PAPER CHROMATOGRAPHY

Paper Chromatography

most common type of chromatography; uses filter paper as a solid support for a liquid medium such as water or alcohol; separates a liquid sample into components according to their different abilities to climb up a strip of paper

structure, molecular size, weight, and polarity of the compound

separation of sample compounds can depend on:

Paper adsorption chromatography and paper partition chromatography

Types of paper chromatography

Paper adsorption chromatography

paper impregnated with silica or alumina acts as adsorbent (stationary phase) and solvent as mobile phase

Paper partition chromatography

moisture/water present in the pores of cellulose fibers present in filter paper acts as stationary phase and another mobile phase is used as solvent

Chromatography

involves physical separation of the different chemical substances in a mixture based on the different speeds at which they travel through a stationary material or medium

high selectivity

chromatographic methods of separation are distinguished by this; ability to separate components of closely similar physical and chemical properties

partition rather than adsorption

Principle of separation

Stationary phase and papers used

Application of sample

Mobile phase

Development technique

Detecting or Visualizing agents

Practical Requirements

Whatman filter paper

contains 98-99% of alpha-cellulose, 0.3-1% beta-cellulose

Nature of sample and solvents used

Based on quantitative and qualitative analysis

Based on thickness of the paper

Factors that govern the choice of paper

Modified papers, Hydrophilic papers, Hydrophobic papers

Other types of papers

Modified papers

acid or base washed filter paper, glass fiber type paper

Hydrophilic papers

papers modified with methanol, formamide, glycol, glycerol, etc.

Hydrophobic papers

acetylation of OH groups leads to hydrophobic nature, hence, can be used for reverse phase chromatography

Polar solvents

commonly employed solvents

Hydrophilic and Hydrophobic

Types of Mobile phase

Hydrophilic Mobile Phase

isopropanol, ammonia, methanol, water

Hydrophobic Mobile Phase

dimethyl ether, cyclohexane, kerosene

Ascending Development, Descending Type, Ascending-Descending Development, Circular/Radial Development, Two-dimensional Development

Types of Development Technique

Ascending Development

like conventional type, the solvent flows against gravity. the spots are kept at the bottom portion of paper and kept in a chamber with mobile phase solvent at the bottom

Descending Type

carried out in a special chamber where the solvent holder is at the top; the spot is kept at the top and the solvent flows down the paper; advantage is that, development is faster

Ascending-Descending Development

a hybrid of the two techniques; only length of separation is increased; first ascending takes place, followed by descending

Circular/Radial Development

spot is kept at the center of a circular paper; solvent flows through a wick at the center and spreads in all directions uniformly

Two-dimensional Development

paper is developed in one direction and after development, the paper is developed in the second direction allowing more compounds to be separated into individual spots; in the second direction, either the same solvent/different solvent system can be used for development

non specific methods (physical methods) and specific methods (chemical methods) or spraying method

Detecting methods

non specific methods (physical methods)

iodine chamber method; UV chamber

Specific methods (chemical methods) or spraying method

ninhydrin in acetone, dragendroff’s reagents, amino acids, alkaloids

Direct Measurement Techniques and Indirect Measurement Techniques

Other detecting techniques

Comparison of visible spots, photo densitometry, fluorimetry, radiotracer method, polarographic & conductometric methods

Comparison of visible spots

rough quantitative measurement; component in a mixture can be carried out by comparing the intensity and size of the spot with a standard substance

Photo densitometry

used for colored compounds; instrument measures quantitatively the density of the spots

Fluorimetry

the compound to be determined by fluorimetry must be fluorescent or convertible into fluorescent derivatives

Radiotracer Method

the compound containing radioactive element is labeled and treated with locating reagent; using Geiger Muller counter

Polarographic & Conductometric methods

used to measure the amount of material in the spot

Retardation factor/Retention factor

Indirect Measurement Techniques

Retardation/Retention factor

ratio between the distance travelled by the solute to the distance travelled by the solvent front; represents the movement or migration of solute relative to the solvent

Temperature

Purity of the solvents used

The quality of the paper, adsorbents and impurities present in the adsorbents

Chamber saturation techniques, method of drying and development

Distance travelled by the solute and solvent

Chemical reaction between the substances being partitioned

pH of the solution

Factors affecting the Rf value

Development, Amount of sample, Touching the chromatography paper

Sources of Error

Development

improper adjustment of the paper in the tanks leads to this error so the paper should be held vertically

Amount of sample

applying too much sample may results in large bands of chemical moving up the plate, making it difficult to accurately measure the distance the chemical can be transported; too much sample will cause the spots to streak together (the compounds will overlap)

Touching the chromatography paper

when you handle this paper, hold it only on one of the long sides, which will be considered the top sheet. the amino acids from your fingers will contaminate the paper and lead to erroneous results if it is touched on the bottom

simple, rapid, inexpensive

Advantages of Paper Chromatography

establishing the vapor solvent equilibrium, stability of solvent mixture

Disadvantages of Paper Chromatography

Separation of mixtures of drugs

Separation of carbohydrates, vitamins, antibiotics, proteins

Identification of drugs

Identification of impurities

Analysis of metabolites of drugs in blood, urine

Applications

Pathology and Forensic Science, Qualitative Analysis

Uses of Paper Chromatography

Pathology and Forensic Science

for investigation if crimes, because this process can be successfully carried out even with very small quantities of material; samples from crime scenes are collected to be analyzed and identified, using this technique

used in DNA and RNA fingerprinting, and to detect the presence of alcohol or chemicals in blood

Qualitative Analysis

one of the methods of qualitative analysis, to analyze or separate the different constituents of a mixture, it is a useful tool for separating polar as well as nonpolar solutes; pharmaceutical companies use this technique to analyze the different compounds in drugs.

Used in the testing of antibiotics and determining the pollutants in water