Paper chromatography

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Paper Chromatography:

1 Paper Chromatography PRESENTED BY, Rucha V.Deshmukh M-Pharm 1 st year Dept. of Pharmaceutics


2 Contents Introduction Principle Types Experimental details Applications References


3 Chromatography A method of separating a mixture of components into individual components through the equilibrium distribution between two phases. Nondestructive separation in which separation of solute occur between a stationary and mobile phase. It is a technique for separating mixtures into their components in order to analyze, identify, purify & quantify the mixture or components.

Paper chromatography:

4 Paper chromatography Paper adsorption chromatography Paper impregnated with silica or alumina as adsorbent (stationary phase ) & solvent as mobile phase. Paper partition chromatography Moisture or water present in the pores of cellulose fibers present in filter paper acts as stationary phase & another mobile phase is used as solvent.

Concept of Paper partition:

5 Concept of Paper partition • Separates component on the basis or their partition coefficients. Type of partition chromatography Substances are distributed between two liquids. Stationary phase is water ,which is held in fibers of paper. Mobile phase is solvent The components of the mixture migrates at different rates and appears as spots.


6 Principle Capillary Action – the movement of liquid within the spaces of a porous material due to the forces of adhesion, cohesion, and surface tension.  The liquid is able to move up the filter paper because its attraction to itself is stronger than the force of gravity. Solubility – the degree to which a material (solute) dissolves into a solvent. This allows different solutes to be separated by different combinations of solvents. Separation of components depends on • Solubility in the mobile phase • Differential affinity

Migration parameter:

7 Migration parameter Distance travelled by solute from the origin R f = Distance travelled by solvent from origin line • R f is Retardation factor • R f value ranges from 0 to 1 • Ideal value 0.3 to 0.8 • Constant for every compound


8 Diagram


9 Types Ascending chromatography Descending chromatography Ascending-Descending chromatography Radial chromatography Two Dimensional

Ascending chromatography:

10 Ascending chromatography Allow the solvent to travel up the paper. Mobile phase at the bottom of the chamber. Samples are applied a few centimeters from the bottom edge of the paper suspended from hook.

Diagram :

11 Diagram

Descending chromatography:

12 Descending chromatography Allow the solvent to travel down the paper. A well sealed glass tank of suitable size and shape. Mobile phase in upper portion. Tank is equilibrated with mobile phase. The sample spotted paper is inserted with the upper end in the trough containing the mobile phase. Development can be continued indefinitely.

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13 Ascending Descending


14 Ascending-Descending Hybrid of ascending and descending. Ascending chromatography is changed to descending by crossing through a glass-rod. Length of separation is increased. First ascending takes place followed by descending development. Ascending Descending spots

Radial chromatography:

15 Radial chromatography Also called as circular chromatography. A circular filter paper is employed. Sample to be analyzed placed at the centre-dry the spots-Fix the paper horizontally on a Petri dish such that the tongue of the paper dips in to solvent. Cover the paper by means of Petri dish. The solvent rises to the tongue moves sufficient distance and the components get separated in the form of concentric circular zones. Instrumentation spots

Two dimensional chromatography:

16 Two dimensional chromatography A square or a rectangular paper is used. Sample is applied to one of the corner. Second development is performed at right angle to the direction of first drug.


17 Diagram

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18 Choice of proper chromatographic technique. Choice of filter paper. Proper developing solvent. Preparation of sample. Spotting Drying of chromatogram. Detecting or Visualising agent Quantitative analysis Qualitative analysis Experimental details of paper chromatography

2.Choice of filter paper:

19 2.Choice of filter paper Whatmann chromatographic papers are used. Coarser & faster –Whatmann 31 ET-apart Rf value. Slow papers- whatmann 20 schleicher & schull 2045, machery nagel 261 & erdrol 208 – for close R f values Heavy papers – whatmann 3 MM, schlelicher & schull 207 – preparative purpose.

Modified papers for paper chromatography:

20 Modified papers for paper chromatography Type Typical uses Carboxylic papers Cationic separation of protonated amines Acetylated papers Metal cations,Steroids,insecticides & pigments Kieselguhr, alumina, zirconia, silica Amines,fatty acids, steroids, triglycerides,vitamins Ion exchange paper Ion exchange paper chromatography

3.Proper developing solvent:

21 3.Proper developing solvent R f value should be between 0.05 – 0.85. Difference between R f value of any two components must be 0.05. Should not undergo any chemical reaction. Should not interfere with detection of spots. Composition of solvent system should not alter with time.

Suitable solvent systems:

22 Suitable solvent systems Stationary phase Mobile phase Water Isopropanol -ammonia-water (9:1:2) Water N-butanol -acetic acid-water (4:1:5) Formamide Chloroform Formamide Benzene Dimethyl formamide Benzene –cyclohexane(1:9) Phenoxy ethanol cyclohexane Kerosene 70% iso propanol

4.Preparation of samples :

23 4.Preparation of samples Sample volume of 10-20µ having as many µ gm of substance is that ideal quantity to be separated. 5.spotting Micro pipettes used for spotting the sample

6.Drying the chromatogram:

24 6.Drying the chromatogram The wet chromatogram after development are dried in special drying cabinets. 7. Detecting / Visualising agent 1 .Iodine chamber- amber/brown Colour. 2 .UV chamber for fluorescent compounds-254 nm or 365 nm Drying cabinet

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25 3.Specific methods- The following reagents are used for spraying… A. Ferric chloride – Phenolic & tannins B .Ninhydrin – Amino acids C .Dragandroff’s reagent – Alkaloids D .2,4-Dinitrophenyl hydrazine- Aldehydes & Ketones E .3,5-Dinitro benzoic acid – Cardiac glycosides

8.Quantitative analysis:

26 8.Quantitative analysis Direct techniques A) By Visual assessment B) By measurement of areas C) By densitometer D) Potentiometry Indirect techniques A) Gravimetric estimation B) UV spectrophotometry C) Colorimetry D) Polarography E) Radioactivity In situ /No destruction of sample

9.Qualitative Analysis:

27 9.Qualitative Analysis Distance travelled by solute from the origin R f = Distance travelled by solvent from origin line Distance travelled by the substance from the origin line R x = Distance travelled by the standard substance x from the origin line


28 Applications Pharmaceutical Company – determine amount of  each chemical found in new product Hospital – detect blood or alcohol levels in a  patient’s blood stream Law Enforcement – to compare a sample found at  a crime scene to samples from suspects Environmental Agency – determine the level of  pollutants in the water supply Manufacturing Plant – to purify a chemical  needed to make a product

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29 Separation of mixtures of drugs of chemical or biological origin , plant extracts. Separation of carbohydrates ,vitamins, antibiotics, proteins, alkaloids, glycosides, amino acids. Identification of impurities DRUG MOBILE PHASE DETECTING AGENT Hydroxocobalamine S butyl alcohol: acetic acid :pot cyanide Elution & measurement of abs at 361nm

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30 DRUG MOBILE PHASE DETECTING AGENT Erythromycin estolate Isobutyl methyl ketone Nutrient agar containing bacillus pumilus Gentamycin Chloroform : methanol: ammonia : water(10:5:3:2) Ninhydrin in pyridine acetone mixture Vancomycin T-methyl alcohol : acetone : water (2:1:2) Nutrient agar containing bacillus subtilis Identification of drugs

Identification of related compounds:

31 Identification of related compounds DRUG MOBILE PHASE DETECTING AGENT Phenformin HCl Ethyl acetate :ethanol :water(6:3:1) Potassium ferricyanide. Sod nitroprusside & NaOH Ergotamine injection Chloroform : methanol P-dimethyl amino benzaldehyde reagent Vitamin A Dioxan :methanol :water(70:15:5) UV 366nm


32 References “ Instrumental Methods Of Chemical Analysis”, Gurdeep R. Chatwal & Sham K.Anand....Himalaya publishing house. “Textbook of Pharmaceutical Analysis”, S. Ravi Shankar…RX publications. “Pharmaceutical Analysis”, volume 2…Instrumental methods by Dr.A V.Kasture. Dr. K R.Mahadik, Dr.S G.Wadodkar,Dr.H N. More.

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33 That's it..... Thank you!!

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