Chromatography is a technique used to separate mixtures based on differences in how components interact with stationary and mobile phases. There are two main types: column chromatography uses a stationary phase packed in a column, while planar chromatography uses a stationary phase coated on a flat surface like a plate or paper. Column chromatography is useful for separating solids and liquids while planar chromatography like thin layer chromatography and paper chromatography can separate mixtures like pigments or amino acids. Both techniques work on the principle that different compounds move through the phases at different rates based on their interactions with the phases.
3. Chromatography
Chromatography is a combination of two words;
* Chromo – Meaning color
* Graphy – representation of something on paper
■ It is a physical method of separation in which the components of a
mixture are separated by differences in their distribution between two
phases,
■ One of which is stationary (stationary phase) while the other (mobile
phase) moves through it in a definite direction. The substances must
interact with the stationary phase to be retained and separated by it.
4. Principle
Chromatography usually consists of mobile phase and stationary phase.
■ The mobile phase refers to the mixture of substances to be separated
dissolved in a liquid or a gas.
■ The stationary phase is a porous solid matrix through which the sample
contained in the mobile phase percolates. The interaction between the
mobile phase and the stationary phase results in the separation of the
compound from the mixture.
7. Column chromatography
Column chromatography is one of the most useful methods for
the separation and purification of both solids and liquids.
■ This is a solid - liquid technique in which the stationary phase
is a solid & mobile phase is a liquid.
8. Principle
■ When a mixture of components dissolved in the mobile phase
is introduced into the column, the individual components
move with different rates depending upon their relative
affinities.
■ The compound with lesser affinity towards stationary phase
moves faster and it is eluted (Extract) out of the column first.
The one with greater affinity towards stationary phase moves
slower down the column and hence it is eluted latter. Thus the
compounds are separated
10. Preparation of column chromatography
■ It consists of a glass tube with bottom portion of the column – packed
with glass wool/cotton wool or may contain asbestos pad,
» Above which adsorbent is packed
» After packing a paper disc kept on the top, so that the adsorbent layer is
not disturbed during the introduction of sample or mobile phase.
11. Packing techniques in C.C
There are two types of preparing the column, they are:
i. Dry packing / dry filling
Ii. Wet packing / wet filling
12. Packing techniques in C.C
Dry PackingTechnique
■ Adsorbent is packed in the column in dry form
■ Fill the solvent, till equilibrium is reached
DEMERIT:Air bubbles are entrapped b/w M.P & S.P→ cracks appear in the adsorbent
layer.
■ After filling tapping can be done to remove void spaces.
13. Packing techniques in C.C
■ Wet PackingTechnique
■ ideal & common techniqueThe material is slurried with solvent and generally added
to the column in portions.
■ S.P settles uniformly & no crack in the column of adsorbent.
■ solid settle down while the solvent remain upward.» this solvent is removed then
again cotton plug is placed.
14.
15. APPLICATIONS
■ Separation of mixture of compounds
■ Purification process
■ Estimation of drugs in
■ Removal of impurities or purification process
■ Isolation of metabolites from biological fluids.
16. Advantages of C.C
■ Any type of mixture can be separated
■ Any quantity of mixture can be separated
■ Wider choice of Mobile Phase
■ Automation is possible
Disadvantages of C.C
■ Time consuming
■ more amount of Mobile Phase are required
■ Automation makes the techniques more complicated & expensive
17.
18. Planner Chromatography
the stationary phase is supported on a flat plate or the interstices of a paper and the
mobile phase moves through the stationary phase by capillary action or by gravity.
Types of planner chromatography
■ Thin layer chromatography
■ Paper chromatography
19. Thin-layer chromatography
Thin-layer chromatography (TLC) is a chromatographic technique that is useful for
separating organic compounds. Because of the simplicity and rapidity ofTLC, it is often
used to monitor the progress of organic reactions and to check the purity of products.
20. Principle
■ Similar to other chromatographic methodsTLC is also based on the principle of
separation.The separation depends on the relative affinity of compounds towards
stationary and mobile phase.The compounds under the influence of mobile phase
(driven by capillary action) travel over the surface of stationary phase. During this
movement the compounds with higher affinity to stationary phase travel slowly while
the others travel faster.Thus separation of components in the mixture is achieved.
■ Once separation occurs individual components are visualized as spots at respective
level of travel on the plate.Their nature or character are identified by means of
suitable detection techniques.
21. Four Stages inTLC
1. SampleApplication -Capillary used to spot solution of each sample.
2. Development -This is when the separation actually occurs.
3. Visualization - viewed under UV light.
4. Interpretation of Result - Comparison of retention factors.
22. 1. Sample Application
A. Draw “guid lines”
lightly with pencil
B. Dissolve solid sample in
solution(MeOH)
C. UseTLC capillary to transfer
and spot dissolved sample
27. Applications
■ Purity of any sample
■ Identification of compounds
■ Separation of carbohydrates
■ Separation of lipids
■ Separation ofTriacylglyceroles
28. Advantages
■ Less equipment is required.
■ Very little time for separation is required.
■ It is more sensitive.
■ The lower detection limit of analytical sample inTLC is approximately
one decimal and very small quantities of sample is sufficient for
analysis.
29. Disadvantages
■ spots are often faint, andTLC is difficult to reproduce. NOT typically
■ In this method the plate length is limited and hence separation takes
place only up to certain length.
■ The separation takes place in an open system or in open condition and
hence there are chances that sample may be affected by the humidity
and temperature
30.
31. Paper Chromatography
■ Paper chromatography is an analytical method that is used to separate
coloured chemicals or substances, especially pigments.This can also be
used in secondary or primary colours in ink experiments.This method
has been largely replaced by thin layer chromatography, but is still a
powerful teaching tool.
32. principle
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.
33. Preparation process
a) Selection of suitable type of development:
This depends on the complexity of the mixture, solvent, paper, etc. But in
paper chromatography are used as they are easy to perform, handle, less
time-consuming and also give chromatogram faster.
b) Selection of suitable filter paper:
Filter paper is selected based on pore size, the quality of the sample to be
separated, and also mode of development
34. Cont….
c) Preparation of sample:
Preparation of sample involves dissolution of sample in suitable solvent used in
making mobile phase.The solvent used should be inert with the sample under
analysis.
d) Spotting of sample on the paper:
Samples are to be spotted at proper position on the paper preferably using a
capillary tube.
d) Development of chromatogram: Sample spotted paper is subjected to
development by immersing it in the mobile phase.The mobile phase moves over
the sample on the paper under the capillary action of paper.
35. Cont…
e) Drying of the paper and detection of the compounds:
Once the development of chromatogram is over, the paper is held carefully at the borders
so as to avoid touching the sample spots and dried using an air drier. Sometimes the
detecting solution is sprayed in the developed paper and dried to identify the sample
chromatogram spots.
f) Determine the Rf value for each coloured spot.
Rf = a/d
36.
37. Applications
■ For separation of amino acids.
■ It is used to determine organic compounds, biochemical 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.
38. Advantages
■ It requires very less quantitative material.
■ It is Cheaper compared to other chromatography methods.
■ Both unknown inorganic as well as organic compounds can be identified by paper
chromatography method.
■ Paper Chromatography do not occupy much space as compared to other analytical
methods.
39. disadvantages
■ Complex mixture cannot be separated by paper chromatography
■ Less accurate then HPLS/HPTLP.
■ It can not used for quantitative analysis.
■ This is one of oldest method
40. Comparison b/w Column and Planner
chromatography
■ It is hypothesized that in particular cases, conventional planar chromatography
provides a more effective and robust system than column chromatography with
regard to separation efficiency and peak distribution of mixtures composed of low-
retarded analytes.
■ Unlike column chromatography, the planar counterpart can work easily as a two-
dimensional (2-D) method. For that reason, this technique has a great ability to
separate a number of analytes that are components of complex biosamples including
blood, urine, tissue, sewage water, or sediments.
