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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
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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
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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.)
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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.
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