Chromatography:

Chromatography is by far the most useful general group of techniques available for the separation of
closely related compounds in a mixture. Here the separation is effected by differences in the equilibrium
distribution of the components between two immiscible phases, viz., the stationary and the mobile
phases. These differences in the equilibrium distribution are a result of nature and degree of interaction
of the components with these two phases. The stationary phase is a porous medium like silica or
alumina, through which the sample mixture percolates under the influence of a moving solvent (the
mobile phase). There are a number of interactions between the sample and the stationary phase and
these have been well exploited to effect the separation of compounds.

Paper Chromatography
Paper chromatography has proved to be very successful in the analysis of chemical compound and lipid
sample in particular.

Nature of the paper:

The paper commonly used consists of highly purified cellulose. Cellulose, a homopolysaccharide of
glucose. Contains several thousand anhydro-glucose units-linked through oxygen atoms. The paper
exhibits weak ion exchange and adsorptive properties. Modified forms of paper have been produced in
which the paper has been impregnated with alumina, silica gel, and ion-exchange resin etc.

The chemical composition of whatmann filter paper no: 1 is: a-cellulose (98 to 99%), b-cellulose (0.3 to
1%), Pentosans (0.4 to 0.8%), Ash (0.07 to 0.1%) & ether soluble matter (0.015 to 0.1%).

Apparatus:

The apparatus required for paper chromatography are

1) Support for paper

2) Solvent trough

3) Airtight chamber

4) Whattmann filter paper number 1

5) Capillary tubes

6) Samples – Amino acids (or) Pigments

7) Solvents

8) Platinum loop

Paper development
There are two main techniques, which may be employed for the development of paper Chromatograms.

1) Ascending techniques

2) Descending techniques

3) Radial development

4) Two-dimensional chromatography

1) Ascending techniques:

The filter paper is then dried and equilibrated by putting it

into on airtight cylindrical jar, which contains an aqueous
solution of a solvent. The most widely applicable solvent
mixture is n-butanol: acetic acid: Water (4:1:5), which is
abbreviated as BAW.

The sheet of paper is supported on a frame with the button

edge in contact with a trough with solvent. The arrangement
is contained in an airtight tank lined with paper saturated
with the solvent to prove a constant atmosphere and separations are carried out in a constant
temperature room. Thus, the solvent will ascend into the paper this process is, therefore, termed
“Ascending Chromatography”

2) Descending techniques:

The end of the filter paper may be put into the solvent mixture contained in a narrow trough mounted
near the top of the container. In this chromatography, the solvent will descend into the paper and this
process is then termed “Descending Chromatography”.

This method is convenient for compounds, which have similar

Rf values since the solvent drips off the bottom of the paper,
thus giving a wider separation.

3) Two dimensional chromatography (3D):

The mixture is separated then the first solvent, which should

be volatile: then after drying, the paper is turned through
900 and separation is carried out in the second solvent.

After locating the migrated unknown sample along with standard known sample, a map is obtained and
comparing their position with a map of known compounds can identify compounds.

Locating the compounds:

Strip is removed when the solvent has migrated over most of the available space. The distance to which
the solvent has run is marked. In most cases, the completed Chromatogram is colorless with no
indication of the presence of any compounds. Such a chromatogram is said as “Undeveloped” for
locating the various compounds. The filter paper strip is first dried, then sprayed with 0.5% Ninhydrin in
acetone and at least heated for a few minutes at 80 to 1000 C. the reaction occurs and the colored spots
appear at the sites of the amino acids, such as Chromatogram is now called “Developed”.

In paper chromatography, the stationary cellulose phase is more polar than the mobile organic phase.

Identifying the compounds:

The ratio of the distance travelled by a component (i.e. amino acid) to that travelled by the solvent
front, both measured from the marked point of the application of the mixture, is called the “Resolution
front (Rf)” value for that component.

Distance from origin run by the compound

Rf = -----------------------------------------------------------------------------

Distance from origin run by the solvent

Detection:

The filter paper strip may be sprayed with ninhydrin and heated so that the colored spots indicating the
location of amino acids may develop. The color densities of these spots may be measured with a
recording transmittance (or) reflectance photometer device.

Ninhydrin test:

Amines (including α-amino acids) react with ninhydrin to give a

coloured product.

It can be used qualitatively (e.g. for chromatographic visualisation) or

quantitatively (e.g. for peptide sequencing).

The α-amino acids typically give a blue-purple product.

Proline, a secondary amine, gives a yellow-orange product.

The test is sensitive enough that ninhydrin can be used for the visualisation of fingerprints.

Applications:

By using this technique

1) To check the control of purity of pharmaceuticals,

2) To the detection of adulterants,

3) To detect the contaminants in foods and drinks,

4) To the study of ripening and fermentation,

5) To the detection of drugs and dopes in animals & humans

6) To the analysis of cosmetics

7) To the analysis of the reaction mixtures in biochemical labs.

 AFFINITY CHROMATOGRAPHY
It is mainly based on the biological affinity (or) biological specificity. This technique mainly requires
previous knowledge of the molecule to be separated a specific ligand will only attach with a specific
molecule.

The materials to be isolated are capable of binding reversibly to a specific ligand i.e., attached to an
insoluble matrix.

Affinity chromatography offers high selectivity, resolution, and capacity in most protein purification
schemes. It is the only technique that has the advantage of utilizing a protein's biological structure or
function for purification. As a result, purifications that would otherwise be time consuming and
complicated, can often be easily achieved with affinity chromatography.

Supporting matrix:

1) Characteristics of Matrix:

The matrix should be inert to other molecules to minimize non-specific adsorption.

It should possess good flow properties.

It should be chemically and mechanically stable at varying pH, ionic strength and denaturating
conditions employed for binding and elution.

It should contain large numbers of suitable chemical groups for ligand attachment.

It should be highly porous a large surface area for attachment of the ligand and allows interaction of the
desired macromolecule with the immobilized ligand.

2) Types:

The particles, which are uniform, spherical and rigid, are used. The most commonly used ones are

a) Agarose b) Polyacrylamide c) Controlled glass beads

a) Agarose:

The agarose beads have most desired features as mentioned above. But it has some advantage when
use the denaturant solution for elution, which have a susceptibility to contraction.

b) Polyacrylamide:

The polyacrylamide bead lacks porosity. This undesirable trait is heightened even further when they are
substituted by ligands.

c) Controlled porosity glass beads:

This bead provides mechanical rigidity and chemical inertness in addition to providing very good flow
rates. High degree of nonspecific protein adsorption is the most serious drawback to these beads, which
avoid to some extent by treatment with “Hexamethyldisilazane”.

3) Ligand selection:

The selection of ligand should have two most important requirements:

Ligand interaction should be less with desired macromolecules.

The ligand should possess functional groups that can be modified to form covalent linkage with the
supporting matrix.

4) Ligand attachment:

Covalent coupling of the ligand to the supporting matrix involve the following steps:

Activation of the matrix functional groups

Covalent attachment of the ligand to the activated functional groups.

i) Activation of the matrix functional groups:

The most common method of activation of polysaccharide supports (agarose) involves treatment with
“CNBr” at alkaline pH (pH=11.0). Usually 300 mg of powdered cyanogens bromide used per ml of packed
gel gives the maximum substitution. The reaction is exothermic and maintains the temperature constant
at 200C at all times. To maintain temperature the pH at 11, the mixture is continuously stirred and an
electrode dipped into it at all times. The pH is maintained by the addition of 2M NaOH. The activated
suspension is now washed with about 20 times the gel volume with a buffer (buffers –Tris, Ammonium
acetate, Glycine) at a pH of 9.5 to 10. Usually just 10 to 15 minutes are required for the reaction to be
completed. Sodium bicarbonate and borate buffers are the usual choice.

ii) Covalent attachment of the ligand to the activated functional groups:

Coupling of amino – containing ligand to CNBr activated support is normally carried out by suspending
the support and the ligand in a basic buffer solution at pH (0.25 M NaHCO3, pH-9.0). The suspension
stirred overnight in a cold room. During this time the ligand is covalently attached to the support
medium.

After the reaction is over, the matrix should wash with 0.1 M solution of pH 9.0 glycine buffer, the
solution destructs the any extra-activated groups.

The number of ligand group bound is usually expressed in terms of capacity per ml of packed matrix
rather than in terms of its dry weight.

Some group specific ligands

5) The ARM:

To avoid the encounter steric repulsion between ligand and activated groups of matrix with
macromolecule, which is used to introduce a spacer between the activated groups of the support and
the ligand. This space is known as “ARM”. The ligand projects out the macromolecule to prevent
repulsion.

E.g.:

1)Hexamethylene, 3,3’-diamino propylamine

2) 1,6-diamino hexane

3) 6-amino-hexanoic acid

4) 1,4-bis-(2,3-epoxypropoxy) butane

These spacer arms have two different functional groups; one to react with the functional groups of the
matrix and the other is to react with ligand.

In organo synthetic procedures “Succinic anhydride” and a “Water soluble carbodiimide” are using to
attaches the ligand.

Practical Procedure:

It is also carried out in the column. In this matrix and ligands are used. Prior to use, the gel (or) matrix
must be converted to the swollen form, done by allowing a known weight of the gel to swell either in
water (or) in a weak salt solution. The greater the porosity, the more will be the time required to reach

equilibrium.
Agarose reacts with cyanogens-Bromide and forms activated complex. Then this activated complex
reacts with epoxide and forms agarose cyanogens bromide-epoxide complex. Epoxide is a spacer arm
attaches the matrix with ligands.

The agarose-CNBr-epoxide complex is filled in column. Then the specific ligand is added. Now the ligand
react with the spacer arm and attaches to it. Then the sample is applied to the column. The specific
ligand attaches with specific molecules. The remaining material will comedown the column, the
attached molecule can be obtained by suitable buffer. The buffer supplement on the gel bed, it
encourages adsorption of the desired molecule the buffer chosen must be supplemented with any
cofactors (e.g.: Metal ions) required for “Ligand-Macromolecule interaction”. The buffer should also
possess a high ionic strength so as to minimize non-specific polyelectrolyte adsorption onto charged
groups in the ligand.

Applications:

 The technique has been used to purify a large variety of macromolecule such as enzymes,
Immunoglobulins, membrane receptors, Nucleic acids and even polysaccharides.
 By using affinity chromatography, Whole cells have been purify include fat cells, T and B-
lymphocytes, Spleen cells, Lymph node cells, Oocytes and chick embryo neural cells.
 Metal chelate affinity chromatography is the logical extension technique. Same molecular
weight protein can be separated by this technique by using the metal ion containing matrix by
chelation, because of their difference in their metal binding ability with proteins.
 By using the “Magnetic gel beads affinity chromatography”, immunoglobulin negative
thymocytes and neuroblastoma cells have been purified by this method. The magnetic gel
beads, usually polyacrylamide (or) agarose have a core made up of Fe3 O4 (Magnetite) and are
chemically coupled to a protein ligand.
 Immobilized enzymes (Solid-state enzymes) are also isolated and purified by this method.
 mRNA can be isolated by this technique.
 Native proteins can be separated from denatured proteins by this technique.
 DNA & RNA can be separated from each other
 Papain and Urease can be separated by this technique.
 THIN LAYER CHROMATOGRAPHY (TLC)

TLC may be either carried out by the adsorption principle (if the thin layer is prepared by an adsorbent
such as “Keiselguhr” (or) “Alumina” (or) by the partition principle (if the layer is prepared by a substance
such as “Silica gel” which hold water like the paper).

Preparation of the layer:

The glass plate should be washed thoroughly & dried before layer application. The material to be used
for layer preparation is a follows:

The selected material is usually mixed with water, it form thick suspension, known as ”Slurry”. This
slurry is applied to a plate surface uniformly with 0.25mm thickness. To this layer mix the binder
“Calcium sulphate” for better adhesion of the stationary phase. The plates are dried after application of
the slurry. If adsorption chromatography is to be performed, the thin layer is activated by heating at
1100 C for several hours.

Compounds Adsorbents Solvent systems

Mono and Disaccharides Kieselguhr.G Ethylacetate/Propanol

(Sod.acetate) (65/35)

Kieselguhr.G Butanol/Acetone/Phosphate
buffer (pH-5.0)
(Sod.Phosphate, pH-5.0)
 (40-50-10)

Amino acids Silica Gel.G 96% ethanol/water

(70/30)

Plant pigments Kieselguhr.G Petroleum ether/Propanol

(99/1)

Procedure:

Chromatographic plates (20X20cm) of 200m thicknesses are prepared by using a suspension of 30 grams
of silica gel G in 63ml of 0.1M Na2CO3 solutions by shaking vigorously for 90 seconds. The silica gel
slurry is applied on to the glass plate in the form of a uniform layer. These plated are activated at 1100C
for 30 minutes immediately prior to use.

Then the samples (5 to 100mL) are applied on the silica plate in the form of small drops at regular
intervals. In this plate these samples are applied as a spot of less than 5 minutes diameter on the lower
right corner of the plates under a stream of warm air.

This plate is introduced into the saturated standard Brinkman developing chamber with the vapor of the
solvent mixture with Chloroform: Methanol: Acetic acid: Water (250:74:19:3 v/v) to dip 4.5 cm of its
bottom.
When solvent migrates about 15cm, plates are dried in air for 15 minutes and develop in the second
dimension (900 rotation clockwise) with CHCl3: CH3OH: 7M NH4OH (230: 90: 50 v/v). The solvent front
is again allowed to stand (or) more about 15cm. Then the plate is dried in air for 5 minutes and exposed
to iodine vapor (or) UV light the sample molecules can be visualized. When a permanent record of
developed plates is desired, plates are sprayed lightly with 10N H2SO4 and then heated at 1100 for 15

minutes.

By calculating the Rf values are can easily identified the molecules present in the mixture.

Detection: Several detection methods are available. They are,

1) Spraying the plate with 25 to 50% H2SO4 in ethanol and heating. This results in charring of most of
the compounds, which show up as Brown spots.

2) Iodine vapours are used extensively as a universal reagent for organic compounds. This iodine spot
disappears rapidly but can be made more permanent by spraying with 0.5% benzidine solution in
absolute ethanol.

Applications:

1) The constituents of the mixture of amino acids, and the constituents of natural lipids and
phospholipids are separated and estimated in a short time.
2) Enzymes, nucleic acids, pigments, sugars can also be separated by using this technique.

3) TLC has often been used to identify drugs, contaminants & Adulterants.

Advanced TLC:

TLC can be automated using forced solvent flow, running the plate in an vacuum-capable chamber to
dry the plate, and recording the finished chromatogram by absorption or fluorescence spectroscopy
with a light source. The ability to program the solvent delivery makes it convenient to do multiple
developments in which the solvent flows for a short period of time, the TLC plate is dried, and the
process is repeated. This method refocuses the spots to acheive higher resolution than in a single run.
See for example: Poole, C. F.; Poole, S. K. "Instrumental Thin-Layer Chromatography," Anal.
Chem. 1994, 66, 27A.

Two-dimensional TLC uses the TLC method twice to separate spots that are unresolved by only one
solvent. After running a sample in one solvent, the TLC plate is removed, dried, rotated 90o, and run in
another solvent. Any of the spots from the first run that contain mixtures can now be separated. The
finished chromatogram is a two-dimensional array of spots.

Gel Filtration Chromatography

This is also known as “Molecular exclusion chromatography” (or) “Molecular sieve chromatography”,
“Size exclusion chromatography” and “Permeation chromatography”

Principle:

In exclusion chromatography the separation of molecules is based up on the size and shape. The
stationary phase is porous bead material and the mobile phase is the solvent system.

The large molecules cannot enter the pores of the beads so they are excluded out and come down
rapidly. Small sized molecules enter the pores of the beads so that their speed is retarded and
comedown slowly. 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 and the pore diameter.
Mathematical relationships about solute behaviour on molecular sieve gels:

1) “The distribution of a solute particle between the inner and

outer solvent (solvent within and outside of the gel bead) is
defined as “Distribution coefficient” (Kd).

Kd= 0 –> Solute molecule is large & excluded out completely

Kd= 1 –> Solute molecule is small, retards it in inner solvents

2) The volume of outer solvent, i.e., the solvent surrounding the

gel beads is indicated as Vo. The technical term for this is “Void
volume”.

The volume of solvent inside gel bead – Inner solvent = Vi

The distribution coefficient = Kd

The effluent volume = Ve

Thus

The volume of inner solvent, Vi, can be calculated if the dry weight of gel employed and the “Water
regain value” of the particular gel are known. Thus,

For two different substances possessing different molecules weights and therefore, different
distribution coefficients (Kd1 & Kd2) the difference in their effluent volumes, Vs is given by

Vs =Ve1-Ve2 = (V0+Kd1.Vi)-(Vo+Kd2.Vi)

Types of gels:

Characteristics of gels:
1) The gel material should be chemically inert.

2) It should preferably contain ravishingly small number of ionic groups.

3) Gel material should provide a wide choice of pore and particle sizes.

4) The gel should have uniform particle & pore sizes.

5) The gel matrix should have high mechanical rigidity.

There are FIVE principle types of media:

a) Sephadex

b) Agarose

c)Polyacrylamide (Bio-gel.P)

d) Styragel e) Porous glass & silica granules

a) Sephadex:

1. It is most popular gel for proteins & most of the biomolecules separation.

2. When”Leuconosto mesenteroids” indulge in sucrose fermentation, large polymers of glucose are the
results. These polymers are known as “Dextrans” are used to prepare sephadex.

3. It is cross-linked polymer.

4. Sephadex gels are insoluble in water, and are stable in bases, weak acids and mild reducing and
oxidizing agents.

5. Sephadex gels are insoluble in water, and are stable in bases, weak acids and mild reducing and
oxidizing agents.

6. Sphadex, which cannot be used to separate biopolymers larger than 300,000 Daltons.

7. Some identified gels serial number gels are G-25; G-50; G-75; G-100; G-200

b) Agarose:

1. Agarose gels are produced from AGAR.

2. They are linear polysaccharides alternating residues of D-Galactose and 3,6-anhydro-L-galactose units.

3. These gels are hydrophilic and are almost completely free of charged groups.

4. Agarose gels, due to their greater porosity, it may be used to separate molecules and participates up
to a molecular weight of several million Daltons.
5. The gels are used in the study of viruses, nucleic acids and polysaccharides.

6. Some commonly used agarose gels are

--> Sepharose 2B

--> Sepharose 4B

--> Sepharose 6B

c) Polyacrylamide:

1. This is very popular medium is produced by polymerizing acrylamide into bead form.

2. polyacrylamide gels can be used to separate molecules of up to 300,000 daltons.

3. This gel is insoluble in water and common organic solvents may be used in the pH range of 2 to 11.

4. Some common gels are,

Bio-gel P 10, Bio-gel P60, Bio-gel P100,

Bio-gel P200, Bio-gel P300

d) Styragel:

1. For completely non-aqueous separations, a gel that will swell in an organic solvent is required.
Styragel provides this option.

2. It is a rigid cross-linked polystyrene gel.

3. The gel structure is unaffected by temperatures as high as 1500 C

4. the gel can be used with such solvent as tetrahydrofuran, cresol, dimethyl sulfoxide, chloroform,
carbon tetrachloride and others.

e) Controlled pore glass beads:

1. These fine glass spheres are manufactured from borosilicate glass to contain large number of pores
within a very narrow size distribution.

2. The glass spheres have a molecular exclusion limit ranging from 3000 to 9 million Daltons.

Procedure:

This is mainly carried out in the columns. The column is filled with beads, these beads contain pores. The
beads are made up of gels. The gels are made up of dextrans (Sephadex), Agarose (Biogel-A) and
Polyacrylamide (Biogel-P).
The sample is discovered in buffer and allowed to flow through the column. Large molecules can not
enter the pores of the beads, they are excluded out and reach down fastly. The small sized molecules
enter into the pores of the beads; their speed is retarded and reaches down slowly. These particles are
analyzed by spectroscopy.

Applications:

1) The main application of gel-filtration is the purification of molecules, viruses, nucleic acids, hormones,
enzymes, proteins, and antibodies and can be separated and purified by this technique.

2) It is also used for the separation of vitamins, steroids, neuropeptides and drugs.

3) Separations are achieved very quickly by this technique.

4) The molecular weight of the molecule can also be determined by this technique.

5) Protein receptor binding can be understood by this technique.

6) This method is especially useful for the separation of 4S and 5S tRNA.

7) It is also the most satisfactory method for separating DNA (from bacteria, usually Gram positive) from
the invariable contaminants, the “Teichoic acid”

ION-EXCHANGE CHROMATOGRAPHY

W.cohn first developed this procedure. The reversible exchange of ions in solution with ions
electrostatically bound to some sort of insoluble support medium.
Principle:

Exchange of ions is the basic principle in this type of Chromatography. In this process two types of
exchangers i.e., cationic and anionic exchangers can be used.

Cationic exchangers possess negatively charged group, and these will attract positively charged cations.
These exchangers are also called “Acidic ion exchange materials”, because their negative charges result
from the ionization of acidic group.

Anionic exchangers have positively charged groups that will attract negatively charged anions. These are
also called “Basic ion exchange” materials.
Types of ion exchange resins:

Two main groups of materials are used to prepare ion exchange resins: Polystyrene and Cellulose.

Resins made from both of these materials differ in their flow properties, ion accessibility and chemicals
and mechanical stability.

1. Polystyrene resins are proposed by polymerization reaction of styrene and divinyl benzene.

A higher concentration of divinyl benzene produces higher cross linkages.

Polystyrene resins are very useful for separating small molecular weight compounds.

Increasing the cross linkage increases the rigidity, reduces swelling, reduces porosity & reduces the
solubility of the polymeric structure.

sulfonic acids are strong acids with good proton dissociation ability. By sulfonation process, acidic
functional groups are easily attached to nearly every aromatic nucleaus.

Resins substituted with sulfonic acid groups are strong cationic exchangers.

To prepare weekly acidic exchanger, carbohydrate groups can be attached to the aromatic rings instead
of sulfonic acid group.

If basic functional groups are introduced, the resin can exchange anions rather than cations. Strong
anion exchangers are prepared with a tertiary amine, yielding a strongly basic quaternary ammonium
group. Weak anionic exchangers can be prepared with secondary amines, yielding a weakly basic
tertiary amine.

2.Cellulose resins have much greater permeability to macromolecular polyelectrolytes and possess
a much lower charge density as compared to polystyrene exchangers.

Carboxymethyl cellulose (CM-cellulose) – Cationic exchanger

DEAE cellulose - Anionic exchanger

Choice of Buffers:

Anionic exchange Chromatography should be carried out with cationic buffers.

Cationic exchange Chromatography should be carried out with anionic buffers.

The pK of the buffer should be as near as possible to the pH at which the system is buffered. This results
in high buffer capacity, which can with stand the local changes of pH in the column easily.
 Buffers PH range

Ammonium acetate 4 to 6

Ammonium formate 3 to 5

Pyridinium formate 3 to 6

Pyridinium acetate 4 to 6

Ammonium carbonate 8 to 10

Practical procedure:

Ion exchange separations are carried out mainly in columns packed with an ion-exchanger. These ionic
exchangers are commercially available. They are made up of styrene and divinyl benzene.

DEAE-cellulose is an anionic exchanger, CM-cellulose is a cationic exchanger. The choice of the

exchanger depends upon the charge of particle to be separated. To separate anions “Anionic exchanger”
is used, to separate cations “Cationic exchanger” is used.

First the column is filled with ion exchanger then the sample is applied followed by the buffer. The tris-
buffer, pyridine buffer, acetate buffer, citrate and phosphate buffers are widely used. The particles
which have high affinity for ion exchanger will come down the column along with buffers. In next step
using corresponding buffer separates the tightly bound particles. Then these particles are analyzed
spectroscopically.
Applications:

1. It is extremely used in the analysis of amino acids. The amino acid “Autoanalyzer” is based on in
exchange principle.

2. To determine the base composition of nucleic acids. Chargaff used this technique for established the
equivalence of Adenine and Thymine; Guanine and Cytosine.

3. This is most effective method for water purification. Complete deionization of water (or) a non-
electrolyte solution is performed by exchanging solute cations for hydrogen ions and solute anions for
hydroxyl ions. This is usually achieved by method is used for softening of drinking water.

4. Proteins are also successfully separated by this technique.

5. It is also used for the separation of many vitamins, other biological amines, and organic acids and
bases.

What is Liquid Chromatography?

The history of Liquid Chromatography (LC) began at early 20th century. In 1906, a Russian
botanist Mikhail Tswett invented LC in order to separate various plant pigments. He injected the
plant extract and petroleum ether through a glass column packed with calcium carbonate.
Since this was done on a glass column, he was able to observe the changes inside the column. At
the beginning, there is only one layer of pigment on the top of the column (Figure 1.a). But as
time passes by, the pigment is separated into four different colored-layers (Figure 1.b). The later
research discovered that those four-layers were (i) bluish green; chlorophyll a, (ii) yellowish
green; chlorophyll b, (iii) yellow; xanthine, and (iv) orange; carotene. The whole process of
separation took several hours and thus it was not a very practical method. This long analysis time
was a part of the reason that LC did not become a popular analytical tool until 1970s, a half
century after Mikhail Tswett's invention.
Below is some terms commonly used in chromatography analysis. Chromatography is a
separation technique and the word chromatography originated from chroma meaning "color" and
graphein meaning "write". Chromatograph is the separation equipment and chromatogram is an
out-put chart obtained from the analysis.

Analytical Technique
(No Image!)
 Chromatograph Chromatograph chromatogram

Figure 2. Images of chromatography, chromatograph, and chromatogram.

HPLC
HPLC stands for High Performance Liquid Chromatography. Before HPLC was available, LC
analysis was carried by gravitational flow of the eluent (the solvent used for LC analysis) thus
required several hours for the analysis to be completed. Even the improvements added in later
time were able to shorten the analysis time slightly. Those classical/initial LC systems are called
"low pressure chromatography" or "column
chromatography". Figure 3. Representation of Tswett's LC analysis.
In 1970s in the US, Jim Waters founded
Waters Corporation and started to sell HPLC instruments. This promoted the use of HPLC in
practical analysis areas. The LC systems that Waters Corporation developed used high-pressure
pump that generates rapid-flow of eluent, and thus resulted in dramatic improvement in the
analysis time. Compared to the “low pressure chromatography” the newer types were called
"high pressure liquid chromatography". Therefore it was used to be thought that HPLC stands for
High Pressure Liquid Chromatography,

High-performance liquid chromatography

Basic Principle:

Separation is based on the analyte’s relative solubility between two liquid phases. HPLC utilizes
different types of stationary phase (typically, hydrophobic saturated carbon chains), a pump that
moves the mobile phase(s) and analyte through the column, and a detector that provides a
characteristic retention time for the analyte. Analyte retention time varies depending on the
temperature of the column, the ratio/composition of solvent(s) used, and the flow rate of the
mobile phase. With HPLC, a pump (rather than gravity) provides the higher pressure required to
propel the mobile phase and analyte through the densely packed column

HPLC – Modes: Normal Phase - Polar stationary phase and non-polar solvent

Reverse Phase - Non-polar stationary phase and a polar solvent.

 Figure 4. Components of HPLC system
Pump
In the earlier state of HPLC development, the pump was the most important part of the system.
The development of HPLC can be said that it was a development of pump system. Pump is
positioned in the most upper stream of the LC system and generates a flow of eluent from the
solvent reservoir to the system. In the earlier stage of LC development, to be able to generate the
high pressure was the one of the most important system requirements. However, nowadays, the
high pressure generation is a "standard" requirement and what is more concerned nowadays is to
be able to provide a consistent pressure at any condition, to provide a controllable and
reproducible flow rate. Since a change in the flow rate can influence the analysis largely.
Most pumps used in current LC systems generate the flow by back-and-forth motion of a motor-
driven piston (reciprocating pumps). Because of this piston motion, it produces "pulses". There
have been large system improvements to reduce this pulsation and the recent pumps create much
less pulse compared to the older ones. However, recent analysis requires very high sensitivity to
quantify a small amount of analytes, and thus even a minor change in the flow rate can influence
the analysis. Therefore, the pumps required for the high sensitivity analysis needs to be highly
precise.

Injector
An injector is placed next to the pump. The simplest method is to use a syringe, and the sample
is introduced to the flow of eluent. Since the precision of LC measurement is largely affected by
the reproducibility of sample injection, the design of injector is an important factor. The most
widely used injection method is based on sampling loops. The use of autosampler (auto-injector)
system is also widely used that allows repeated injections in a set scheduled-timing.

Column
The separation is performed inside the column; therefore, it can be said that the column is the
heart of an LC system. The theory of chromatography column has not changed since Tswett's
time; however there has been continuous improvement in column development. The recent
columns are often prepared in stainless steel housing, instead of glass columns used in Tswett's
experiment. The packing material generally used is silica or polymer gels compared to calcium
carbonate used by Tswett.
The eluent used for LC varies from acidic to basic solvents. Most column housing is made of
stainless steel, since stainless is tolerant towards a large variety of solvents. However, for the
analysis of some analytes such as biomolecules and ionic compounds, contact with metal is not
desired, thus a polyether ether ketone (PEEK) column housing is used instead.

Detector
Separation of analytes is performed inside the column, whereas a detector is used to observe the
obtained separation. The composition of the eluent is consistent when no analyte is present.
While the presence of analyte changes the composition of the eluent. What detector does is to
measure these differences. This difference is monitored as a form of electronic signal. There are
different types of detectors available. Different detector types are explained in Lesson 6.

Recorder
The change in eluent detected by a detector is in the form of electronic signal, and thus it is still
not visible to our eyes. In older days, pen (paper)-chart recorder was popularly used. Nowadays,
computer based data processor (integrator) is more common. There are various types of data
processors; examples include a simple system consisting of in-built printer and word processor,
and a personal computer type consisting of display monitor, keyboard, and printer. Also there are
software that are specifically designed for LC system. It provides not only data acquisition, but
features like peak-fitting, base line correction, automatic concentration calculation, molecular
weight determination, etc…

The components introduced so far are the basics of LC system. Below are some optional
equipment used with the basic LC system.

Degasser
The eluent used for LC analysis may contain gases such as oxygen that are non-visible to our
eyes. When gas is present in the eluent, this is detected as a noise and causes unstable baseline.
Generally used method includes sparging (bubbling of inert gas), use of aspirator, distillation
system, and/or heating and stirring. However, the method is not convenient and also when the
solvent is left for a certain time period (e.g., during the long analysis), gas will dissolve back
gradually. Degasser uses special polymer membrane tubing to remove gases. The numerous very
small pores on the surface of the polymer tube allow the air to go through while preventing any
liquid to go through the pore. By placing this tubing under low pressure container, it created
pressure differences inside and outside the tubing (higher inside the tubing). This difference let
the dissolved gas to move through the pores and remove the gas. Compared to classical batch
type degassing, the degasser can be used on-line, it is more convenient and efficient. Many of
new HPLC unit system contain a degasser.

Column Heater
The LC separation is often largely influenced by the column temperature. In order to obtain
repeatable results, it is important to keep the consistent temperature conditions. Also for some
analysis, such as sugar and organic acid, better resolutions can be obtained at elevated
temperature (50 to 80°C). It is also important to keep stable temperature to obtain repeatable
results even it is analyzed at around room temperature. There are possibilities that small different
of temperature causes different separation results. Thus columns are generally kept inside the
column oven (column heater).

Peaks: