Supplementary Material (ESI) for Analyst
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Supplementary Information
Moving affinity boundary electrophoresis
and its selective isolation of histidine in
urine
Jia Meng,
a, b
Wei Zhang, a, b Cheng-Xi Cao, a * Liu-Yin Fan, a * Jin Wu a and Qiu-Ling Wang a
Supporting Information of Experimental Section
Table S1. The analytical conditions used for the standard amino acid analyses of urine sample.
Time
(min)
%B1
%B2
%B3
%B4
%B5
%B6
Pump 1
Flow rate
(mL/min)
Column
Temp
%R1
%R2
%R3
Pump 2
Flow rate
(mL/min)
0.0
2.0
21.5
21.6
33.5
33.6
36.5
43.5
43.6
50.5
50.6
68.4
69.5
69.6
75.0
75.1
82.0
82.1
92.5
100
0
0
0
0
0
0.350
38
30
50
50
0
0.300
100
80
70
10
0
20
30
90
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
a
60
40
10
0
0
0
90
100
100
0
0
0
0
100
0
0
0
0
0
0
0
0
0
0
0
0
70
45
0
60
60
0
0
0
0
0
0
0
0
20
100
0
0
0
0
0
0
40
40
100
100
80
0
0
0
0
0
0
0
0
0
0
0
0
70
Laboratory of Analytical Biochemistry & Bio-separation, Key Laboratory of Microbiology of Educational
Ministry, School of Life Science and Biotechnology, Shanghai Jiao Tong University, Shanghai, 200240, China.
E-mail: cxcao@sjtu.edu.cn; Fax: +86 21-3420 5820; Tel: +86 21-3420 5682; lyfan@sjtu.edu.cn; +86 21-3420
5682.
b
The first two authors contributed equally to the paper.
Supplementary Material (ESI) for Analyst
This journal is (C) The Royal Society of Chemistry 2010
99.5
99.6
112.5
112.6
116.0
116.1
121.5
121.6
125.0
126.0
126.1
148.0
0
0
0
0
20
0
0
0
0
100
0
0
0
0
0
0
0
0
80
100
100
0
0
0
0
0
0
0
0
0
0
0
0
100
50
0
50
0
0
100
0
50
0
50
100
0
100
0
38
100
Analysis
Condition
0
0
B1: PF-1
B2: PF-2
B3: PF-3
B4: PF-4
B5: H2O
B6: PF-RG
0
0
0
R1: Nin
R2: Nin-Buffer
R3: 5% Ethanol
Table S2. The composition of buffer solutions used for the standard amino acid analyses of urine
sample.
Name
Vessel (buffer)
Lithium concentration (N)
1. Distilled water (approx.)
2. Lithium citrate (4 H2O)
3. Lithium chloride
4. Citric acid (H2O)
5. Lithium hydroxide
6. Ethanol
7. Thiodiglycol
8. Benzyl alcohol
9. Brig-35*
10. pH (nominal)
11. Total (adjust)
12. Caprylic acid
PF-1
B1
0.09
700 mL
5.73 g
1.24 g
19.90 g
____
30.0 mL
5.0 mL
____
4.0 mL
2.8
1.0 L
0.1 mL
*Dissolve 25 g into 100 mL of distilled water.
PF-2
B2
0.255
700 mL
9.80 g
6.36 g
12.00 g
____
30.0 mL
5.0 mL
____
4.0 mL
3.7
1.0 L
0.1 mL
PF-3
B3
0.721
700 mL
8.79 g
26.62 g
11.27 g
____
100.0 mL
____
3.0 mL
4.0 mL
3.6
1.0 L
0.1 mL
PF-4
PF-RG
B4
1.00
700 mL
9.80 g
38.15 g
3.30 g
____
____
____
____
4.0 mL
4.1
1.0 L
0.1 mL
B6
0.20
700 mL
____
____
____
____
30.0 mL
____
____
4.0 mL
____
1.0 L
0.1 mL
Supplementary Material (ESI) for Analyst
This journal is (C) The Royal Society of Chemistry 2010
Table S3. The composition of Ninhydrin reagents used for the standard amino acid analyses of
urine sample.
Vessel
Step 1
1
2
3
4
5
R1 (ninhydrin)
R2 for ninhydrin
buffer solution
R3 for 5% of Ethanol
1
2
3
4
5
6
1
2
3
Reagent
Measurement
Propylene glycol monomethyl ether
Ninhydrin
Nitrogen bubbling dissolution
Sodium borohydride
Nitrogen bubbling
Density
Distilled water
Lithium acetate dehydrate
Glacial acetic acid
Propylene glycol monomethyl ether
Total
Nitrogen bubbling
Density
Distilled water
Ethanol
Total
Density
979 mL
39 g
5 min minimum
81 mg
30 min minimum
0.96
336 mL
204 g
123 mL
401 mL
1000 mL
10 min minimum
0.96
900 mL
50 mL
1000 mL
1.00
Supporting Information of Results and Discussions
Histamine
N
HN
NH2
COOH
CH2
Trp
N
H
CH
NH2
Supplementary Material (ESI) for Analyst
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O
H2N
Cys
CH
C
OH
CH2
SH
Fig. S1. Chemical structures of Histamine, Trp and Cys that can offer solitary electronic pairs to
metal ion of Ni(II).
0.0250
Pk #
A
0.0225
0.0200
0.0175
0.0150
AU
26
0.0125
0.0100
4
5
0.0075
6
1
7
16
10 11
8
9
1213 14 15
1718
23
1920 21 22
28
24 25
30
29
27
0.0050
2
0.0025
3
0.0000
0
2
4
6
8
10
12
14
16
18
20
22
24
26
28
16
18
20
22
24
26
28
Minutes
0.10
B
0.09
0.08
0.07
0.06
AU
0.05
0.04
0.03
0.02
0.01
0
2
4
6
8
10
12
14
Minutes
Supplementary Material (ESI) for Analyst
This journal is (C) The Royal Society of Chemistry 2010
0.0350
Pk #
C
0.0325
0.0300
31
0.0275
12
0.0250
20
0.0225
AU
0.0200
53
33
19
26
0.0175
11
25
21
0.0150
9
0.0100
1
7
2
24
29
28
23
22
1618
17
8
36
39
14
15
10
0.0125
30
13
35
32
34 37
38
27
48
44
40
43
42
41
46
49 51
45 47 50
52
54
3
5
0.0075
6
4
0.0050
3
4
5
6
7
8
9
10
11
12
13
14
15
Minutes
D
Pk #
0.024
0.022
62
0.020
59
53
0.018
AU
0.016
0.014
68
0.012
58
91
61
92
75
0.010
60
55
54
56
63
64
6566
57
83
71
69
67
70
73
72
74
76
82
77
78
79
80
88
81
84
86
85
89 90
93
87
0.008
15
16
17
18
19
20
21
22
23
24
25
26
27
28
Minutes
Fig. S2. Comparison of urine profiling achieved with (A) conventional CZE, (B) 0-28 min
MRB-induced stacking, (C) 2.5-15.5 min MRB-induced stacking followed by CZE separation,
and (D) 15-28 min MRB-induced stacking. In electrophoregram A, the urine sample was prepared
in the running buffer and injected for 5 s at 0.2 psi. An insert is included where the minor peaks
are enlarged, but it should be noted that the scale of the insert is different from that of
electrophoregram A and B. In electrophoregram B and C, the sample was prepared in 25 mM
Gly-HCl (pH 2.5) and injected for 10 s at 1.4 psi. An insert is also included to better illustrate the
stacking effect. Other conditions are the same in both runs: 50 mM pH 12.3 Gly-NaOH running
buffer, 15 kV, capillary 75 μm i.d. × 375 μm o.d. × 60.2 cm length (50 cm to the detector), 214 nm
and 24 ℃.
Supplementary Material (ESI) for Analyst
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10
E
6
mAU
peak of T rp
F
8
D
4
plateau of T rp
C
B
2
plateau of T rp
A
0
0
2
4
6
8
10
12
14
T im e/m in
Fig. S3. Capture efficiency of Trp by the MAB-ACE method under different pH value of running
buffer with 1.0 mM sodium chloride: (A) pH 5.7, (B) pH 6.0, (C) pH 6.3, (D) pH 6.6, (E) pH 6.9
and (F) pH 7.2. Conditions: 10 μM Trp in 50 mM pH 5.7-7.2 running buffer with 1.0 mM NaCl
in whole capillary, 2.0 mM Ni(II) in 50 mM pH 5.7~7.2 running buffer with 1.0 mM NaCl in the
anodic vial, 50 mM pH 5.7~7.2 running buffer with 1.0 mM NaCl in the cathodic vial. The other
conditions were the same as those in Fig. 3.
A
20
B
20
20
peak of His
10
10
mAU
5
5
His plateau
0
4
5
no His plateau
6 7 8
Time/min
9
C
15
mAU
15
mAU
15
peak of His
Trp flat
5 His+Trp
Trp plateau
0
plateau
no Trp plateau
6
7 8 9
Time/min
10
10
Trp flat
0
4
5
6 7 8
Time/min
9 10
Fig. S4. Selective capture of MAB-ACE to His rather than Trp: (A) electropherogram of 50 μM
His; (B) electropherogram of 10 μM Trp; (C) electropherogram of 50 μM His and 10 μM Trp in
the whole capillary. Conditions: 2.0 mM Ni(II) in 50 mM pH 6.0 running buffer with 1.0 mM
NaCl in the anodic vial. The other conditions are the same as those in Fig. 3.
Supplementary Material (ESI) for Analyst
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0.05
A
0.04
0.03
45 ¡æ
0.02
40 ¡æ
AU
35 ¡æ
0.01
30 ¡æ
25 ¡æ
0.00
20 ¡æ
-0.01
-0.02
3.0
15 ¡æ
3.5
4.0
4.5
5.0
5.5
6.0
6.5
7.0
7.5
8.0
8.5
9.0
9.5
10.0
10.5
11.0
11.5
12.0
Minutes
0.05
B
0.04
0.03
0.02
45 ¡æ
AU
40 ¡æ
0.01
35 ¡æ
30 ¡æ
0.00
25 ¡æ
20 ¡æ
-0.01
15 ¡æ
-0.02
3.0
3.5
4.0
4.5
5.0
5.5
6.0
6.5
7.0
7.5
8.0
8.5
9.0
9.5
10.0
10.5
11.0
11.5
12.0
Minutes
Fig. S5. The affection of temperature on the affinity interaction between His and Ni(II). An
apparatus of CE (P/ACE MDQ, Beckman Coulter Co., Fullerton, CA, USA) with temperature
control was employed in the experiment, and the wavelength were set at (A) 214 nm, and (B) 200
nm, respectively. Conditions: 50 μM His in 50 mM pH 6.0 running buffer with 1.0 mM NaCl in
whole capillary, 2.0 mM Ni(II) in 50 mM pH 6.0 running buffer with 1.0 mM NaCl in the anodic
vial. The other conditions are the same as those in Fig. 3.