Chromatography Basic

1. BY: LOKESH Sv.M.R.P

2. 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.

3. Paper Chromatography
 Coloumn Chromatography
HPLC(high performance
Liquid Chromatography)
Thin-Layer Chromatography

4.  The principle involved is partition chromatography wherein the
substances are distributed or partitioned between liquid phases.
One phase is the water, which is held in the pores of the filter
paper used; and other is the mobile phase which moves over the
paper. The compounds in the mixture get separated due to
differences in their affinity towards water (in stationary phase)
and mobile phase solvents during the movement of mobile phase
under the capillary action of pores in the paper.
 The principle can also be adsorption chromatography between
solid and liquid phases, wherein the stationary phase is the solid
surface of the paper and the liquid phase is of the mobile phase.
But most of the applications of paper chromatography work on
the principle of partition chromatography, i.e., partitioned
between to liquid phases.

5. Ascending chromatography
 Descending chromatography
Ascending- descending mode
Radial mode

6.  Ascending chromatography: As the name indicates, the chromatogram ascends.
Here, the development of paper occurs due to the solvent movement or upward
travel on the paper.
 The solvent reservoir is at the bottom of the beaker. The paper tip with sample spots
just dips into the solvent at the bottom so that spots remain well above the solvent.

7.  Descending chromatography: Here, the development of paper occurs due to
solvent travel downwards on the paper.
 The solvent reservoir is at the top. The movement of the solvent is assisted by
gravity besides the capillary action.

8.  Ascending- descending mode: Here solvent first
travels upwards and then downwards on the paper.

9.  4. Radial mode: Here, the solvent moves from the center (mid-point)
towards the periphery of circular chromatography paper. The entire
system is kept in a covered Petri dish for the development of the
chromatogram.
 The wick at the center of paper dips into the mobile phase in a petri
dish, by which the solvent drains on to the paper and moves the
sample radially to form the sample spots of different compounds as
concentric rings

10.  Uses and Applications of Paper Chromatography
 Paper chromatography is specially used for the
separation of a mixture having polar and non-
polar compounds.
 For separation of amino acids.
 It is used to determine organic compounds,
biochemicals in urine, etc.
 In the pharma sector, it is used for the
determination of hormones, drugs, etc.
 Sometimes it is used for evaluation of inorganic
compounds like salts and complexes.

11.  When the mobile phase along with the mixture that needs to be
separated is introduced from the top of the column, the
movement of the individual components of the mixture is at
different rates. The components with lower adsorption and
affinity to stationary phase travel faster when compared to the
greater adsorption and affinity with the stationary phase. The
components that move fast are removed first whereas the
components that move slow are eluted out last.
 The adsorption of solute molecules to the column occurs in a
reversible manner. The rate of the movement of the components
is expressed as:
 Rf = the distance travelled by solute/ the distance travelled by
solvent
 Rf is the retardation factor.

12.  Before starting with the Column Chromatography Experiment let us understand the different phases
involved.
 Mobile phase – This phase is made up of solvents and it performs the following functions:
 It acts as a solvent – sample mixture can be introduced in the column.
 It acts as a developing agent – helps in the separation of components in the sample to form bands.
 It acts as an eluting agent – the components that are separated during the experiment are removed
from the column
 Some examples of solvents used as mobile phase based on their polarity are – ethanol, acetone,
water, acetic acid, pyridine, etc.
 Stationary phase – It is a solid material which should have good adsorption property and meet the
conditions given below:
 Shape and size of particle: Particles should have uniform shape and size in the range of 60 – 200μ in
diameter.
 Stability and inertness of particles: high mechanical stability and chemically inert. Also, no reaction
with acids or bases or any other solvents used during the experiment.
 It should be colourless, inexpensive and readily available.
 Should allow free flow of mobile phase
 It should be suitable for the separation of mixtures of various compounds.

13.  The stationary phase is made wet with the help of solvent as the upper level of the mobile
phase and the stationary phase should match. The mobile phase or eluent is either solvent or
mixture of solvents. In the first step the compound mixture that needs to be separated, is
added from the top of the column without disturbing the top level. The tap is turned on and
the adsorption process on the surface of silica begins.
 Without disturbing the stationary phase solvent mixture is added slowly by touching the sides
of the glass column. The solvent is added throughout the experiment as per the requirement.
 The tap is turned on to initiate the movement of compounds in the mixture. The movement is
based on the polarity of molecules in the sample. The non-polar components move at a
greater speed when compared to the polar components.
 For example, a compound mixture consists of three different compounds viz red, blue, green
then their order based on polarity will be as follows blue>red>green
 As the polarity of the green compound is less, it will move first. When it arrives at the end of
the column it is collected in a clean test tube. After this, the red compound is collected and at
last blue compound is collected. All these are collected in separate test tubes.

15.  Column Chromatography is used to isolate
active ingredients.
 It is very helpful in Separating compound
mixtures.
 It is used to determine drug estimation from
drug formulations
 It is used to remove impurities.
 Used to isolation metabolites from biological
fluids.

16.  What Is Thin Layer Chromatography?
 Thin Layer Chromatography is a technique used to isolate
non-volatile mixtures. The experiment is conducted on a
sheet of aluminium foil, plastic, or glass which is coated
with a thin layer of adsorbent material. The material
usually used is aluminium oxide, cellulose, or silica gel.
 On completion of the separation, each component appears
as spots separated vertically. Each spot has a retention
factor (Rf) expressed as:
 Rf = dist. travelled by sample / dist. travelled by solvent
 The factors affecting retardation factor are the solvent
system, amount of material spotted, absorbent and
temperature. TLC is one of the fastest, least expensive,
simplest and easiest chromatography technique.

17. Principle
Like other chromatographic techniques, thin
layerchromatography (TLC) depends on the separation
principle. The separation relies on the relative affinity
of compounds towards both the phases. The compounds in the
mobile phase move over the surface of the stationary phase. The
movement occurs in such a way that the compounds which have a
higher affinity to the stationary phase move slowly while the other
compounds travel fast. Therefore, the separation of the mixture is
attained. On completion of the separation process, the individual
components from the mixture appear as spots at respective levels
on the plates. Their character and nature are identified by suitable
detection techniques.

19.  Before starting with the Thin Layer Chromatography Experiment let us
understand the different components required to conduct the procedure along
with the phases involved.
 Thin Layer Chromatography Plates – ready-made plates are used which are
chemically inert and stable. The stationary phase is applied on its surface in the
form of a thin layer. The stationary phase on the plate has a fine particle size
and also has a uniform thickness.
 Thin Layer Chromatography Chamber – Chamber is used to develop plates. It
is responsible to keep a steady environment inside which will help in
developing spots. Also, it prevents the solvent evaporation and keeps the entire
process dust-free.
 Thin Layer Chromatography Mobile phase – Mobile phase is the one that
moves and consists of a solvent mixture or a solvent. This phase should be
particulate-free. The higher the quality of purity the development of spots is
better.
 Thin Layer Chromatography Filter Paper – It has to be placed inside the
chamber. It is moistened in the mobile phase.

20.  Thin Layer Chromatography Experiment
 The stationary phase that is applied to the plate is made to dry
and stabilize.
 To apply sample spots, thin marks are made at the bottom of the
plate with the help of a pencil.
 Apply sample solutions to the marked spots.
 Pour the mobile phase into the TLC chamber and to maintain
equal humidity, place a moistened filter paper in the mobile
phase.
 Place the plate in the TLC chamber and close it with a lid. It is
kept in such a way that the sample faces the mobile phase.
 Immerse the plate for development. Remember to keep the
sample spots well above the level of the mobile phase. Do not
immerse it in the solvent.
 Wait till the development of spots. Once the spots are developed,
take out the plates and dry them. The sample spots can be
observed under a UV light chamber.

21. • The qualitative testing of Various medicines such as sedatives, local anaesthetics,
anticonvulsant tranquilisers, analgesics, antihistamines, steroids, hypnotics is done
by TLC.
• TLC is extremely useful in Biochemical analysis such as separation or isolation of
biochemical metabolites from its blood plasma, urine, body fluids, serum, etc.
• Thin layer chromatography can be used to identify natural products like essential
oils or volatile oil, fixed oil, glycosides, waxes, alkaloids, etc
• It is widely used in separating multicomponent pharmaceutical formulations.
• It is used to purify of any sample and direct comparison is done between the sample
and the authentic sample
• It is used in the food industry, to separate and identify colours, sweetening agent,
and preservatives
• It is used in the cosmetic industry.
• It is used to study if a reaction is complete.

22.  Thin Layer Chromatography plates do not have longer
stationary phase.
 When compared to other chromatographic techniques the
length of separation is limited.
 The results generated from TLC are difficult to reproduce.
 Since TLC operates as an open system, some factors such
as humidity and temperature can be consequences to the
final outcome of the chromatogram.
 The detection limit is high and therefore if you want a lower
detection limit, you cannot use TLC.
 It is only a qualitative analysis technique and not
quantitative.

23.  High performance liquid chromatography (HPLC) is
basically a highly improved form of column liquid
chromatography.
 Instead of a solvent being allowed to drip through a column
under gravity, it is forced through under high pressures of
up to 400 atmospheres. That makes it much faster.
 All chromatographic separations, including HPLC operate
under the same basic principle; separation of a sample into
its constituent parts because of the difference in the relative
affinities of different molecules for the mobile phase and
the stationary phase used in the separation.

24. Types of HPLC
There are following variants of HPLC, depending upon the phase system (stationary) in the process :
1. Normal Phase HPLC
This method separates analytes on the basis of polarity. NP-HPLC uses polar stationary phase and
non-polar mobile phase. Therefore, the stationary phase is usually silica and typical mobile phases
are hexane, methylene chloride, chloroform, diethyl ether, and mixtures of these.
Polar samples are thus retained on the polar surface of the column packing longer than less polar
materials.
2. Reverse Phase HPLC
The stationary phase is nonpolar (hydrophobic) in nature, while the mobile phase is a polar liquid,
such as mixtures of water and methanol or acetonitrile. It works on the principle of hydrophobic
interactions hence the more nonpolar the material is, the longer it will be retained.
3. Size-exclusion HPLC
The column is filled with material having precisely controlled pore sizes, and the particles are
separated according to its their molecular size. Larger molecules are rapidly washed through the
column; smaller molecules penetrate inside the porous of the packing particles and elute later.
4. Ion-Exchange HPLC
The stationary phase has an ionically charged surface of opposite charge to the sample ions. This
technique is used almost exclusively with ionic or ionizable samples.
The stronger the charge on the sample, the stronger it will be attracted to the ionic surface and thus,
the longer it will take to elute. The mobile phase is an aqueous buffer, where both pH and ionic
strength are used to control elution time.

26. 1. Solvent Resorvoir
Mobile phase contents are contained in a glass resorvoir. The mobile phase, or solvent, in HPLC is usually a
mixture of polar and non-polar liquid components whose respective concentrations are varied depending on
the composition of the sample.
2. Pump
A pump aspirates the mobile phase from the solvent resorvoir and forces it through the system’s column and
detecter. Depending on a number of factors including column dimensions, particle size of the stationary
phase, the flow rate and composition of the mobile phase, operating pressures of up to 42000 kPa (about
6000 psi) can be generated.
3. Sample Injector
The injector can be a single injection or an automated injection system. An injector for an HPLC system
should provide injection of the liquid sample within the range of 0.1-100 mL of volume with high
reproducibility and under high pressure (up to 4000 psi).
4. Columns
Columns are usually made of polished stainless steel, are between 50 and 300 mm long and have an internal
diameter of between 2 and 5 mm. They are commonly filled with a stationary phase with a particle size of
3–10 µm.
Columns with internal diameters of less than 2 mm are often referred to as microbore columns. Ideally the
temperature of the mobile phase and the column should be kept constant during an analysis.
5. Detector
The HPLC detector, located at the end of the column detect the analytes as they elute from the
chromatographic column. Commonly used detectors are UV-spectroscopy, fluorescence, mass-
spectrometric and electrochemical detectors.
6. Data Collection Devices
Signals from the detector may be collected on chart recorders or electronic integrators that vary in complexity
and in their ability to process, store and reprocess chromatographic data. The computer integrates the
response of the detector to each component and places it into a chromatograph that is easy to read and
interpret.

27.  Applications of HPLC
◦ Pharmaceutical Applications
1. To control drug stability.
2. Tablet dissolution study of pharmaceutical dosages form.
3. Pharmaceutical quality control.

◦ Environmental Applications
1. Detection of phenolic compounds in drinking water.
2. Bio-monitoring of pollutants.

◦ Applications in Forensics
1. Quantification of drugs in biological samples.
2. Identification of steroids in blood, urine etc.
3. Forensic analysis of textile dyes.
4. Determination of cocaine and other drugs of abuse in blood, urine etc.

◦ Food and Flavour
1. Measurement of Quality of soft drinks and water.
2. Sugar analysis in fruit juices.
3. Analysis of polycyclic compounds in vegetables.
4. Preservative analysis.

◦ Applications in Clinical Tests
1. Urine analysis, antibiotics analysis in blood.
2. Analysis of bilirubin, biliverdin in hepatic disorders.
3. Detection of endogenous Neuropeptides in extracellular fluid of brain etc.
