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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
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
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.
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.
Figure 5
sticking to the glass plate. The plates are prepared by spreading slurry of
work.
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
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
different methods:
2. Spraying the plate with 50% sulphuric acid and heating so,
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:
Non-polar solvents:
Non-destructive techniques:
Destructive techniques
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
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.
For silica gel TLC: The plate is sprayed with a solution of 300 mg of
pH 12.0) and
the separation depend on the particular plate used. Whatman K5 silica gel
Advantages of TLC.
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.
gravity aids the solvent flow and with substances of relatively low
mobility. The solvent can run off the paper. Giving a longer path for
ascent technique.
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
solvent trough of the descending tank (Fig. 1). Which has been
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,
lipids.)
cm is used. A pencil line 20-25 mm from the bottom is drawn across the
paper
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
screw top, gallon jar or a cylinder with a ground-glass edge and a glass
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,
RGlc or for amino acids, the standard might be glycine and the ratio would
be RGly
in which the sample is spotted in the lower left-hand corner and irrigated
the solvent dried, turned 90, and irrigated in the second dimension with
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
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
vertical sections of the location standards are cut out and developed to
reveal the positions of the compounds. After drying, these standards are
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.
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
occurs by capillary flow of the water down the paper strip into a baker.
radioactive compounds are cut out and placed into a liquid scintillation
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.
• Analyze
Separate • Identify
• Purify
• Quantify
Mixture Components
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
Stationary Phase
Separation
Mobile Phase
Mixture Components
Components Affinity to Stationary Phase Affinity to Mobile Phase
Blue ---------------- Insoluble in Mobile Phase
Black
Red
Yellow
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%
0 20 50 70 100
% % %
Concentration of Isopropanol
% %
Alternative Experiments
Protein purification Chromatography
aggregate:
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
Dr.Ehab Aboueladab (Assistance. Prof.of Biochemistry, Mansoura University) صفحة5
Protein purification Chromatography
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:
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.
Dialysis
off", with the protein solution, and placing the filled tubing in a large
easily from the ratio of the volumes inside and outside of the bag.
Dialysis requires a few hours, after which the bag may be
including salt ions, metal ions and cofactors, pass through the membrane,
which retains only macromolecules. Neither tightly bound metal ions and
volume. The volume of the contents of the bag must be measured after
gravity flow or for use with low pressure peristaltic pumps, ion exchange
Since these carbohydrates are compressible, they are not used in higher-
pressure systems, and more rigid inert phases such as TSK (a polyether-
were used by MOORE and STEIN in their famous amino acid analyzer.
molecules, but have given way to the ion exchange polysaccharides for
the solid supports depend to some extent on the chemistry of the support
nitrogen atoms are almost universally used for anion exchange media.
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.,
above their pK4. Methods for determining the optimal pH for separation
stationary phases than simple salts because they possess more negative
protein, but the charge density that determines the affinity. More
solutions are generally desalted, then loaded onto a column packed with a
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
having a charge of the same sign as the protein can act as a displacing ion
the charge densities of the proteins still on the column. However, the
Such a gradient can cover a range from 0 to 1 M NaCl over the volume of
wide range of affinities for the column can be eluted in a single run. The
much more rapidly in the column than the protein (the protein is retained
enzyme actually appears at the bottom of the column is much higher than
chloride in the fraction in which the activity appears) is much too strong
moves down the column. To exercise maximum control over the system,
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
chromatography should have the same charge as the column, i.e., cations
Tris (for instance) buffers are used for anion exchange. It is necessary
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.
1. Group separations:
distribution analysis.
Gel filtration medium is packed into a column to form a packed bed. The
been chosen for their chemical and physical stability, and inertness (lack
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
free labels are easily separated. Samples can be prepared for storage or
separation mode
is often used in protein purification schemes for desalting and
buffer exchange
bed) can be applied at high flow rates using broad, short columns. Figure
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
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
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
(packed bed). Molecules with partial access to the pores of the matrix
elute from
the column in order of decreasing size. Small molecules such as salts that
have full access to the pores move down the column, but do not separate
from each other. These molecules usually elute just before one total
column volume, Vt, of buffer has passed through the column, Fig. 4.
Separation examples
Fig. 6.
Columns : a) HiLoad 16/60 Superdex 75 prep grade
b) HiLoad 16/60 Superdex 200 prep grade
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
partition coefficient Kav against the log of the molecular weight for a
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
Ve – V0
Kav =--------------
Vt – V0
On semilogarithmic graph paper, plot the Kav value for each protein
weight (on the logarithmic scale). Draw the straight line which best fits
the points on the graph. Then, Calculate the corresponding Kav for the
calibration curve.
Sephadex:
Rapid group separation of high and low molecular weight
salt and other small contaminants away from molecules that are greater
salts or additives for storage. Then, end product of required high level
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
epichlorohydrin to give gel beads with different pore sizes Fig.2. Cross-
Chemicals, lnc., (Uppsala, Sweden), and sold under the trade name
numbers refer to the amount of water gained when the beads are
different pore sizes. This gives gels that have capabilities of separating
smallest peptide or globular protein that will not enter the gel pore.
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
California, as the Bio-Gel P series. Like the Sephadex G series. the Bio-
Sephadex G series See Table. 1 for the exclusion limits and properties of
Agarose is a gel material with pore sizes larger than cross-linked dextran
method is used for both preparative and analytical purposes. The latter
Then, from a plot of log molecular weight versus elution volume, the
and other small molecules from high molecular weight biomolecules. The
sample containing the biomolecules and the salt is passed over a gel
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,
bound negative charges are called cation exchangers and those with
ions for the anion exchangers and metal ions for the cation exchangers.
which are to be adsorbed on the exchangers also have net charges which
let us say that the molecules to he adsorbed from solution have a negative
When (Na+ X-) molecules in solution interact with the ion exchanger, the
but opposite process would take place for positively charged molecules
Thus the cation exchangers will bind positively charged molecules from
solution and the anion exchangers will bind negatively charged molecules
from solution.
CM –cellulose
swell the exchangers so that they become fully accessible to the charged
exchanger (Table. 1), and is allowed to stand at least 30 minutes but not
stirred into 15 volumes of the "second treatment" and allowed to stand for
an additional 30 minutes. The second treatment is repeated and the
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
then titrated with the basic component of the buffer to the desired pH,
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
Three general methods are used for eluting molecules from the
exchanger:
weakened (i.e., the pH is lowered for an anion exchanger and raised for a
cation exchanger),
ligand.
cytochrome C will now has a net zero charge and will pass rapidly
molecules
insoluble inert polymer to which the ligand has been covalently attached.
having affinity for the ligand pass through the column. Wide molecules
that have specific affinity for the ligand are bound and retained on the
The inert solid supports are the same materials discussed in the
it also must have a chemical group that can be modified for covalent
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.
bromide. The products that arc formed upon coupling of the activated
group on the ligand can then be coupled to the free aldehyde group.
(Equation 1.0)
where
molecule
electrical field (E) and charge (q) but inversely on a counteracting force
some experiments are run under conditions of constant current (where the
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
(Equation 2.0)
(Equation 3.0)
medium and shielding of the molecules by buffer ions. This means that
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
Method of Electrophoresis
acids and carbohydrates, and polyacrylamide and agarose gels are widely
below.
features in electrophoresis:
matrix, and
D) Physical stability of the matrix that gels can be prepared with
filtration, large molecules migrate through the matrix faster than small
molecules The opposite is the case for gel electrophoresis, where there
large molecules.
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
for a column gel. Glass tubes (10 cm X 6 mm l.d.) are filled with a
gel column is inserted between two separate buffer reservoirs. The upper
reservoir usually contains the cathode and the lower the anode. Gel
polymers are anionic; hence, they move down toward the anode. The
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.
use than several individual column gels. Slab gels also offer the
Figure 2.0.
The polyacrylamide slab is prepared between two glass plates that are
cm are often required. A plastic "comb" inserted into the top of the slab
polymerization, the comb is carefully removed and the wells are rinsed
acrylamide. The gel plate is clamped into place between two buffer
reservoirs, a sample is loaded into each well, and voltage is applied. For
and initiators also require special handling and are unstable- In addition,
(SDS-PAGE)
molecules are treated with SDS, the detergent disrupts the secondary,
The bound detergent molecules carrying negative charges mask the native
molecular size the larger molecules are retarded by the molecular sieving
effect of the gel, and the smaller molecules have greater mobility
Figure 5.0
gel electrophoresis finds its greatest use in characterizing the sizes and
200,000, but these gels do not set well and are very fragile because of
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
warm electrophoresis buffer. After cooling the gel mixture to 50°C, the
polyacrylamide. Gels with less than 0.5% agarose are rather fragile and
orange-red bands where nucleic acids are present. The mobility of nucleic
Figure 6.0
(form I), nicked circular (form II), and linear (form III). The small,
followed by the rodlike, linear form III molecules. The extended, circular
they have zero net charge) are positively charged and migrate to a
medium is identical to the pHI of a protein, the protein has a net charge
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
Figure 7.0
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
less than the pHI of the protein and the pH in region 2 is greater than the
migrates, it will encounter an increasing pH, which will influence its net
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
coincide with the pHI of the protein and can be measured with
migrate toward the anode. In this case, the net charge on P molecules will
eventually come to rest in a sharp band; that is, they will "focus" at a
pHI values just as proteins do and establish a pH gradient that is stable for
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
(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
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
larger pore sizes must be prepared. Such gels can be prepared with a
add strength. Precast gels for isoelectric focusing are also commercially
available. The protein sample can be loaded on the gel in either of two
proteins occurs. Very small protein samples can be separated by IER. For
------------Negative--------------------------o-----------------------Positive-----------------------