1. System setting.
(a) Column System: TSK gel G6000 PW column connected
in series with a TSK gel G5000 PW column and a TSK gel
PWH guard column.
Table 5
Dilutions for preparing the ADH calibration curve for TNBS method
Amino groups (nmol/mL)
ADH (nmol/mL)
ADH std
100 nmol/mL (μL)
Water (μL)
0
0
0
500
24
12
60
440
48
24
120
380
96
48
240
260
144
72
360
140
200
100
500
0
Table 6
Dilutions for preparing the ADH calibration curve for free ADH quantification by RP-HPLC
ADH
ADH 0.4 μg/mL (μL)
Water (μL)
μg/mL
nmol/mL
0.024
0.14
30
470
0.052
0.30
65
435
0.100
0.57
125
375
0.200
1.15
250
250
0.400
2.30
500
0
Table 7
Detailed chromatographic elution conditions for free ADH quantification by RP-HPLC
Time (min)
Flow rate (mL/min)
Water (%)
ACN (%)
0.0
1
35
65
7.5
1
35
65
7.6
1
5
95
20.0
1
5
95
20.1
1
35
65
50.0
1
35
65
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