Paper Chromatography PDF

## PAPER CHROMATOGRAPHY The name "chromatography" comes from the Greek word "khroma" meaning colour. The technique was first used to separate colours, pigments and dyes. Nowadays, it can be used to separate colourless substances. Paper chromatography is a very useful technique for separating small...

## PAPER CHROMATOGRAPHY The name "chromatography" comes from the Greek word "khroma" meaning colour. The technique was first used to separate colours, pigments and dyes. Nowadays, it can be used to separate colourless substances. Paper chromatography is a very useful technique for separating small quantities of very similar samples. The chromatography paper with the separated components is called a chromatogram. ### Question II: How do we distinguish whether a sample is pure or impure using paper chromatogram? A pure sample gives only one spot on a chromatogram Paper chromatography is only one type of chromatography, other chromatography techniques include gas chromatography, thin layer chromatography etc. The big advantage of chromatography is that it can be used to identify very tiny amounts of substances. Some chromatography can detect less than 1 picogram (or 10^-12 g) of substance. ### Applications of chromatography techniques: * To separate and identify types of sugars (such as sucrose, maltose, and glucose) in fruit juice. * To separate and identify types amino acids in protein samples. * To detect illegal drugs used to colour drinks/food. * To detect illegal chemicals used in food. * To detect excessive use of drugs by athletes eg anabolic steroids, beta blockers found in urine samples * Screening on national service personnel to detect drugs through urine samples ### Principle of Chromatography: The dyes move up at different rate because of the difference in: 1. solubility in the solvent 2. absorbance on the paper The greater the solubility of a solute in the mobile solvent, the faster the solute moves up the paper the higher is the spot on the paper. As different solutes have different solubility, they move up the paper at different rates. Thus, a mixture of solutes can be separated. Some substances in the mixture are more adsorbed onto the surface of the paper. As the mobile solvent travels up, the more strongly adsorbed substances are held back onto the paper and thus travel slower while the less strongly adsorbed move on ahead. As a result, a separation takes place. (Note: Do not confuse the term "adsorbed" with "absorbed".) * Adsorbed - attracted to the surface of the paper * Absorbed- penetration into entire body eg water in sponge ### Mobile solvent: usually organic solvent (comes from living things, contains carbon) e.g ethanol, butanol or a mixture of butanol, ethanoic acid and water. The decision on which solvent to use depends on type of substance being separated. ### Water: Water dissolves solutes which are less soluble in organic solvent while solutes are less soluble in water if they dissolve easily in organic solvent. Solute that is strongly adsorbed onto a chromatographic paper dissolves better in water than in mobile organic solvent. ### Procedure: 1. A base line (starting line) is drawn on a piece of chromatography paper with a pencil. ### Question III: Why? an ink pen is not used because the ink is a mixture which can be dissolved in certain solvents and cause unwanted spots. This will interfere with the wanted, separated spots.) 2. The starting point must be a small spot. Spot some of the mixture on the pencil line. Allow the spot to dry. Repeat by spotting 2 or 3 times on same spot. ### Question IV: Why? Otherwise, the ink spot will smudge and become big. The separated dyes will later produce big spots that overlap each other, leading to bad separation. 3. Place the paper in a boiling tube or beaker with some solvent. Hold the paper upright using cork pieces. ### Question V: Why? The spots will then travel vertically upwards and not deviate to the sides 4. The solvent level should be below the pencil line. ### Question VI: Why? Otherwise, the ink mixture spot will immerse and subsequently dissolve in the solvent. It will not travel up the paper readily. 5. Place set-up in a test tube stand to avoid tilting and disturbance of solvent and solute movement. Cover the container. There should be no wind current that could cause rapid evaporation of organic solvent which is normally volatile. 6. The solvent is allowed to move up the paper by capillary action. The ink and dyes dissolve in the solvent and also move up the paper at different speeds and hence different distances. 7. Remove the chromatography paper when the solvent has reach near to the top edge of the paper. Use pencil to draw a line on the location where the solvent stops. This line is called the solvent front. 8. The resulting chromatography paper with separated spots of colours on it is called a **chromatogram**. ### Identifying of unknown sample from chromatogram: * The retention factor (Rf value) = distance travelled by the solute / distance travelled by the solvent front * Rf value is constant if the temperature is constant and solvent is fixed. * After the spots are located and their Rf values are measured, their identity can be obtained by comparing to the Rf values known pure substances. * Each dye/ sugar/ amino acid has its own Rf. It differs with different solvent. * Rf <1. Eg Glucose = 0.57. * Can solvent front vary in different experiment? Yes * Rf is a constant by assuming same solvent is used. In different experiments run at different length of time, distance traveled by dye changes proportionately with distance traveled by solvent to give a constant Rf. ### Separating colourless substances: 1. A locating agent can be sprayed on the chromatogram of colourless substances to show where they are on the paper. A locating agent is a chemical that will react with the colourless substances to produce visible coloured product. * Examples: ninhydrin spray can react with colourless amino acids to produce a purple stain; spray of silver nitrate solution followed by sodium hydroxide make colourless sugars appears brown. 2. Some substances appear colourless in ordinary light but fluorescence under ultra-violet light. Hence ultra-violet light can be used to locate colourless spots on the chromatogram.