Chromatography
Chromatography is a combination of laboratory techniques that are used for separating
the mixture components.
General Principles
Adsorption
Partition
Theories of Chromatography
Plate Theory
Rate or Kinetic Theory
Development of Chromatogram
Frontal Analysis
Elution Analysis
Gradient elution
Displacement Analysis
COLUMN CHROMATOGRAPHY
Adsorption column chromatography
Partition Column Chromatography
Ion Exchange Chromatography
Gel Chromatography
Methodology
Selection of column
election of adsorbents and solvents
Preparation of column
Application of sample
Shahnawaz Ansari
19PH09
Elution procedures
Detectors
Wet Packing or Slurry Method
Dry Packing
Isocratic Elution
Stepwise or Fractional Elution
Gradient Elution
Advantages
1) It can be used to separate any type of mixture.
2) It can be used to separate any quantity of mixture.
3) A wide range of mobile phases can be used.
4) The sample can be separated and reused.
5) Automation is also possible.
Disadvantages
1) It is a time-consuming method.
2) It is an expensive method as it utilises a large amount of solvent.
3) Automation is possible and this makes the technique complicated and more expensive.
Applications
Analytical Uses
Separation of Geometrical Isomers
Separation of Diastereomers
Separation of Tautomeric Mixtures
Separation of Racemates
:THIN LAYER CHROMATOGRAPHY
1) Normal Phase TLC: In this type, the stationary phase is polar and the mobile phase
is non-polar.
2) Reverse Phase TLC: In this type, the stationary phase is non -polar and the mobile
phase is polar.
Advantages
Simple Equipment
Short Development Time
Wide Choice of Stationary Phase
Early Recovery of Separated Components
Separation Effects
Easy Visualisation of Separated Compounds
Sensitivity
Variable Thickness of Thin Layers
Chemically Inert Stationary Phase
Disadvantages
1) There is a no longer stationary available in TLC plates, therefore, its separation
length is insufficient in comparison to other chromatographic techniques.
2) The results obtained are difficult to reproduce.
3) Only soluble components of the mixtures can be separated.
4) It is not automatic.
5) It works in the open system, thus can get affected by temperature and humidity.
METHODOLOGY
1) Selection of coating materials or adsorbents,
2) Preparation of plates,
3) Activation of adsorbent,
4) Purification of silica gel G layers,
5) Marking the plate and spotting of the sample,
6) Preparation of the development tank,
7) Selection of solvent system,
8) Plate development,
9) Detection of components - Rf value,
10) Evaluation of the chromatogram, and
11) Recovery of components.
Types of Development in Thin Layer Chromatography
Ascending Development
Descending Development
Horizontal Development
Stepwise and Multiple/Repeated Development
Two-Dimensional Development
Preparative TLC
Gradient Elution
Reverse Phase TLC
APPLICATIONS
Qualitative Estimation
Quantitative Estimation
As a Check on Processes
In Organic Chemistry
Applications of TLC for Separation of Inorganic Ions
Separation of Amino Acids
Separation of Vitamins by Thin -layer Chromatography
Photodensitometry or Visual Comparison
Measurement of Spot Areas
PAPER CHROMATOGRAPHY
Principle
Paper Partition Chromatography
Paper Adsorption Chromatography
Development Techniques
Descending chromatography
Ascending chromatography
Ascending-descending chromatography
Radial paper chromatography
Two-dimensional chromatography
Methodology
Choice of the proper chromatographic technique
Choice of the filter paper
Choice of solvent
Preparation of sample
Spotting
Drying the chromatogram
Detection of spots
Calculation of RF values
Advantages
It needs fewer quantitative materials to carry a separation.
It requires less time for the separation of compounds.
It requires a small amount of sample for analysis.
It is very convenient to use.
Its setup can be installed even in a small space.
Disadvantages
1) It cannot be used to separate volatile substances.
2) It is not compatible with large amounts of sample.
3) It is not useful for quantitative analysis of the compounds.
4) It cannot be used to separate complex mixture.
5) It has less accuracy as compared to HPLC or HPTLC methods.
6) In this technique, the data cannot be saved for long periods.
Applications
1) Separate plant pigments, e.g., chlorophyll a and b,
2) Determine sequence in the DNA and RNA molecules,
3) Determine sequence of the amino acid in proteins,
4) Detect forensic samples,
5) Separate sugar molecules,
6) Separate vitamins,
7) Separate antibiotics,
8) Analyse drug metabolites in blood and urine samples,
9) Detect unknown compounds, and
10) Determine the insecticides in food components.