1. Lab Activity 7 IUG, Fall 2012 Dr. Tarek Zaida IUG, Fall 2012 Dr. Tarek Zaida 1

2. Chromatography 2

3. Definition A physical method of separation in which the components to be separated are distributed between two phases: A stationary phase and a mobile phase that moves in a definite direction. Definition A physical method of separation in which the components to be separated are distributed between two phases: A stationary phase and a mobile phase that moves in a definite direction. 3

4. Types of Chromatography Types of chromatography Based on stationary phase Column Planar Based on mobile phase Liquid Gas 4

5. Planar Chromatography A separation technique in which the stationary phase serves as a plane. The plane can be either a paper (paper chromatography) or a layer of solid particles spread on a support such as a glass- or a plastic- plate (thin layer chromatography). A separation technique in which the stationary phase serves as a plane. The plane can be either a paper (paper chromatography) or a layer of solid particles spread on a support such as a glass- or a plastic- plate (thin layer chromatography). 5

6. Thin Layer Chromatography (TLC) Principle  Different compounds in sample mixture travel different distances according to how strongly they interact with the stationary phase as compared to the mobile phase.  The specific Retention factor (R f ) of each chemical can be used to aid in the identification of an unknown substance. Principle  Different compounds in sample mixture travel different distances according to how strongly they interact with the stationary phase as compared to the mobile phase.  The specific Retention factor (R f ) of each chemical can be used to aid in the identification of an unknown substance. 6

7. Measuring R f 7

8. Determination of amino acids using thin layer chromatography Development of Ruhemann’s purple from ninhydrin and amino acid. 8

9. Instruments, chemicals and glassware Eluent1. [Mix n-butanol, acetic acid (purity 98 – 100 %) and distilled water in volume ratio 5:1:5. Stir for 10 minutes, then let the layers separate. Use upper layer as eluent]. Developing Solution: Dissolve 0.3 g of ninhydrin in 100 ml n- butanol. Add 3 ml of glacial acetic acid. 0.2% in 10% isopropyl alcohol solutions of diferrent amino acids (e.g. leucine, methionine, alanine and serine) and mixture of these amino acids. Chromatographic paper Elution chamber, Glass capillaries for spotting the samples. Drying oven at ~ 60° C. Instruments, chemicals and glassware Eluent1. [Mix n-butanol, acetic acid (purity 98 – 100 %) and distilled water in volume ratio 5:1:5. Stir for 10 minutes, then let the layers separate. Use upper layer as eluent]. Developing Solution: Dissolve 0.3 g of ninhydrin in 100 ml n- butanol. Add 3 ml of glacial acetic acid. 0.2% in 10% isopropyl alcohol solutions of diferrent amino acids (e.g. leucine, methionine, alanine and serine) and mixture of these amino acids. Chromatographic paper Elution chamber, Glass capillaries for spotting the samples. Drying oven at ~ 60° C. 9

10. Procedure 1.Rubber gloves must be used during this work to avoid contamination of chromatographic paper with amino acids from skin, and for protecting skin from solvents and ninhydrin while working with the sprayer or sprayed paper. 2.While the paper is being prepared for chromatographic analysis it should be kept on a piece of filter paper. 3.Mark the starting line to the paper - 8-9 mm from the edge of the plate - with graphite pencil (very slight line!). Also mark the locations where the samples will be spotted. The distance between neighboring spots should be about 8 mm and the spots should be at least 5 mm away from the paper’s edge. Usually the spot of unknown substance is applied to the center of the starting line. 1.Rubber gloves must be used during this work to avoid contamination of chromatographic paper with amino acids from skin, and for protecting skin from solvents and ninhydrin while working with the sprayer or sprayed paper. 2.While the paper is being prepared for chromatographic analysis it should be kept on a piece of filter paper. 3.Mark the starting line to the paper - 8-9 mm from the edge of the plate - with graphite pencil (very slight line!). Also mark the locations where the samples will be spotted. The distance between neighboring spots should be about 8 mm and the spots should be at least 5 mm away from the paper’s edge. Usually the spot of unknown substance is applied to the center of the starting line. 10

11. 4.Before applying samples to the paper and filling the elution chamber fit the length of chromatographic paper with the height of elution chamber. 5.The spots of individual amino acids and sample solutions are applied to the chromatographic paper. Use separate clean and dry glass capillary for each solution. Dip the capillary into solution – some solution is drawn into the capillary. With the filled capillary touch the prepared location on chromatographic paper. The spot on the paper should not be bigger than 2-3 mm. (You can exercise spotting on a sheet of filter paper.) 6.After application of samples let the spots dry. Meanwhile measure with a graduated test-tube 5 ml of eluent into the elution chamber. Cover the chamber with lids and let the chamber atmosphere saturate with eluent vapors for at least 10 min. 4.Before applying samples to the paper and filling the elution chamber fit the length of chromatographic paper with the height of elution chamber. 5.The spots of individual amino acids and sample solutions are applied to the chromatographic paper. Use separate clean and dry glass capillary for each solution. Dip the capillary into solution – some solution is drawn into the capillary. With the filled capillary touch the prepared location on chromatographic paper. The spot on the paper should not be bigger than 2-3 mm. (You can exercise spotting on a sheet of filter paper.) 6.After application of samples let the spots dry. Meanwhile measure with a graduated test-tube 5 ml of eluent into the elution chamber. Cover the chamber with lids and let the chamber atmosphere saturate with eluent vapors for at least 10 min. 11

12. 7.Elution is stopped when the solvent front has traveled up the plate until 7-10 mm from the lid. 8.Remove the paper from elution chamber and place it on a sheet of filter paper. After 2-3 minutes mark the eluent front with pencil and dry the paper in oven. 9.When the paper is dry, take it into the fume hood and spray it with solution of ninhydrin until the paper is slightly damp. Chromatographic paper and the paper supporting it should lie at 45° angle while spraying. The chromatographic paper is again put in the drying oven (60 C) for 15 min to speed up the reactions. 7.Elution is stopped when the solvent front has traveled up the plate until 7-10 mm from the lid. 8.Remove the paper from elution chamber and place it on a sheet of filter paper. After 2-3 minutes mark the eluent front with pencil and dry the paper in oven. 9.When the paper is dry, take it into the fume hood and spray it with solution of ninhydrin until the paper is slightly damp. Chromatographic paper and the paper supporting it should lie at 45° angle while spraying. The chromatographic paper is again put in the drying oven (60 C) for 15 min to speed up the reactions. 12

13. 10.Remove chromatographic paper from drying in the oven, draw the contours and centers of the chromatographic bands. Calculate R F values by the method described above. 11.Compare retention of standard substances and components in sample and determine which amino acids were present in the sample. 10.Remove chromatographic paper from drying in the oven, draw the contours and centers of the chromatographic bands. Calculate R F values by the method described above. 11.Compare retention of standard substances and components in sample and determine which amino acids were present in the sample. 13

14. TLC For Sugars The procedure will be repeated with sugars, with the difference that the spots at the end will be developed by using Aniline hydrogen phthalate instead of Ninhydrine. Chromatographs will be dried in oven at 100C. The procedure will be repeated with sugars, with the difference that the spots at the end will be developed by using Aniline hydrogen phthalate instead of Ninhydrine. Chromatographs will be dried in oven at 100C. 14

15. quiz 1. Write down the reactions on which the determination of vitamin C is based. 15

16. 2. The principle of kjeldahl method? 16

17. 3. If you were given a food sample that weighs 10 g. How would be the percentage of protein in this sample if you found that its nitrogen content was 200 mg? 17