chromatography introduction and paper chromatography

1. AMRUTHA JOSE
PHARMACY PRACTICE
M.PHARM
CHROMATOGRAPHY-PAPER CHROMATOGRAPHY

2. Chromatography, Color writing. "Chroma" is a Greek roots prefix for color and "graphy" is a
Greek roots suffix for writing. It is used to analyze, identify, purify & quantify the compounds.
Chromatography is defined as a method of separating a mixture of components in to individual
components through equilibrium distribution between two phases.
Mixture Components
 Analyze
 Identify
 Purify
 Quantify

3. The components to be separated are distributed between two phases, a stationary and mobile
phase.
Chromatographic technique is based on the different interactions of compounds
with two phases, a mobile phase and a stationary phase, as the compounds travel through
a supporting medium.
Components:
 mobile phase: a solvent that flows through the supporting medium
 stationary phase: a layer or coating on the supporting medium that
interacts with the analytes
 supporting medium: a solid surface on which the stationary phase is bound
or coated

4. Applications of chromatography
In any chemical or bio-processing industry, the need to separate and purify a product from a
complex mixture is a necessary and important step in the production line. This separation of
mixtures is useful to us in various ways. For examples,
 Pharmaceutical industryuses chromatographyto isolatepenicillin and other antibiotics.
 Proteins can even beseparated into amino acids.
 Chromatographyis also used in crime sceneinvestigation for DNAand RNA sequencing.
 In manyscientificstudies to identifyunknown organicand inorganiccompounds.
 Government laboratories used to check dyes in food and vegetables contained tiny
amounts of pesticides and herbicides.

5. Types of Chromatography
There are many forms of chromatography, but all forms work on the same principle:
1.BASED ON STATIONARY PHASE AND MOBILE PHASE.
a). Solid –Liquid Chromatography
Examples:
Coloumn chromatography with silica gel
Thin Layer Chromatography
High Performance Liquid Chromatography
b). Liquid –Liquid Chromatography
Examples:
Paper Chromatography
Column Partition Chromatography
c). Gas-Solid Chromatography
d). Gas-Liquid Chromatography

6. 2. BASED ON PRINCIPLE OF SEPERATION.
Partition Chromatography which includes a liquid film coated in an inert suitable
support.
Partition chromatography depends on the partitioning behavior of substance between two
immiscible liquids. When a mixture of solute is shaken with two immiscible liquids, most
of the solute distribute themselves between the liq depending up on their partition
coefficient.Partition coefficient is the ratio of concentration of substance in each phase.
Kd =concentration in solvent A / concentration in solvent B
Adsorption Chromatography where adsorption takes place at solid liquid interphase.in
sussessful separation,the components show different degrees of affinity for the solid
support.components with least affinity move down the column at a faster rate and are
eluted from the end of the column before those with the greatest affinity for the
adsorbent.

7. 3. BASED ON MODES OF SEPERATION.
1).Normal phase chromatography
 Stationary phase is polar, mobile phase is non-polar
 Non-polar compounds travel faster and eluted first
 Polar compounds retained for longer time in the column
 Not advantageous in pharmaceutical field
 Silica or alumina possess polar sites that interact with polar molecules

8. 2).Reverse phase chromatography
 Non-polar stationary phase, polar mobile phase
 Glass or silica is used as a base on to which the organic material is bonded
 Silica gel esterified with alcohols to form silicate esters ,Si-OH groups on silica gel can
be silanized with organo chloro silanes

9. OTHER TYPES OF CHROMATOGRAPHY
1. Ion Exchange Chromatography
(which is reversible step) which includes ionic groups (ionic means holding different
charges) which are attached to an inert material; this method is used in purifying water
for example & the competition will be between the sample (water considered mobile
phase also) & the stationary phase directly.
2. Gel Chromatography
(also called molecular sieving/Gel filtration/Gel permeation/Molecular exclusion) which
depends on a suspension of a polymer having a suitable pore size (like agar) & is an
important method for some analysis kinds such as separating hormones, enzymes &
biological fluids; AGAR itself is a polymer with pores, so small particles will enter into
the pores & might leave only in case it found a larger pore to enter in it.
3.Chiral chromatography
Separation of enantiomers .Either the mobile phase or the stationary phase must be made
chiral
4. Fast protein liquid chromatography
Applied to several chromatographic techniques which are used to purify proteins
For preparing large scale batches of a purified product

10. The Theory of Chromatography
There are two theories to explain chromatography
Plate theory
Rate theory
PLATE THEORY
The col umn is made up of large number o f parallel layers or theoretical plates and when the
mobile phase travel down the column, the components distribute themselves between the
stationary phase and mobile phase in accordance with their partition coefficient. The rate o f
movement of mobile is assumed to be such that dynamic equilibrium is established with in each
plate. The components move down the column at a definite rate depending on the r ate of
movement of mobile phase.
RATE THEORY:
The rate theory explains the effect of variables such as mobile phase velocity and adsorbabilities
which determin the width of an elution band.It also effects the time taken by a solute to make it
appear at the end of the column.

11. Paper chromatography
Paper chromatography is an analytical technique for separating and identifying both
colored (e.g. pigments) and colorless (e.g. amino acids) mixtures.

12. In paper chromatography, the stationary phase is a very uniform absorbent paper. Cellulose
(non polar) in the form of paper sheets makes an ideal support medium where water is adsorbed to
the cellulose fibers and forms the stationary hydrophilic phase .Cellulose is a polymer of the simple
sugar, glucose.
The key point about cellulose is that the polymer chains have -OH groups
sticking out all around them..The cellulose fibers attract water vapour from the
atmosphere as well as any water that was present when the paper was made.
You can therefore think of paper as being cellulose fibres with a very thin layer of
water molecules bound to the surface.

13. Non-polar molecules in the mixture that you are trying to separate will have
little attraction for the water molecules attached to the cellulose, and so will
spend most of their time dissolved in the moving solvent. Molecules like this
will therefore travel a long way up the paper carried by the solvent. They will
have relatively high Rf values.
On the other hand, polar molecules will have a high attraction for the water
molecules and much less for the non-polar solvent. They will therefore tend
to dissolve in the thin layer of water around the cellulose fibers much more
than in the moving solvent.
Because they spend more time dissolved in the stationary phase and less
time in the mobile phase, they aren't going to travel very fast up the paper.

14. Choice of paper
Whatman chromatography papers are the most widely used papers for
chromatography worldwide. This acceptance and usage reflect the purity,
high quality, and consistency of Whatman papers. These qualities are relied
upon by chromatographers and are essential to successful, reproducible
chromatography. Whatman chromatography paper media are made from
specially selected cotton cellulose. They are rigorously quality controlled for
characteristics important to the chromatographer and to ensure uniformity within the grade.
• Whatman No. 1 MM is the paper most frequently used for analytical purpose.
• Whatman No. 3 MM is a thick paper is best employed for separating large quantities of
material but the resolution is inferior to Whatman No. 1.
• Whatman Nos. 4 and 5 are convenient for a rapid separation, although the spots are less well
defined. The paper may be impregnated with a buffer solution before use or chemically
modified by acetylation.

15. Choice of Solvent
It is depend on the mixture investigated. If the compounds move close to the solvent front in
solvent A then they are too soluble, while if they are crowded around the origin in solvent B then
they are not sufficiently soluble. Therefore, a suitable solvent would be an appropriate mixture of
both solvent A & solvent B, so that the Rf values of the components of the mixture are spread
across the length of the paper.
The pH may also be important in a particular separation because many solvents contain acetic
acid or ammonia to create a strongly acidic or basic environment.
Spotting the paper
The sample (10-20 ul) is spotted onto the paper by the capillary glass
in contact with the paper so that the spot is no larger than 1 mm in
diameter, then raise the capillary and do it this procedure in all samples [1]
.

16. Development
A. Ascending paper chromatography
Solvent running up the paper or TLC by capillary action. It is most
employed and has the advantage that separation can be carried out in two
dimensions.Ascending is simple and inexpensive compared with descending
and usually gives more uniform migration with less diffusion of the sample
"spots".
Eg:Sodium Iodo hippurate injection tested for radiochemical purity.
Solvent
B.Descending paper chromatography
In descending paper chromatography, the chromatogram is held
vertically, and the spot of dye is drooped on the top of the chromatogram.
Solvent drips off the bottom of the paper by gravity. It is convenient for
compound which has similar Rf values. Descending chromatography is
faster because gravity helps the solvent flow but it’s difficult to set the
apparatus.
Eg: To identify Vancomycin Hydrochloride.
Solvent

17. . Ascending – descending chromatography
Hybrid of the above two techniques
Upper part of the ascending chromatography can be folded over a glass rod allowing the
ascending development to change over in to the descending after crossing the glass rod.
D.Radial paper chromatography
o Circular paper chromatography
o Circular filter paper is used
o Sample placed at the center
o Dry the spot
o Paper is fixed horizontally on the petridish possessing the solvent
o Tongue/wick of the paper dips in to the solvent
o Solvent rises through the tongue /wick
o When solvent front moved through large distance, the components gets separated
in the form of concentric circular zones
Eg: Liothyronie Sodium tested for DiIodothyronine.

18. Two dimensional chromatography
A square / rectangular paper is used
The sample is applied to one of the sides
The second development was performed at right angle to the direction of the first run.

19. Detection of spots
Most biological compounds are colorless and are visualized by:
1. Spraying the paper by specific reagents, for example, Ninhydrin (Triketohydrindane hydrate).
2. Dipping in a solution of the reagent in a volatile solvent, for example, Iodine vapors.
3. Fluorescence compounds can be visualized with ultraviolet light.
4. Radioactive spots can be located with a detector, or the chromatogram can be pressed against
X-ray film for minutes or hours to expose the film.
Experimental details for quantitative analysis
Isolation of separated components from paper chromatography
 Cut out the appropriate part of the filter paper
 Soak it in the minimum quantity of the solvent
 Separation of substance from the spot- elution
Direct technique:light is passed through the spot using a photodensitometer and transmitted
light is measured. By determining the optical density of both standard and test,quantity of
substance is calculated.
Indirect technique: The spot is out and are eleuted with a polar solvent.vigorous shaking
followed by centrifugation yields a clear layer of spectrophotometric examination.

20. Retention factor (Rf)
Rf allows identification of individual compound in a mixture when compared to one or more
standard compounds under absolutely identical conditions to that of the test compound . When
comparing two different compounds run under identical conditions, the compound with the
larger Rf is less polar because it interacts less strongly with the polar adsorbent on the paper.
Specifically, the retention fraction is defined as the fractional distance the spot moves compared
to the distance travelled by the solvent front. The Rf is constant for a particular compound as
long as you keep everything else constant - the solvent system, the stationary phase, temperature,
amount of material spotted, pH- for example. It corresponds to a physical property of the
compound. Spots with the same Rf values within experimental error and the same appearance are
likely to be the same compound.
For each compound it can be worked out using the formula:
Distance from baseline to spot, x
Distance from baseline to solvent, y
Rf =

21. R f value depends on
• The solvent employed
• The medium used for separation
• The nature of the mixture
• Temperature
• The size of the vessel in which the operation is carried out
Applications
o P C applied to the separation of many organic and biochemical products
o Identification of drugs
o Identification of foreign substances in drugs
o Investigation of DNA and RNA sequence for forensic investigation.
o Analysis of metabolites of drugs in blood, urine
o Analysis of food colouring and plant pigments