Question

what is the prediction of the unknown amino acids mixture based on the strip from thin layer chromatography sheet?

The unknown mixtures can be ala plus asp, ala plus asp plus lys, lys plus asp or ala plus lys.

anion exchange resin was used

Tubes 2,3, 6 and 7 had a spot appear that was light pink

H-) to the column followed by a sufficient amount of the slurry of Dowex-l-CI (anion exchange) to give a column height of 5 inches 3. Allow the resin time to settle (5-10 minutes). Allow the phosphate buffer to run through the column slowly as the resin settles, but do NOT let the liquid fall below the surface of the resin. 4. Close the valve. 5. You will be provided with one of four mixtures of amino acids containing either: ala plus asp, ala plus asp plus lys, lys plus asp, or ala plus lys ( you will not know the makeup prior to running the column 6. Add 1 ml of the amino acid mixture to the column. 7. Let the solution run into the resin until the surface of the liquid falls just below the top of the resin and then stop the flow. 8. Carefully add 10 mls of 0.005M NaH2PO4 buffer pH 7.5 to the column, being careful not to disturb the top of the resin. 9. Release the clamp and collect five 2 ml fractions by letting the eluate run into the test tubes previously marked at a volume of 2 ml. After the last fraction has been collected clamp the tubing 10.carefully add 14 ml of 0.1 M HCI to the column. 9nh an N.B. Add the first ml of HCI in 4 x 0.25 ml portions, allow each portion to run into the column before adding the next > Then carefully top up with the remaining 13 ml of HCI 11.Collect seven fractions as above in 2 ml aliquots o Resin can be removed from the buret and placed in beaker as designated by the TA when time permits 12. Using pH paper test each of the seven fractions collected after changing the eluting solvent to HCI to determine the first tube in which the eluate has an acid pH. 13. In order to identify the fractions which contain amino acids carefully spot 20 μ1 of the first 5 fractions and 40 μ1 of the second seven fractions onto a strip of a cellulose thin layer chromatography sheet 14.Keep the spot as small as possible by adding only 5 ul at a time (use the automatic pipettes) and allowing the spot to dry betweern additions. Drying may be hastened by using a hair dryer. Spotting may be done at the same time that you are collecting. The TA will spray the sheet with ninhydrin reagent (USE THE FUME HOOD) and place strip in glass Petri dish or beaker in the oven (in S133) at 2500F for 10 min. Amino acids should develop a pinkish-purple color 15 Label the tubes from the elution that contain the amino acids (record results in your manual). B. Thin layer chromatography 1. Prepare a thin layer plate of cellulose MN 300 for chromatography by carefully marking 7 equally spaced dots 2 cm from the bottom of the plate. N.B.:WORK ON A CLEAN SURFACE AND DO NOT TOUCH THE PAPER WITH YOUR HANDS EXCEPT ON THE EDGES 2. Spot the 2 fractions containing the highest concentration of amino acids obtained from the ion exchange column (use 12 ul of the sample from the fraction eluted with phosphate buffer and use 20 μ1 of the fraction eluted with HCI.) in adjacent spots at the origin and carefully mark the fraction number in pencil. Were amino acids present in both sets of eluents? > In order to keep the spots small apply no more than 2 μ1 at a time and dry with a hair dryer between applications. 3. In adjacent columns spot 5 ul of each of the standard amino acio solutions, lys, asp and ala (1 mg/ml). 2 groups (4 STUDENTS) should share each chromatographic plate. Hand the chromatogram in to the TA They will be run in the next lab period. Solvent frent Development chamber 苕 Developing solvenit Origin Iine Shown above is the general procedure for the separation of the amino acids from the origin. Consult your lecture notes for further information DAY 2 4. Chromatograms will be handed back at the start of the lab period. 5. They should be placed as soon as possible in chromatographic tanks which will have been pre-equilibrated with the following solvent system (likely done for you by the technician prior to the start of the lab) propanol 40 99% formic acid Solvent: : water (pH 2.5) :

Answer 1

Dowex ICI is the anion exchanger resin which is mentioned in the above procedure

It's alanine plus lysine . Since it's an anion exchange aspartic acid is acidic in nature. It will not be well suited fir this