Definition
Chromatography
is the ability to separate molecules using partitioning characteristics of
molecule to remain in a stationary phase versus a mobile phase. Once a molecule
is separated from the mixture, it can be isolated and quantified.
The separation
is based on the partitioning between the mobile and stationary phase
Basic Chromatographic terminology
Chromatograph:
Instrument employed for a chromatography.
Stationary
phase: Phase that stays in place inside the column. Can be a particular solid
or gel-based packing (LC) or a highly viscous liquid coated on the inside of
the column (GC).
Mobile phase:
Solvent moving through the column, either a liquid in LC or gas in GC.
Eluent: Fluid
entering a column.
Eluate: Fluid
exiting the column.
Elution: The
process of passing the mobile phase through the column.
Chromatogram:
Graph showing detector response as a function of a time.
Flow rate: How
much mobile phase passed / minute (ml/min).
Linear velocity:
Distance passed by mobile phase per 1 min in the column (cm/min).
Types of
chromatography on the basis of interaction of the analyte with stationary phase
• Adsorption –
of solute on surface of stationary phase;
for polar non-ionic compounds
• Ion Exchange –
attraction of ions of opposite charges; for ionic compounds anions or cations
• Partition -
based on the relative solubility of analyte in mobile and stationary phases
• Size Exclusion
(gel filtration, gel permeation) – separates molecules by size; sieving - not
real interaction, small molecules travel longer
• Affinity –
specific interactions like a particular antibody to protein
Partition
coefficient KD
Based on
thermodynamic equilibrium
Ratio of Analyte
KD=CS/CM
CS = concentration in stationary phase
CM = concentration in mobile phase
The same
principle as Liquid Liquid Extraction
THIN LAYER CHROMATOGRAPHY
Introduction
Thin
layer chromatography was first discovered by Izmailov and Shraiber in
1938.Further Stahl (1958) perfected the method and developed equipment and
standardized adsorbents for the preparation of uniform layers on glass plates.
And this technique, chromatography using thin layers of an adsorbent held on a
glass plate or other supporting medium is called as thin layer chromatography.
Experimental Setup
Preparation of thin layers on plates
Coating
of glass plates with adsorbent layer is made by spreading, pouring, spraying or
dipping. The adsorbent layer may be solid or loose. For solid layers, a uniform
layer of adsorbent material could be applied to a lean glass plate with an
applicator. The applicator used for the preparation of 0.25 mm layer thickness
film is the Stahl’s original applicator. Sometimes a binder such as calcium
sulphate is added to the adsorbent in small quantities before coating. Loose
layers may be prepared by dipping the plates in the suspension, spraying a thin
suspension or pouring of suspension on to the plate.
Application of sample on the chromo plates
The
application of sample on the chromo plates are similar to those used in paper
chromatography. In T.L.C. 0.1 – 1 %
solution of the sample are applied to the plates with the help of capillaries,
micropipettes and micro syringes. The sample solution could be applied as
single sports in a row along one side of the plate, about 2 cm from the edge.
Choice of adsorbent
The
commonly used adsorbents in TLC are silica gel, alumina, Kieselguhr and
powdered cellulose. Silica gel is the most widely used adsorbent. It is
slightly acidic in nature. Alumina is basic, and Kieselguhr is a natural
adsorbent. Layers of about 0.25 mm thick can be prepared by spreading aqueous
slurry of the adsorbent with a commercial applicator on glass plates. Thick
layers are air dried for about ten minutes and then activated by heating in an
oven at 110°C for 2 hours.
Chromatographic
adsorbents. The order in the table is approximate, since it depends upon the
substance being adsorbed, and the solvent used for elution.
Most Strongly
Adsorbent Alumina Al2O3
Charcoal C
Florisil MgO/SiO2 (anhydrous)
Least Strongly
Adsorbent Silica gel SiO2
Choice of solvents
The
choice of solvent depends on the nature of substances to be separated and the
material on which the separation is to be carried out. Polar solvents are
preferred, because it results in better separation. And a combination of two
solvents too gives better results. Hence the solvent mixture which is highly
preferred for its efficiency is n-hexane-diethyl ether, acetic acid in the
ratio 90:10:1.
Eluting solvents
for chromatography
Least Eluting
Power (alumina as adsorbent)
Petroleum ether (hexane; pentane)
Cyclohexane
Carbon tetrachloride
Benzene
DichIoromethane
Chloroform
Ether (anhydrous)
Ethyl acetate (anhydrous)
Acetone (anhydrous)
Ethanol
Methanol
Water
Pyridine
Greatest Eluting
Power (alumina as adsorbent) Organic acids
Developing chamber
The
thin layer chromatoplates are usually developed by placing them on edges in the
jar containing a 0.5– 10 cm layer of the solvent. The jar is then covered with
an air tight lid. After the developing solvent ascends for about 10-12 cm above
the origin, the plate is taken out of the jar.
The
relationship between the distance traveled by the solvent front and the
substance is usually expressed as the Rf
value:
Rf value =
distance traveled by substance /
distance traveled by solvent front
The
Rf values are strongly dependent upon the nature of the adsorbent and
solvent. Therefore, experimental Rf
values and literature values do not often agree very well. In order to determine whether an unknown
substance is the same as a substance of known structure, it is necessary to run
the two substances side by side in the same chromatogram, preferably at the
same concentration.
Application of the Sample
The
sample to be separated is generally applied as a small spot (1 to 2 mm
diameters) of solution about 1 cm from the end of the plate opposite the
handle. The addition may be made with a
micropipet prepared by heating and drawing out a melting point capillary. As small a sample as possible should be used,
since this will minimize tailing and overlap of spots; the lower limit is the
ability to visualize the spots in the developed chromatogram. If the sample solution is very dilute, make
several small applications in the same place, allowing the solvent to evaporate
between additions. Do not disturb the
adsorbent when you make the spots, since this will result in an uneven flow of
the solvent. The starting position can
be indicated by making a small mark near the edge of the plate.
Detecting reagents
Detecting
reagents used in paper chromatography may be used in T.L.C. also. Iodine
dissolved in an organic solvent could be sprayed to deduct many components.
Sulphuric acid also forms complex which are colored and visible in day and all
light.
Development and detection
The
various development techniques available include ascending development,
horizontal development, multiple development, stepwise development, gradient
development, continuous development and two dimensional development. Normally
ascending development is commonly used. During ascending development the sample
is spotted at one end of the plate and then developed by ascending technique.
The plates are usually placed vertically in a container saturated with developer
vapor and solvent.
General Procedure
§ A plate made of
glass is coated with a loose powder or with slurry of an adsorbent. This slurry
adheres to the surface of the glass plate as a thin layer.
§ The unknown
substance and reference materials are dissolved in water or an organic solvent
and the solution is applied in a row of spot 1-2 cm from the edge of the plate,
with micropipette or microsyringe.
§ The
chromatoplate is placed in a jar containing the jar is lined inside with filter
paper which acts as a wick and saturates the atmosphere of the jar with the
vapors of the solvent. The jar is covered with an air tight lid.
§ As the solvent
ascends by capillary action, the sample gets resolved into fractions. When the
solvent front has migrated about 75% of the plate, the plate is then dried and
sprayed with to iodine vapor.
§ The position of
the fractions would be indicated as brown spots.
Types of TLC
§ Adsorption TLC:
Scientist Kucharezyk (1963) has used adsorption TLC for the fractionation and
analysis of petroleum products. Further it has been used for analysis of waxes
and fats, for analysis of essential oils, analysis of carotenoids, steroids,
fat soluble vitamins and certain alkaloids by different scientists.
§ Ion exchange
TLC: This type of TLC has been used for the separation of ionic compounds from
non ionic compounds. It has been used for the fractionation of short chain
carboxylic acids, sugars, amino acids, nucleotides and detergents.
§ The other types
of TLC include the Partition T.L.C. and Reverse phase partition T.L.C.
Advantages of TLC
·
The
technique helps in the separation of even microgram of the substances.
·
The
separation is very sharp. It is used to study various biological changes, to
study fractionation of large number compounds, to analyze urine and blood
samples.
TLC has following advantages over paper chromatography:
·
greater speed
·
greater sensitivity
for many substances than paper
·
Small sample
requirement
·
Usually sharper
preparation
·
Different kind of
reagents can be applied without damaging the plate
ION EXCHANGE CHROMATOGRAPHY
Introduction
Adsorption
chromatography depends upon interactions of different types between solute
molecules and ligands immobilized on a chromatography matrix. The first type of
interaction to be successfully employed for the separation of macromolecules
was that between charged solute molecules and oppositely charged moieties
covalently linked to a chromatography matrix. The technique of ion exchange
chromatography is based on this interaction. Ion exchange is probably the most
frequently used chromatographic technique for the separation and purification
of proteins, polypeptides, nucleic acids, polynucleotides, and other charged
biomoleules. The reasons for the success of ion exchange are its widespread
applicability, its high resolving power, its high capacity, and the simplicity
and controllability of the method.
The theory of ion exchange
Purification
using ion exchange chromatography depends upon the reversible adsorption of
charged solute molecules to immobilized ion exchange groups of opposite charge.
Most ion exchange experiments are performed in five main stages. These steps
are illustrated schematically.
§ The first stage
is equilibration in which the ion exchanger is brought to a starting state, in
terms of pH and ionic strength, which allows the binding of the desired solute
molecules. The exchanger groups are associated at this time with exchangeable
counter-ions (usually simple anions or cations, such as chloride or sodium).
§ The second stage
is sample application and adsorption, in which solute molecules carrying the
appropriate charge displace counter-ions and bind reversibly to the gel.
Unbound substances can be washed out 332 Ion Exchange Technologies from the
exchanger bed using starting buffer.
§ In the third
stage, substances are removed from the column by changing to elution conditions
unfavourable for ionic bonding of the solute molecules. This normally involves
increasing the ionic strength of the eluting buffer or changing its pH, (i.e)
desorption is achieved by the introduction of an increasing salt concentration
gradient and solute molecules are released from the column in the order of
their strengths of binding, the most weakly bound substances being eluted
first.
The
principle of ion exchange chromatography (salt gradient elution).
§ The fourth and
fifth stages are the removal from the column of substances not eluted under the
previous experimental conditions and re-equilibration at the starting
conditions for the next purification.
Separation is
obtained since different substances have different degrees of interaction with
the ion exchanger due to differences in their charges, charge densities and
distribution of charge on their surfaces. These interactions can be controlled
by varying conditions such as ionic strength and pH. The differences in charge
properties of biological compounds are often considerable, and since ion
exchange chromatography is capable of separating species with very minor
differences in properties, e.g. two proteins differing by only one charged
amino acid, it is a very powerful separation technique.
In ion exchange chromatography one can choose
whether to bind the substances of interest and allow the contaminants to pass
through the column, or to bind the contaminants and allow the substance of
interest to pass through. Generally, the first method is more useful since it
allows a greater degree of fractionation and concentrates the substances of
interest. In addition to the ion exchange effect, other types of binding may
occur. These effects are small and are mainly due to Vander Waals forces and
non-polar interactions.
The matrix
An
ion exchanger consists of an insoluble matrix to which charged groups have been
covalently bound. The charged groups are associated with mobile counter ions.
These counter-ions can be reversibly exchanged with other ions of the same
charge without altering the matrix. It is possible to have both positively and
negatively charged exchangers. Positively charged exchangers have negatively
charged counter-ions (anions) available for exchange and are called anion
exchangers. Negatively charged exchangers have positively charged counter-ions
(cations) and are termed cation exchangers. The matrix may be based on
inorganic compounds, synthetic resins or polysaccharides. The characteristics
of the matrix determine its chromatographic properties such as efficiency,
capacity and recovery as well as its chemical stability, mechanical strength
and flow properties.
Ion
exchanger types.
The
nature of the matrix will also affect its behavior towards biological
substances and the maintenance of biological activity. The first ion exchangers
were synthetic resins designed for applications such as demineralization, water
treatment, and recovery of ions from wastes. Such ion exchangers consist of
hydrophobic polymer matrices highly substituted with ionic groups, and have
very high capacities for small ions. Due to their low permeability these
matrices have low capacities for proteins and other macromolecules. In
addition, the extremely high charge density gives very strong binding and the
hydrophobic matrix tends to denature labile biological materials. Thus despite
their excellent flow properties and capacities for small ions, these types of ion
exchange are unsuitable for use with biological samples. The first ion
exchangers designed for use with biological substances were the cellulose ion
exchangers. Because of the hydrophilic nature of cellulose, these exchangers
had little tendency to denature proteins. Unfortunately, many cellulose ion
exchangers had low capacities (otherwise the cellulose became soluble in water)
and had 334 Ion Exchange Technologies poor flow properties due to their
irregular shape. Ion exchangers based on dextran (Sephadex), followed by those
based on agarose (Sepharose CL-6B) and cross-linked cellulose (DEAE Sephacel)
were the first ion exchange matrices to combine a spherical form with high
porosity, leading to improved flow properties and high capacities for macromolecules.
The presence of charged groups is a fundamental property of an ion exchanger.
The type of group determines the type and strength of the ion exchanger; their
total number and availability determines the capacity. There is a variety of
groups which have been chosen for use in ion exchangers; some of these are
shown as follows
Anion exchangers Functional group
Diethylaminoethyl
(DEAE) -O-CH2-CH2-N+H(CH2CH3)2
Quaternary
aminoethyl (QAE) -O-CH2-CH2-N+(C2H5)2-CH2-CHOH-CH3
Quaternary
ammonium (Q)
-O-CH2-CHOH-CH2-O-CH2-CHOH-CH2-N+(CH3)3
Cation exchangers
Functional group
Carboxymethyl
(CM) -
O-CH2-COO
Sulphopropyl
(SP)
-O-CH2-CHOH-CH2-O-CH2-CH2-CH2SO3
Methyl
sulphonate (S)
-O-CH2-CHOH-CH2-O-CH2-CHOH-CH2SO3
Sulphonic
and quaternary amino groups are used to form strong ion exchangers; the other
groups form weak ion exchangers. The terms strong and weak refer to the extent
of variation of ionization with pH and not the strength of binding. Strong ion
exchangers are completely ionized over a wide pH range. Whereas with weak ion
exchangers, the degree of dissociation and thus exchange capacity varies much
more markedly with pH.
Some properties of strong ion exchangers are:
§ Sample loading
capacity does not decrease at high or low pH values due to loss of charge from
the ion exchanger.
§ A very simple
mechanism of interaction exists between the ion exchanger and the solute.
§ Ion exchange
experiments are more controllable since the charge characteristics of the media
do not change with changes in pH.
Resolution in ion exchange chromatography
The
result of an ion exchange experiment, as with any other chromatographic
separation, is often expressed as the resolution between the peaks of interest.
The resolution is defined as the distance between peak maxima compared with
average base width of the two peaks. Elution volumes and peak widths should be
measured with the same units to give a dimensionless value to the resolution.
Capacity
The
capacity of an ion exchanger is a quantitative measure of its ability to take
up exchangeable counter-ions and is therefore of major importance. The capacity
may be expressed as total ionic capacity, available capacity or dynamic
capacity. The total ionic capacity is the number of charged substituent groups
per gram dry ion exchanger or per ml swollen gel. Total capacity can be
measured by titration with a strong acid or base. The actual amount of protein
which can be bound to an ion exchanger, under defined experimental conditions,
is referred to as the available capacity for the gel. If the defined conditions
include the flow rate at which the gel was operated, the amount bound is
referred to as the dynamic capacity for the ion exchanger. Available and
dynamic capacities depend upon the properties of the protein.
The
properties of the ion exchanger are the chosen experimental conditions. The
properties of the protein which determine the available or dynamic capacity on
a particular ion exchange matrix are its molecular size and its charge/pH
relationship. The capacity of an ion exchanger is thus different for different
protein.
Choice of exchanger group
Substances
are bound to ion exchangers when they carry a net charge opposite to that of
the ion exchanger. This binding is electrostatic and reversible. In the case of
substances which carry only one type of charged group the choice of ion
exchanger is clear-cut. Substances which carry both positively and negatively
charged groups, however, are termed 336 Ion Exchange Technologies amphoteric
and the net charge which they carry depends on pH. Consequently at a certain pH
value an amphoteric substance will have zero net charge. This value is termed
the isoelectric point (pI) and at this point substances will bind to neither
anion or cation exchangers. The net charge of protein as a function of pH.
The
pH of the buffer thus determines the charge on amphoteric molecules during the
experiment. In principle therefore, one could use either an anion or a cation
exchanger to bind amphoteric samples by selecting the appropriate pH. In
practice however, the choice is based on which exchanger type and pH give the
best separation of the molecules of interest, within the constraints of their
pH stability.
Many biological
macromolecules become denatured or lose activity outside a certain pH range and
thus the choice of ion exchanger may be limited by the stability of the sample.
Below its isoelectric point a protein has a net positive charge and can
therefore adsorb to cation exchangers. Above its pI the protein has a net
negative charge and can be adsorbed to anion exchangers. However, it is only
stable in the range pH 5-8 and so an anion exchanger has to be used.
§ If the sample
components are most stable below their pI’s, a cation exchanger should be used.
§ If they are most
stable above their pI’s, an anion exchanger should be used.
§ If stability is
high over a wide pH range on both sides of pI, either type of ion exchanger can
be used.
Determination of starting conditions
The isoelectric point
The
starting buffer pH is chosen so that substances to be bound to the exchanger
are charged. The starting pH should be at least 1 pH unit above the isoelectric
point for anion exchangers or at least 1 pH unit below the isoelectric point
for cation exchangers to facilitate adequate binding. Substances begin to
dissociate from ion exchangers about 0.5 pH units from their isoelectric points
at ionic strength 0.1 M. If the isoelectric point of the sample is unknown, a
simple test can be performed to determine which starting pH can be used. The
range of pH 5-9 for anion and pH 4-8 for cation exchangers, with 0.5 pH unit
intervals between tubes.
Choice of buffer
As
with the choice of ion exchanger, there are a number of variables which have to
be considered. These include:
1. The choice of
buffer pH and ionic strength.
2. The choice of
buffering substance.
It should be
pointed out, however, that in many applications the optimum separation may be
achieved by choosing conditions so that major and troublesome contaminants are
bound to the exchanger while the substance of interest is eluted during the
wash phase. This procedure is sometimes referred to as “starting state
elution”.
Note:
Concentration of sample does not occur with starting state elution. The highest
ionic strength which permits binding of the selected substances and the lowest
ionic strength that causes their elution should normally be used as the
starting and final ionic strengths in subsequent column experiments (i.e. the
starting and limiting buffers for gradient elution). A third and higher ionic
strength buffer is frequently employed as a wash step before column
regeneration and re-use.
The
required concentration of the start buffer will vary depending on the nature of
the buffering substance. In the majority of cases a starting ionic strength of
at least 10 mM is required to ensure adequate buffering capacity. Salts also
play a role in stabilizing protein structures in solution and so it is
important that the ionic strength should not be so low that protein
denaturation or precipitation occurs.
Choice of buffer substance
If
the buffering ions carry a charge opposite to that of the functional groups of
the ion exchanger they will take part in the ion exchange process and cause
local disturbances in pH. It is preferable, therefore, to use buffering ions
with the same charge sign as the substituent groups on the ion exchanger. There
are of course exceptions to this rule as 338 Ion Exchange Technologies
illustrated by the frequency with which phosphate buffers are cited in the
literature in connection with anion exchangers. In those instances when a
buffering ion which interacts with the ionic groups on the matrix is used,
extra care must be taken to ensure that the system has come to equilibrium
before application of sample.
Column chromatography
Good
results in column chromatography are not solely dependent on the correct choice
of gel media. The design of the column and good packing technique are also
important in realizing the full separation pot Sential of any gel. The material
used in the construction of the column should be chosen to prevent destruction
of labile biological substances and minimize non-specific binding to exposed
surfaces. The bed support should be designed so it is easily exchangeable to
restore column performance whenever contamination and/or blockage in the column
occur. Bed supports made from coarse sintered glass or glass wool cannot be
recommended because they soon become clogged, are difficult to clean and cause artifacts.
The pressure specifications of the column have to match the back-pressure
generated in the packed bed when run at optimal flow rate. This is particularly
important when using high performance media with small bead size. All are easy
to dismantle and reassemble to allow thorough cleaning, which is a particularly
important aspect when handling biological samples. As for most adsorptive, high
selectivity techniques, ion exchange chromatography is normally carried out in
short columns. A typical ion exchange column is packed to a bed height of 5-15
cm. Once the separation parameters have been determined, scale-up is easily
achieved by increasing the column diameter.
Quantity and preparation of ion exchanger
The
amount of ion exchanger required for a given experiment depends on the amount
of sample to be chromatographed and on the available or dynamic capacity of the
ion exchanger for the sample substances. For the best resolution in ion
exchange chromatography, it is not usually advisable to use more than 10-20% of
this capacity, although this value can be exceeded if resolution is adequate.
Preparation of the ion exchanger having chosen the appropriate ion exchanger
and starting buffer it is essential that the exchanger is brought to
equilibrium with start buffer before sample application. To prepare the gel,
the supernatant is decanted and replaced with starting buffer to a ratio of
approximately 75% settled gel to 25% buffer. If large amounts of ion exchangers
are to be equilibrated with a weak buffer, the ion exchanger should first be
equilibrated with a 10 times concentrated buffer solution at the correct pH,
and then with a few volumes of starting buffer.
Sample preparation
The
amount of sample which can be applied to a column depends on the dynamic
capacity of the ion exchanger and the degree of resolution required. For the
best resolution it is not usually advisable to use more than 10-20% of this
capacity. Information on the available capacities for the different exchangers
is given in the relevant product sections. The ionic composition should be the
same as that of the starting buffer. If it is not, it can be changed by gel
filtration on Sephadex G-25 using Desalting Columns, dialysis, diafiltration or
possibly by addition of concentrated start buffer. If the ion exchanger is to
be developed with the starting buffer (isocratic elution), the sample volume is
important and should be limited to between 1 and 5% of the bed volume. If
however, the ion exchanger is to be developed with a gradient, starting
conditions are normally chosen so that all important substances are adsorbed at
the top of the bed. In this case, the sample mass applied is of far greater
importance than the sample volume. This means that large volumes of dilute
solutions, such as pooled fractions from a preceding gel filtration step or a
cell culture supernatant can be applied directly to the ion exchanger without
prior concentration. Ion exchange thus serves as a useful means of
concentrating a sample in addition to fractionating it. If contaminants are to
be adsorbed, and the component of interest is allowed to pass straight through,
then the sample volume is less important than the amount of contaminant which
is present. Under these conditions there will be no concentration of the
purified component, rather some degree of dilution due to diffusion. The
viscosity may limit the quantity of sample that can be applied to a column. A
high sample viscosity causes instability of the zone and an irregular flow
pattern. The critical variable is the viscosity of the sample relative to the
eluent. This corresponds to a protein concentration of approximately 5%.
Approximate relative viscosities can be quickly estimated by comparing emptying
times from a pipette.
Sample load
When
the selectivity parameters have been defined to achieve the most optimal
balance between resolution, capacity, speed and recovery, in ion exchange
chromatography, as for most other adsorption techniques, there are then
basically two alternative routes to follow for optimization of sample load and
flow rate to achieve highest possible productivity in the system. In a typical
capture situation the sample will be applied to the column, non bound substances
will be washed out from the column and the compound of interest will be eluted
from the column with a simple step elution procedure. The difference in eluting
strength, between the different steps will usually be large, i.e. it will be
possible to elute one group of compounds while the others are still retained on
the column. In this mode, the entire bed volume can be utilized for sample
binding and the prime consideration when optimising for highest possible
productivity is to define the highest possible sample load over the shortest
possible sample application time with acceptable loss in yield.
Flow rate
The
maximum flow rate that can be applied in any particular ion exchange
chromatography step will differ between different parts of the chromatographic
cycle. Since low molecular weight substances show high diffusion rates,
i.e. transported rapidly between the
mobile phase and stationary phase, the flow rate during equilibration, washing
and regeneration procedures is limited primarily by the rigidity of the
chromatography media and by system constraints regarding pressure
specification. Larger molecules, i.e. the substances to be separated during the
Chromatographic run, show a lower diffusion rate which will limit the flow rate
that can be applied during sample adsorption and desorption. In a typical
capture situation, the flow rate during sample application has to be controlled
so that the residence time in the column allows for a complete binding without
leakage in the flow through fraction. Maximum flow rate is defined by running
the frontal analysis test (break-through) referred to above at a number of
different flow
rates. Optimal conditions will depend on the requirements for speed and
capacity in the system. If speed, i.e. sample application time, is critical due
to proteolysis or other deterimental effects in the feed material, a higher
flow rate may have to be used on the expense of the binding capacity in terms
of amount of sample that can be applied per volume of media. If speed is not a
big issue, binding capacity can be increased on the expense of flow rate which
will reduce the scale of work in the final production process.
Elution
If
starting conditions are chosen such that only unwanted substances in the sample
are adsorbed, then no change in elution conditions is required since the
substance of interest passes straight through the column. Similarly no changes
are required if sample components are differentially retarded and separated
under starting conditions. This procedure is termed isocratic elution, and the
column is said to be developed under starting conditions. Isocratic elution can
be useful since no gradient apparatus is required for the run and, if all
retarded substances elute, regeneration is not required. Normally, however,
separation and elution are achieved by selectively decreasing the affinity of
the solute molecules for the charged groups on the gel by continuously changing
either buffer pH or ionic strength or possibly both. This procedure is termed
gradient elution.
Change of pH and ionic strength
The
net charge on a molecule depends on pH. Thus altering the pH towards the
isoelectric point of a substance causes it to lose its net charge, desorb, and
elute from the ion exchanger. At low ionic strengths, competition for charged
groups on the ion exchanger is at a minimum and substances are bound strongly.
Increasing the ionic strength increases competition and reduces the interaction
between the ion exchanger and the sample substances, resulting in their elution.
Regeneration
After
each cycle, bound substances must be washed out from the column to restore the
original function of the media. Ion exchange adsorbents can normally be
regenerated after each run by washing with a salt solution until an ionic strength
of about 2 M has been reached. This should remove any substances bound by ionic
forces. The salt should contain the counter-ion to the ion exchanger to
facilitate equilibration. To prevent a slow build up of contaminants on the
column over time, more rigorous cleaning protocols may have to be applied on a
regular basis.
Applications
§ Ion exchange has
proven to be one of the major methods of fractionation of labile biological
substances.
§ In the
development of modern high performance media for purification, ion exchange
chromatography has played a major role in the separation and purification of
biomolecules and contributed significantly to our understanding of biological
processes.
§ Analytical and
preparative applications from the research laboratory. Ion exchange
Chromatography has played a role in the purification of thousands of enzymes,
and using modern matrices with optimized separation conditions gives extremely
high recoveries.
§ Normally the
isoforms of an enzyme have approximately the same molecular weight. This makes
their separation impossible by gel filtration. However, the small differences
in charge properties resulting from altered amino acid composition enable the
separation of isoenzymes using ion exchange chromatography. Ion exchange is
frequently used for the purification of immunoglobulins.
§ Ion exchange
chromatography also has many important applications in the field of industrial
and pilot scale preparations. Many blood products such as albumin and IgG as
well as the products of recombinant DNA technology, such as growth factors and
Pharmaceutically important enzymes are purified using this technique.
Analytical applications of ion exchange chromatography are to be found in
diverse areas such as quality control of purified products or process
monitoring in biotechnology.
§ Other areas of
application include food research where FPLC ion exchange can be used in the
clinical research where ion exchange chromatography has been used in studies
such as the relationship between post-partum depression and -endorphin
secretion and the correlation of a chromatogram of the urine from patients
exhibiting tubular proteinuria, due to acute pyelonephritis, severe burns or
renal transplants, shows distinct peaks corresponding to 2-microglobulin,
retinol binding protein and 1-acid glycoprotein.
GEL FILTERATION CHROMATOGRAPHY
Gel filteration
chromatography is a separation method dependent upon molecular size. The method
is also known as molecular sieve, gel permeation, or molecular exclusion
chromatography.
The advantages are
1. separation of
a labile molecular species.
2.100% solute
recovery
3. excellent
reproducibity
4. comparatively
short time & relatively inexpensive.
5. powerful separation
procedure.
Principle
A
column of gel beads or porous glass granules is allowed to attain equilibrium
with a solvent suitable for the molecules to be separated. If the mixture of
molecules of different size is placed on the top of such as equilibrated
column, the larger molecules pass through the interstitial spaces between the
beads. This is because the pores of the gel have smaller diameter than what is
needed for the large molecules to enter.
Large molecules move down the column
with little resistance. The small molecules enter the pores & are thereby
effectively removed from the stream of the eluting solvent. These molecules are
thus retarded.
The degree of retardation of a
molecule is proportional to the time it spends inside the gel pores, which is a
function of the molecule’s size & the pore diameter.
Types of gels
A gel filtration
medium should possess the following characteristics:
1. The gel material should
be chemically inert.
2. It should preferably
contain vanishingly small number of ionic groups.
3. Gel material should
provide a wide choice of pore & particle sizes.
4. A given gel should have
uniform particle & pore sizes.
5. The gel matrix should
have high mechanical rigidity.
There are five
principal types of media that fulfill the criteria to quite some extent. They
include (i) cross-linked dextrons, (ii) agarose, (iii) polyacrylamide, (iv)
porous glass and silica granules Bio-glass porasil, and (v) polystyrene, other
gels, which have been used for gel filtration include macroreticular polyvinyl
acetate, microparticulate aluminas and silica’s and cellulose packings in bead
form.
Sephadex
For proteins and most of the
bio-molecules, sephadex is by far the most popular of all the gels. When Leuconostoc mesenteroids indulge in
sucrose fermentation, large polymers of glucose are the result. These polymers,
known as dextrans, are used to prepare sephadex. Each of the glucose residues
in the polymer possesses three hydroxyl groups giving the dextran a polar
character. The agent used for cross-linking dextran polymers is epichlorhydrin,
CH2—CHCH2Cl
O
Pore size is
controlled by the molecular weight of the dextran and the amount of
epichlorhydrin used in the preparation. Sephadex gels are insoluble in water
and are stable in bases, weak acids and mild reducing and oxidizing agents. In
addition to water in which they are normally used, sephadex gels will swell in
glycol, dimethylsulfoxide, and formamide.
Polyacrylamide
This very popular medium is produced
by polymerizing acrylamide into bead form. Polyacrylamide gels can be used to
separate molecules of up to 3,00,000daltons. However, the large pored gels used
for such separations lack in mechanical rigidity. These beads also tend to
become compressed in the column. Polyacrylamide is insoluble in water and
common organic solvents and may be used in the pH range of 2-11. these gels
however, are unstable to bases due to hydrolysis of amide groups.
Agarose
Agarose gels are produced from agar.
They are linear polysaccharides of alternating residues of D-galactose and
3.6-anhydro-L-galactose units, and owe their gelling properties to hydrogen
bonding of both inter and intra molecular type. These gels are hydrophilic and
are almost completely free of charged groups. They are therefore almost
completely inert to the being separated. Thus, wide use has been made of these
gels in the study of viruses, nucleic acids, and polysaccharides. Freezing
temperatures and temperatures higher than 30oC cause alterations in
the gel structure. The chromatography has therefore to be performed between 0o
and 30oC.
Styragel
For completely non-aqueous
separations, a gel that will swell in an organic solvent is required. Styragel
provides this option. It is a rigid linked polystyrene gel which can be
prepared in a range of different porosities. The gel structure is unaffected by
temperatures as high as 150oC. The gel can be used with such
solvents as tetrahydrofuran, benzene, trichlorobenzene, perchloroethylene,
cresol, dimethylsulphoxide, chloroform, carbon tetrachloride and others.
Controlled pore glass beads
These fine glass spheres are
manufactured from borosilicate glass to contain large numbesr of pores within a
very narrow size distribution. High flow rates are permitted by their total
rigidity. The glass beads are treated with hexamethyldisilazane. These glass
spheres have a molecular exclusion limit ranging from 3000 to 9 million
Daltons.
Technique
Gel permeation chromatography can be
performed either by column or thin layer chromatographic techniques.
Column preparation
Prior to use, the gel must be
converted to the swollen form. This may be done by allowing a known weight of
the gel to swell either in water or in a weak salt solution. Porous glass
granules, on the other hand, need not be hydrated at all. The gel bed is
supported in the column on a glass wool plug or nylon net and the previously
swollen gel is added in the form of slurry and allowed to settle. Air bubbles
must be removed by connecting the column to a vacuum pump and the level of the
liquid must never be allowed to go lower than the top of the bed. Sample is
applied in a manner indicated previously. The volume of sample that should be
applied varies as per the column size and the type of the gel used. The eluant
is steadily added and the effluent collected in various fractions to be
analyzed. Knowledge of the effluent volume of a particular compound is useful
for the calculation of its distribution coefficient, which might be useful for
molecular weight determination. Elution is usually carried out under a constant
hydrostatic pressure head to achieve a constant flow rate.
Detection
The common detection methods include
collecting and analyzing fractions and continuous methods with flow cells in
which ultraviolet absorption, refractive index or radioactivity is measured.
Thin layer gel chromatography
Determann and Johanson and Rymo (1962)
showed that gel permeation chromatography could be conducted using thin layer
of gel. For clinical use, thin layer gel filtration is ideal since very small
sample volume is required for this technique. In this technique, a layer of
hydrated gel is applied to the plate. The plate is not dried at all and is
placed in an airtight container at an angle of 20o. The plate is
connected to reservoirs at either end by means of filter paper bridges.
Equilibrium must be carried out for at least 12 hours. Such equilibrium serves
to normalize the ratio between the stationary and mobile phase volumes. The
sample may be applied either as a spot or as a band. The plate may then be
developed for a suitable time and the separated components detected by suitable
means.
TLG is used mainly for the study of
hydrophilic substances such as proteins, peptides, nucleic acid, enzymes etc.
TLG has numerous applications in
clinical immunology & immunochemistry.
Applications
§ Gel permeation
chromatography is chiefly used for the purpose of separation of biological
molecules leading to their ultimate purification.
§ Proteins,
enzymes, hormones, antibodies, nucleicacids, polysaccharides, & even
viruses. Gel filtration used for separation of such low molecular weight
compounds as amino acids, small peptides, & oligonucleotides. It is also
the most satisfactory method for separating DNA & RNA (from bacteria,
usually Gram positive) from the invariable contaminants, the teichoic acids.
§ One of the
advantages of this method of desalting is that the macromolecules are eluted
with essentially no dilution.
§ Gel permeation
chromatography is used for determination of molecular weight of macromolecules.
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