CHEM 109C – Zhang CLAS, F10 Amino Acids II: Separation - KEY 1. Explain the order of elution with a buffer pH = 4 of each pair through a column containing Dowex 50. Dowex 50 is a cation-exchange resin, so it binds cations most strongly. a. Asp and Ser O O - O O @ pH 4, Asp = - + NH3 overall negative and Ser = O HO O - + NH3 overall neutral. Since Dowex 50 will bind cations more strongly, it will repel anions and Asp will elute first. b. Tyr and Phe O O - + NH3 @ pH 4, Tyr = HO overall neutral and polar O O - + NH3 and Phe = overall neutral and nonpolar. Since Dowex 50 resin has a nonpolar nature, more nonpolar molecules like Phe will elute first. 2. Give the order for amino acid elution with a buffer pH = 4 from a column of Dowex 1 for a mixture of His, Ser, Asp, Val. Dowex 1 is an anion-exchange resin, so it binds anions most strongly @ pH 4, O O - O + O Asp = NH3 CH3 overall negative and polar → elutes fourth/last O H3C O + Val = - NH3 - overall neutral and nonpolar → elutes third Page 1 of 4 CHEM 109C – Zhang CLAS, F10 Amino Acids II: Separation - KEY O HO O - + NH3 Ser = overall neutral and polar → elutes second O H+ N O - + NH3 N H His = overall positive and polar → elutes first 3. Identify the location and type of charge on the hexapeptide Lys-Ser-Asp-Cys-His-Tyr pKa = 10.79 NH2 pKa = 3.86 OH NH N O O NH H2N pKa = 8.95 pKa = 6.04 O O NH NH NH NH O OH pKa = 2.20 O HO O HS pKa = 8.35 OH pKa = 10.07 a. At pH = 5 overall (+) + NH3 O - NH + NH O O NH + H3N NH NH O HO O NH NH O O O - O HS OH b. At pH = 7 overall neutral Page 2 of 4 CHEM 109C – Zhang CLAS, F10 Amino Acids II: Separation - KEY + NH3 O - NH N O O NH + O O NH H3N NH NH O NH O HO O - O HS OH c. At pH = 9 overall (-2) + NH3 O - NH N O O NH H2N NH NH O HO O NH NH O O S - O - O OH 4. (22.61) A mixture of 15 amino acids gave the fingerprint down below after electrophoresis and chromatography. Identify the spots. (Hint 1: Pro reacts with ninhydrin to produce a yellow color (looks white); Phe and Tyr produce a yellowgreen color (looks grey). Hint 2: Count the number of spots before you start.) Page 3 of 4 CHEM 109C – Zhang CLAS, F10 Amino Acids II: Separation - KEY From Hint 2: number of spots = 14 There are 15 amino acids, so 2 of the amino acids didn’t separate and must be very similar. EX. Leu and Ile From Hint 1: The white spot is Pro, the two grey spots are Phe and Tyr, the more polar amino acid (Tyr) should be closer to the origin). At pH = 5 Overall (-) Overall neutral Overall (+) Polar Nonpolar Asp Ala Arg Arg Trp Glu Gly Ser Ile Ile Asp Leu Leu Glu Val Met Thr Met Ser Ala Thr Gly Trp Val Dec. ↓ Dec. ↓ Cathode (attracts (+) amino acids) Arg Gly Ala Leu,Ile Thr Ser Pro Glu Trp Tyr Val Met Phe More nonpolar amino acids, interact more with the mobile phase and move more. Asp Anode (attracts (-) amino acids) * I differ a little from the solutions manual in the placement of Trp and Leu, Ile: Trp being an aromatic compound and more soluble in organic solvent I expect to interact more with the mobile phase than Leu, Ile. Page 4 of 4