low salt buffer
- dialysis or gel filtration can be used to remove residual
precipitant (if necessary)
Often used in two steps:
• Salt out some impurities w/ lower concentration that does not
precipitate POI
• Use higher concentration for selective precipitation of POI
Protein purification
Prior requirements for devising a purification method:
• Source tissue
– Known or likely to contain “high” levels of POI
– Available in suitable (preparative) quantities
Possible (& potentially misleading) alternative: clone gene &
use expression
system
• Exogenous system may not recapitulate protein
processing/covalent modifications
• POI may function as part of a complex (w/ addn’l subunits
derived from other
genes)
• Assay method that is:
– Specific for POI & relatively insensitive to other components
in extracts
– Linear and quantitative:
• measured activity should be proportional to amount of POI
• Amount of activity is expressed in “units”:
– e.g., amount of S � P per time
– “katal” (kat) is preferred unit: moles per second
– If POI is not enzyme, assay may be based on binding (direct or
indirect)
– Suitably sensitive
– Optimized with respect to pH, ionic strength, temp., substrate
or ligand
concentration, etc.
The polymerization reaction of acrylamide and methylenebisacrylamide
The three major forms of alanine occurring in titrations between pH 1 and 14
4. F. F. Runge, Farbenchemie, I and II (1834, 1843). 5. F. F. Runge, Ann. Phys. Chem. XVII, 31, 65 (1834); XVIII, 32, 78 (1834). 6. F. F. Runge, Farbenchemie III, 1850. 7. F. Goppelsroeder, Zeit. Anal. Chem. 7, 195 (1868). 8. C. Sch¨onbein, J. Chem. Soc. 33, 304–306 (1878). 9. D. T. Day, Proc. Am. Philos. Soc. 36, 112 (1897). 10. D. T. Day, Congr. Intern. P´etrole Paris 1, 53 (1900). 11. D. T. Day, Science 17, 1007 (1903). 12. M. Twsett, Ber. Deut. Bot. Ges. XXIV 316 (1906). 13. M. Twsett, Ber. Deut. Bot. Ges. XXIV 384 (1906). 14. M. Twsett, Ber. Deut. Bot. Ges. XXV 71–74 (1907). 15. R. Kuhn, A. Wunterstein, and E. Lederer, Hoppe-Seyler’s Z. Physiol. Chem. 197, 141–160 (1931). 16. A. Tiselius, Ark. Kemi. Mineral. Geol. 14B(22) (1940). 17. J. N. Wilson, J. Am. Chem. Soc. 62, 1583–1591 (1940). 18. A. Tiselius, Ark. Kemi. Mineral. Geol. 15B(6) (1941). 19. A. J. P. Martin and R. L. M. Synge, Biochem. J. (Lond.) 35, 1358 (1941). 20. R. Consden, A. H. Gordon, and A. J. P. Martin, Biochem. J. 38, 224–232 (1944). 21. S. Claesson, Arkiv. Kemi. Mineral. Geol. 23A(1) (1946). 22. A. J. P. Martin, Biochem. Soc. Symp. 3, 4–15 (1949). 23. E. Cremer and F. Prior, Z. Elektrochem. 55, 66 (1951); E. Cremer and R. Muller, ZElektrochem. 55, 217 (1951). 24. C. S. G. Phillips, J. Griffiths, and D. H. Jones, Analyst 77, 897 (1952). 25. A. T. James and A. J. P. Martin, Biochem. J. 50, 679–690 (1952). 26. E. Glueckauf, in Ion Exchange and Its Applications, Society of the Chemical Industry, London, 1955, pp.34–36. 27. J. J. van Deemter, F. J. Zuiderweg, and A. Klinkenberg, Chem. Eng. Sci. 5, 271–289 (1956). 28. M. J. E. Golay, in Gas Chromatography, V. J. Coates, H. J. Noebels, and I. S. Fagerson, eds., Academic Press, New York, 1958, pp. 1–13. 29. J. C. Giddings, Dynamics of Chromatography, Part I, Principles and Theory, Marcel Dekker, New York, 1965, pp. 13–26.