Separation of biomolecules by paper Saher Islam Chromatography

Separation of biomolecules by paper Saher Islam

Chromatography is a method for separating mixtures into their components based on physical and/or chemical properties of the components. Developed around 1903 by Russian Mikhail Semenovich Tswett in which he separated plant pigments on “diatomaceous earth” with alcohol

Uses for Chromatography can be used to: Qualitatively analyze the components of a mixture Qualitatively identify the components of a mixture using known compounds Quantitatively determine the amount of a component in a mixture using standard samples Purify individual components by separating them from the other compounds in a mixture

The Basics Mixture is placed on stationary phase Mobile phase passes over the stationary phase Mobile phase dissolves the components Mobile phase carries the individual components a certain distance through the stationary phase, depending on their attraction to both of the phases

Paper Chromatography • Paper chromatography (PC) is a type of a planar chromatography whereby chromatography procedures are run on a specialized paper. • PC is considered to be the simplest and most widely used of the chromatographic techniques because of its applicability to isolation, identification and quantitative determination of organic and inorganic compounds. • Paper chromatography was invented by two British biochemists, Archer John Porter Martin (1910 -) and Richard Laurence Millington Synge (1914 -).

Paper Chromatography

Principles of Paper Chromatography • 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. Solutes dissolve into solvents that have similar properties. This allows different solutes to be separated by different combinations of solvents. • Separation of components depends on both their solubility in the mobile phase and their differential affinity to the mobile phase and the stationary phase.

Principles of Paper Chromatography • The principle of separation is mainly partition rather than adsorption. Substances are distributed between a stationary phase and mobile phase.

Principles of Paper Chromatography • Cellulose layers in filter paper contain moisture which acts as stationary phase. • Organic solvents/buffers are used as mobile phase. • The developing solution travels up the stationary phase carrying the sample with it. • Components of the sample will separate readily according to how strongly they adsorb onto the stationary phase versus how readily they dissolve in the mobile phase.

Instrumentation of Paper Chromatography • • Stationary phase & papers used Mobile phase Developing Chamber Detecting or Visualizing agents 1. STATIONARY PHASE AND PAPERS Whatman filter papers of different grades like No. 1, No. 2, No. 3, No. 4, No. 20, No. 42 etc In general the paper contains 98 -99% of α-cellulose, 0. 3 – 1% β -cellulose. Other 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. Impregnation of silica, alumna, or ion exchange resins can also be made.

Instrumentation of Paper Chromatography 2. PAPER CHROMATOGRAPHY MOBILE PHASE Pure solvents, buffer solutions or mixture of solvents can be used. Examples. Hydrophilic mobile phase Isopropanol: ammonia: water 9: 1: 2 Methanol : water 4: 1 N-butanol : glacial acetic acid : water 4: 1: 5 Hydrophobic mobile phases dimethyl ether: cyclohexane kerosene : 70% isopropanol The commonly employed solvents are the polar solvents, but the choice depends on the nature of the substance to be separated. If pure solvents do not give satisfactory separation, a mixture of solvents of suitable polarity may be applied.

Instrumentation of Paper Chromatography 3. CHROMATOGRAPHIC CHAMBER • The chromatographic chambers are made up of many materials like glass, plastic or stainless steel. Glass tanks are preferred most. • They are available invarious dimensional size depending upon paper length and development type. • The chamber atmosphere should be saturated with solvent vapor

Steps in Paper Chromatography In paper chromatography, the sample mixture is applied to a piece of filter paper, the edge of the paper is immersed in a solvent, and the solvent moves up the paper by capillary action. The basic steps include: Selection of Solid Support Fine quality cellulose paper with defined porosity, high resolution, negligible diffusion of sample and favouring good rate of movement of solvent. Selection of Mobile Phase Different combinations of organic and inorganic solvents may be used depending on the analyte. Example. Butanol: Acetic acid: Water (12: 3: 5) is suitable solvent for separating amino-acids.

Steps in Paper Chromatography Saturation of Tank The inner wall of the tank is wrapped with the filter paper before solvent is placed in the tank to achieve better resolution. Sample Preparation and Loading If solid sample is used, it is dissolved in a suitable solvent. Sample (2 -20 ul) is added on the base line as a spot using a micropipette and air dried to prevent the diffusion. Development of the Chromatogram Sample loaded filter paper is dipped carefully into the solvent not more than a height of 1 cm and waited until the solvent front reaches near the edge of the paper.

Steps in Paper Chromatography: Different types of development techniques 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 This is 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. In this method solvent moves from top to bottom so it is called descending chromatography.

Steps in Paper Chromatography: Different types of development techniques Two Dimensional injected sample is separated by passing through two different separation stages CIRCULAR / RADIAL DEVELOPMENT • Spot is kept at the centre of a circular paper. • The solvent flows through a wick at the centre & spreads in all directions uniformly. .

Steps in Paper Chromatography Drying of Chromatogram After the development, the solvent front is marked and the left to dry in a dry cabinet or oven. Detection • Colourless analytes detected by staining with reagents such as iodine vapour, ninhydrin etc. • Radiolabeled and fluorescently labeled analytes detected by measuring radioactivity and florescence respectively.

Rf Values • Some compounds in a mixture travel almost as far as the solvent does; some stay much closer to the base line. • The distance travelled relative to the solvent is a constant for a particular compound as long as other parameters such as the type of paper and the exact composition of the solvent are constant. .

Rf Values • The distance travelled relative to the solvent is called the Rf value. • Thus, in order to obtain a measure of the extent of movement of a component in a paper chromatography experiment, “Rf value” is calculated for each separated component in the developed chromatogram. • An Rf value is a number that is defined as distance traveled by the component from application point.

Preparing the Chromatography Paper • Obtain an 7 x 8 cm of chromatography paper • With a pencil, draw an origin line 1 cm above the bottom edge of the strip. Near the top, ID the chromatogram • Mark the origin line with a lane mark each cm. No lane can be closer than about 1 cm from the edge

Preparing the Chromatography Paper • ID each lane and record what each lane is spotted with • Place a spot from each solution on the origin • More than one strip may be required to analyze all of the solutions

Developing the Chromatograms • Add chromatographic solvent to a sufficiently large beaker to a depth of less than 1 cm • Place the spotted paper in the beaker carefully • Ensure the solution does not go above the origin line • Develop the chromatogram until the solvent front is about 2 cm from the top of the paper

Developing the Chromatograms • Remove the chromatogram and place on a paper towel, trace the solvent front with pencil, and let the chromatogram dry in the oven for a few minutes. • After the chromatogram is dry, calculate the retention factors, Rf, and tabulate the values • Determine the dye content of each of the “unknowns”

Applications of Paper Chromatography • To check the control of purity of pharmaceuticals • For detection of adulterants • Detect the contaminants in foods and drinks • For the detection of drugs and dopes in animals & humans • In analysis of cosmetics • Analysis of the reaction mixtures in biochemical labs

Advantages of Paper Chromatography • • Simple Rapid Paper Chromatography requires very less quantitative material Paper Chromatography is cheaper compared to other chromatography methods Both unknown inorganic as well as organic compounds can be identified by paper chromatography method Paper chromatography does not occupy much space compared to other analytical methods or equipment Excellent resolving power

Limitations of Paper Chromatography • Large quantity of sample cannot be applied on paper chromatography. • In quantitative analysis paper chromatography is not effective. • Complex mixture cannot be separated by paper chromatography. • Less Accurate compared to HPLC or HPTLC

Links • https: //www. youtube. com/watch? v=Td. J 57 SQ 6 GAQ • https: //www. youtube. com/watch? v=mz_xc. Nr TK_U