CHROMATOGRAPHY

TECHNIQUES
BY SREEJALA.T (B1218002)
What is chromatography?
• Chromatography is a laboratory technique for the separation of a mixture. The mixture is
dissolved in a fluid called the mobile phase, which carries it through a structure holding
another material called the stationary phase. The various constituents of the mixture travel
at different speeds, causing them to separate. 
• Chromatography may be preparative or analytical.
The purpose of preparative chromatography is to separate the components of a
mixture for later use, and is thus a form of purification.
Analytical chromatography is done normally with smaller amounts of material and is
for establishing the presence or measuring the relative proportions of analytes in a mixture.
• Chromatography was first employed in Russia by the Italian-born scientist Mikhail Tsvet in
1900.(He worked for separation of plant pigments)
• New types of chromatography developed during the 1930s and 1940s made the technique
useful for many separation process.
• Chromatography technique developed substantially as a result of the work of 
Archer John Porter Martin and Richard Laurence Millington Synge during the 1940s and 1950s,
for which they won the 1952 Nobel Prize in Chemistry. They established the principles and basic
techniques of partition chromatography, and their work encouraged the rapid development of
several chromatographic methods: paper chromatography, gas chromatography, HPLC etc.
• A generalized definition was provided by a special committee of the International Union of
Pure and Applied Chemistry which regards chromatography as ‘ a method, used primarily for
separation of the components of a sample, in which the components are distributed between
two phases, one of which is stationary while the other moves. The stationary phase may be a
solid, liquid supported on a solid, or a gel. The stationary phase may be packed in a column,
spread as a layer, or distributed as a film. The mobile phase may be gaseous or liquid.'
• Analyte : substance to be separated during chromatography. It is also normally what is needed
from the mixture.
• Chromatograph : equipment that enables a sophisticated separation
• Chromatography :  physical method of separation between two phases, one stationary (stationary
phase), the other (the mobile phase)
•  eluate, eluent, eluite : eluate is the mobile phase leaving the column. This is also called effluent.
 eluent is the solvent that carries the analyte. eluite is the analyte, the eluted solute.
• eluotropic series is a list of solvents ranked according to their eluting power
•  retention time : time it takes for a particular analyte to pass through the system
 Partition chromatography
• Partition chromatography is based on differences in retention factor, k, and distribution
coefficients, Kd, of the analytes using liquid stationary and mobile phases.
• In this; a special paper called chromatography paper is used that contains liquid trapped in it, which
acts as a stationary phase.
• It can be subdivided into liquid–liquid chromatography, in which the liquid stationary phase is
attached to a supporting matrix by purely physical means, and bonded-phase liquid
chromatography, in which the stationary phase is covalently attached to the matrix.
• An example of liquid–liquid chromatography : water stationary phase is supported by a cellulose,
starch or silica matrix. An organosilane, such as octadecyl, is the most accepted type of bonded
phase. 
 Paper chromatography:

• • Paper is made up of highly pure

The paper used for chromatography can be common
filter paper (Whatmann #1 is popular) cellulose impregnated with alumina,
silica, resins etc.
• A small, concentrated spot of the sample is applied to
chromatography paper about 1cm from the base,
usually using a capillary tube for maximum precision.
This sample is absorbed onto the paper and forms
interactions with it.
• The paper is placed inside the solvent system (ethanol
or water). The paper is placed such that the solvent
level is below the spot. The paper is placed in a sealed
container.
• The solvent rises up the paper and flows over the spot.
• Used for separation of amino acids, sugars, sugar
derivatives etc.
• The developed paper strip is called chromatogram. The colourless compounds may
be observed either under uv light.
• The Rf is calculated by : distance travelled by solute
distance travelled by solvent
if Rf = 1; then the solution has no affinity for stationary phase and traveles with
the solvent front.
if Rf = 0; the then solute remains in stationary phase and thus is immobile.
• The more soluble a molecule is; the higher it will travel. (If the molecule is polar and
the solvent is non polar the it is insoluble or less soluble and travels less.)
• TYPES:
a) Ascending: sample and solvent move up. Used for both organic and inorganic
substances. Most polar substance will be at the bottom of the paper.
b) Descending: sample and solvent move down based on gravity. Most polar will be
at the top of the paper.
c) Radial
d) Two-dimentional
 Advantages and disadvantages:

• It is simple, easy, rapid and efficient.

• Can be applied to even microgram quantities of the sample.
• Used for separation of amino acids, oligopeptides, sugars, glycosides, purines and
pyrimidines, steroids, vitamins etc.
• Separation time : 14-16 hrs.
• Not preferred for protein separation because they are not soluble in many solvent
systems and may also be denatured by them.
• It is inferior to TLC in resolving power.
• Overloading, fluctuation in temperature near chamber opening, fast movement of
sample etc. effect the separation.
significance
• Determination of plant pigments, detection of the pesticides or insecticides in food,
monitoring organic reactions etc.
• Separation time is very less when compared to paper chromatography. It takes only
3-4 hours.
• TLC has the advantage that the corrosive reagents like sulphuric acid can also be used
which pose a limitation for the paper chromatography.
• It can analyze multiple samples in a single run.
 • The column is made up of stainless steel
wire coiled up to fit in the column and is
Gas liquid chromatography finely packed with diatomaceous earth.
• Involves a sample being vapourised and This is coated with hot boiling liquid
injected onto the head of the usually waxy polymer.
chromatographic column. The sample is
transported through the column by the
flow of inert, gaseous mobile phase. The
column itself contains a liquid stationary
phase which is adsorbed onto the
surface of an inert solid.
• The injector is contained in an oven to
maintain the temperature of the
vaporized sample. The carrier gas is
usually helium.
• Column temperature: 50-250 oc . It is cooler
than the injector. The column starts with an
initial low temperature at the start phase and
later made cooler.
• One of the 3 things happen when a sample is
injected:
a) It may condense on the stationary phase.
b) It may dissolve in the liquid on the surface of
stationary phase.
c) It may remain in the gas phase.
• Retention time is the time taken by the
sample to come out of the column. The more
the compound is soluble in the liquid phase
the more time out takes to come out of the
column.
Adsorption chromatography
• In this technique the separation is based on differences in adsorption at the surface of the solid
stationary medium.
• The adsorbents such as silica gel, charcoal powder and calcium hydroxyapatite are packed into a
column in a glass tube. This serves as the stationary phase. The sample mixture in a solvent is
loaded on this column. The individual components get differentially adsorbed onto the
adsorbent.
• The individual components get differentially adsorbed onto the adsorbent. The individual
compounds come out of the column at different rates which may be separately collected and
identified. An automated column chromatography apparatus- fraction collector is frequently
used now-a-days.
Column chromatography

•  It is used to obtain pure chemical compounds from a mixture of compounds on a scale
from micrograms up to kilograms using large industrial columns. The classical
preparative chromatography column is a glass tube with a diameter from 5 to 50 mm
and a height of 50 cm to 1 m with a tap at the bottom.
• Slurry is prepared of the eluent with the stationary phase powder and then carefully
poured into the column. Care must be taken to avoid air bubbles. A solution of the
organic material is pipetted on top of the stationary phase.
• This layer is usually topped with a small layer of sand or with cotton or glass wool to
protect the shape of the organic layer from the velocity of newly added eluent. Eluent is
slowly passed through the column to advance the organic material. Often a spherical
eluent reservoir or an eluent-filled and stoppered separating funnel is put on top of the
column.
• Separation is based on interaction of the
absorbate with the absorbent.
• The absorbent is the stationary phase.
• The solute binds to the absorbent by Vander
walls interactions.
• Typical absorbents are silica based resins,
alumina etc. that are very rigid.
• Based on elution there are 2 techniques:
isocratic: same solvent composition
gradient :solvent system is changed
constantly in increasing polarity strength.eg.
First benzene, then chloroform, then ethyl
acetate etc.
• Some of the factors like column dimention, particle size of column, activity of the
absorbent, temperature of column etc effect the efficiency.
• It is used for separation of mixed compounds, purification process, estimation and
isolation of compounds, separation of diastereomers.
• The advantages are that any mixture in any quantity can be separated and automation is
possible.
• The disadvantages is that it is time consuming, more amount of mobile phase is
required, and is costlier and complicated.
Thin layer chromatography

• Used for separation of non-volatile mixtures.

• Performed on a sheet of plastic, glass, or aluminium foil which is coated with a thin layer of
absorbent usually silica.
• The absorbent layer is the ‘stationary phase’.
• The solvent system used is generally hexane or ethyl acetate.
• The procedure and principle is similar to that of a paper chromatography, with a faster run
and higher separation.
• Used to monitor the progress of a reaction, identify compounds present in a given
mixture, and determine the purity of a substance. Specific examples of these applications
include: analyzing  fatty acids, detection of pesticides or insecticides in food and water,
analyzing the dye composition of fibers in forensics, assaying the radiochemical purity of 
radiopharmaceuticals, or identification of medicinal plants and their constituents.
• TLC plates are prepared by mixing the
adsorbent, such as silica gel, with a small
amount of inert binder like calcium sulfate
(gypsum) and water. This mixture is spread
as a thick slurry on an unreactive carrier
sheet, usually glass, thick aluminum foil, or
plastic. The resultant plate is dried
and activated by heating in an oven for
thirty minutes at 110 °C. The thickness of
the absorbent layer is typically around 0.1
– 0.25 mm for analytical purposes and
around 0.5 – 2.0 mm for preparative TLC.
• when the stationary phase is polar and the
mobile phase is non-polar, the method
is normal-phase as opposed to reverse-
phase

• A number of enhancements can be made
to the original method to automate the
different steps, to increase the resolution
achieved with TLC and to allow more
accurate quantitative analysis. This
method is referred to as HPTLC, or "high-
performance TLC". HPTLC typically uses
thinner layers of stationary phase and
smaller sample volumes, thus reducing the
loss of resolution due to diffusion.
• The molecules are separated based on the
functional group present on the analyte
and stationary phase, nature and
composition of mobile phase, etc.
• K’ is the capacity factor. It is the ratio of
the quantities of solute distributed
between the mobile and stationary
phases, or the ratio of the respective
times the substance spends in the two
phases
k’= retention time in stationary phase
retention time in mobile phase
k’= 1-Rf
Rf
• Flow constant B = Zf 2
t
Zf is the distance between solvent front and
the solvent level and t is development time.
• Some factors like nature of absorbent, mobile phase, activity and thickness of layer,
temperature, dipping zone, etc effect separation rate.

• It is used for separation of all natural products like amino acids, glycols, alkaloids,
peptides, antibiotics, vitamins,etc.

• HPTLC: steps: prewashing  conditioning(at1200c 15mins)  sample application 

solvent system  detector  chromatogram.
Ion exchange chromatography
• Ion exchange chromatography involves the separation of ionizable molecules based
on their total charge. This technique enables the separation of similar types of
molecules that would be difficult to separate by other techniques because the charge
carried by the molecule of interest can be readily manipulated by changing buffer pH.
• It can be used for almost any kind of charged molecule including large proteins, small
nucleotides and amino acids.
• The solution to be injected is usually called a sample, and the individually separated
components are called analytes.
• However, ion chromatography must be done in conditions that are one unit away from
the isoelectric point of a protein.
• on exchange chromatography is commonly used to separate charged biological
molecules such as proteins, peptides, amino acids, or nucleotides
• Types :
a)anion exchanger ;
b)cation exchanger

• Buffer: Anion exchanger — 0.5–1.5 pH units greater than the pI of the protein of interest

Cation exchanger — 0.5–1.5 pH units less than the pI of the protein of interest
In a buffer with a pH greater than the pI of the protein of interest, the protein will carry a
net negative charge; therefore, a positively charged anion exchange resin is chosen to
capture this protein. For example, if an anion exchange resin is chosen, all proteins that are
negatively charged at the loading buffer pH will bind to the positively charged column
resin. 
example: for cation exchanger: phosphate buffer of ph: 6.7-7.6 is used and for an anionic
exchanger; tris buffer of ph:7.5-8 is used.
• Cation exchange chromatography retains positively charged cations because the
stationary phase displays a negatively charged functional group:
R-X-C+ + M+B-  R-X-M+ + C+ + B-
• Anion exchange chromatography retains anions using positively charged functional
group
• Resins: for anion exchanger; DEAE cellulose, DEAE sephadex etc.
For cation exchanger; CM-sephadex gel, CM-cellulose etc.
• Some ion exchanger matrix must be activated. This can be done by rehydrating the dry/
powder form
If the charged groups on the ion exchanger are titratable, e.g. secondary or tertiary amines as
DEAE (diethylaminoethyl), the ion exchanger is said to be ‘weak’ whereas if the charge of the
ion exchanger is independent of pH over the range commonly used, as QAE (quaternary
aminoethyl), the exchanger is said to be ‘strong’.
• Anion-exchange chromatography
Acetate < formate < chloride < bromide < sulphate < citrate
• Cation-exchange chromatography
Lithium < sodium < ammonium < potassium < magnesium < calcium
Applications
• Protein purification, to study protein-DNA interactions, softening of water, prepare
de-ionized water, purification of rare earth metals from nuclear wastes, concentrating
a sample, separation of lanthanides and organometallics, sugar separation, amino acid
separation etc.
• The major disadvantage is column efficiency is less and stability and reusability is less
after repeated uses.
Hydrophobic Interaction Chromatography
• It is similar to an ion exchange except that the adsorption is based on interaction of
hydrophobic compounds.
• This technique was initially termed as ‘salting out’.
• The sample containing hydrophilic and hydrophobic regions (aminoacids) are applied to
HIC column.
• The proteins in the cell are shielded with a water molecule forming a hydrated shell.
• The salt in buffer reduces the solvation of sample solutes. As solvation decreases, the
hydrophobic regions that are exposed get adsorbed by the media.
• The more hydrophobic, the less salt is needed to promote binding.
• The elution is done by adding a buffer containing salt in very small amount.
• Some of the salts used are :
ammonium sulfate, sodium
sulfate, sodium chloride,
potassium chloride,
ammonium acetate etc.
• The ligands used in HIC has
Alkyl or aryl group
Gel filtration
• This is extremely useful in separating ribosomes, viruses, nucleic acids and proteins
depending on their particle sizes, shape and molecular weight.
• This chromatography distributes the protein or analyte, based on their size by passing
through a porous beads. The column is packed with the beads containing pores to
allow entry of molecules based on their sizes.
• This technique is also referred to as molecular exclusion chromatography.
• The choice of matrix for the gel filtration depends on the mol.wt and preassure limit of
the equipment.
• It is poured into the glass tube and allow the beads to settle without trapping air bubble
within the column.
• The sample is prepared in the mobile phase and it should be free of suspended particle
to avoid clogging of the column. The most recommended method to apply the sample
is to inject the sample with a syringe.
• The column is then equilibrated with mobile phase to regenerate the column. The
column can be store at 400 C in the presence of 20% alcohol containing 0.05% sodium
azide.
• The molecular weight and size of a protein is related to the shape of the molecule and
the relationship between molecular weight (M) and radius
Determination of native molecular weight of a protein using gel filtration
chromatography:
• The molecular weight and size of a protein is related to the shape of the molecule and the
relationship between molecular weight (M) and radius
• R ∝ Ma
• here “a” is a constant and it depends on shape of the molecule, a=1 for Rod, a=0.5 for coils
and a=0.33 for spherical molecules.
• Vt=Vg+Vi+Vo Vg is the volume of gel matrix, Vi is the pore volume and Vo is the void
volume. (Ve) the elution volume is related to the void volume and the distribution
coefficient Kd as given below
Ve=Vo+KdVi Kd= (ve-vo)/ vt.
Analyte with 1.Kd=0, or Ve=Vo, these analytes will be completely excluded from the
column. 2. Analyte with Kd=1 or Ve=Vo+Vi, these analytes will be completely in the pore of
the column. 3. Analyte with Kd>1, in this situation analyte will adsorb to the column matrix.
• For analytes with two different relative masses and kd values; k’d and k’’d;
ve = (k’d-k’’d)vi
• A plot of Kd versus log mol wt is given in Figure and it will allow us to calculate the
molecular weight of the unknown analyte.
• Study protein-ligand interaction,
protein folding, used for desalting,
used for quality testing, purification,
Sugars proteins and peptides separation,
• Resolution can be increased by reducing sample volume, smaller gel beads usage,
connecting the column in series, and reducing the flow rate.

• some of the general uses are desalting of protein samples, mol.wt. estimation of
proteins, protein fractionation.

• This method has short analysis time, well defined separation, good sensitivity, little
amount of mobile phase is required, flow rate can be set.
Affinity chromatography
• The principle of affinity chromatography is based on the property of specific and
non-covalent binding of proteins to other molecules, referred to as substrates or
cofactors.
• The affinity chromatography works on the principle of mutual recognition forces
between a ligand and receptor. (electrostatic, hydrogen bonding, vander waal
interaction). A mutual interaction between a ligand (L) and receptor (R) forms ligand-
receptor complex (RL) with a dissociation constant Kd, which is expressed as follows
R + L ⇋ RL
𝐊𝐝 = [𝐑][𝐋]/ [𝐑𝐋]
• The desired protein captured by the ligands, some reagents that can break protein
ligand interactions can also be employed for the separation by elution.
Importance
• Affinity chromatography is useful for the purification of enzymes, vitamins, nucleic
acids, drugs , hormone receptors, antibodies etc.
• Highly specific, reproducible, easy to perform, high purity yield.
• Bio-affinity chromatography- In this type of affinity chromatography, biomolecules are
used as receptor present on matrix and it exploit the biological affinity phenomenon such
as antibody-antigen. In addition, enzyme-substrate or enzymeinhibitor is also belong to
this class. Ex. GST-Glutathione.
• Pseudo-affinity chromatography-In this affinity chromatography, a non-biological
molecule is used as receptor on matrix to exploit the separation and purification of
biomolecules. There are two specific example to this class:
a) Dye-affinity chromatography-In this method, matrix is coupled to the reactive dye and
the matrix bound dye has specificity towards a particular enzyme. For ex. Cibacron Blue
F3G-A dye coupled to the dextran matrix has strong affinity towards dehydrogenases.
b) Metal-affinity chromatography-In this method, transition metals such as Fe2+, Ni2+ or
Zn2+ is coupled to the matrix and the matrix bound metal form multidentate complex
with protein containing poly-his tag (6x His). The affinity of protein for matrix bound
metal is different and these differences are been exploited in metal affinity
chromatography to purify the protein.
• Covalent chromatography- The matrix in covalent chromatography has immobilized
thio group which forms covalent linkage with the free thiol group containing protein
present in the mixture (Figure 34.3). After a washing step to remove non-specifically
bound protein, a mobile phase containing compound with reducing thio group is passed
to elute the bound protein. The thio group containing compound present in mobile phase
breaks the disulphide bond between protein and matrix thio group to release the protein
in the mobile phase
• eg of some matrices: heparin is used as receptor for DNA binding site, and lectin is a
receptor for glycoprotein
• Affinity column material packed in a column and equilibrate with a buffer containing
high salt (0.5M NaCl) to reduce the non-specific interaction of protein with the
analyte.
• -The sample is prepared in the mobile phase and it should be free of suspended
particle to avoid clogging of the column. The most recommended method to apply the
sample is to inject the sample with a syringe.
• After the elution of analyte, affinity column requires a regeneration step to use next
time. column is washed with 6M urea or guanidine hydrochloride to remove all non-
specifically bound protein. The column is then equiliberated with mobile phase to
regenerate the column. The column can be store at 40 C in the presence of 20% alchol
containing 0.05% sodium azide.
• Used for purification of biomolecules, study of protein protein interaction, to
perform enzymatic assay, immune purification etc.
HPLC
• High Performance Liquid Chromatography (HPLC) is a form of column chromatography that
pumps a sample mixture or analyte in a solvent (known as the mobile phase) at high pressure
through a column with chromatographic packing material (stationary phase).
• The sample is carried by a moving carrier gas stream of helium or nitrogen. HPLC has the ability
to separate, and identify compounds that are present in any sample that can be dissolved in a
liquid in trace concentrations .Because of this versatility, HPLC is used in a variety of industrial
and scientific applications, such as pharmaceutical, environmental, forensics, and chemicals.
• Although can be used with any type of chromatographic method, the major separation modes of
HPLC are:
• 1.  Size exclusion chromatography
• 2.  Ion exchange chromatography
• 3.  Normal phase and adsorption chromatography
• 4.  Reversed-phase chromatography
• High Performance Liquid Chromatography (HPLC) is a form of column chromatography that
pumps a sample mixture or analyte in a solvent (known as the mobile phase) at high pressure
through a column with chromatographic packing material (stationary phase). The sample is
carried by a moving carrier gas stream of helium or nitrogen. HPLC has the ability to separate,
and identify compounds that are present in any sample that can be dissolved in a liquid in trace
concentrations as low as parts per trillion. Because of this versatility, HPLC is used in a variety of
industrial and scientific applications, such as pharmaceutical, environmental, forensics, and
chemicals.
• Pump force the liquid into columns and the
injector injects the sample. The column is made
Up of homogenous small particles
(smaller particles; more the surface area.)
• The major advantage is speed, high resolution, sensitivity, accuracy, column can be
reused without repacking, easy automation and adaptability to large scale.

• The disadvantage is the cost and complexity.

• Used for drug analysis, to purify plant extracts , separate and purify proteins, to
separate thermally unstable compounds like trinitrotoluene etc.
Modes of chromatography
• Batch chromatography: column packed with resin is used. In this form different buffers
are added in a series of steps for the better separation of the compounds.
• Multicolumn chromatography: (stimulated moving bed) allow for semi-continuous
operation of a chromatography process. This is highly used in other industries but are at
beginning to be considered for biopharmaceutical process.
• Expanded bed chromatography: feed material is pumped into the bottom of the
column, forcing the particles to get fluidized (expansion of column) . This allows crude
materials like unclarified cell supernatants to be directly loaded into the column. The
benefit of this method is that clarification and initial purification can be done in a single
step.
Resolution
• A resolution factor of 1.5 yields, in practice, a complete separation of two solutes
reference
• http://
www.biologydiscussion.com/biochemistry/chromatography-techniques/top-12-types-of-chromatographic-techniques-bi
ochemistry/12730
• www.slideshare.net
• Biochemistry and molecular biology by Wilson and walker.
• nptel.ac.in
• Handbook of process chromatography
• www.bio-rad.com
• en.wikipedia.org
• http://
hiq.linde-gas.com/en/analytical_methods/liquid_chromatography/high_performance_liquid_chromatography.html
• https://cstools.asme.org/csconnect/FileUpload.cfm?View=yes&ID=49597
• Chromatography for the Bioprocessing Industry - ASME
• https://cstools.asme.org/csconnect/FileUpload.cfm?View=yes&ID=49597