Ultisil XB-C4-Peptide Side Chain Analysis Method Validation Report.
Test Report
Chromatography conditions:
Column 1 |
Xtimate XB C4（4.6*250mm*5μm，300A）(SN:60220601601，LN:2803.15） |
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Column 2 |
Xtimate XB C4 （4.6*250mm*5μm，300A）(SN:60230403506，LN:2803.16) |
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Column 3 |
Xtimate XB C4 （4.6*250mm*5μm，300A）(SN:60230403507，LN:2803.16) |
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Mobile Phase |
Phase A: 0.01% formic acid aqueous solution Phase B: chromatographically pure acetonitrile |
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Column Temperature |
40℃ |
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Detector |
UV |
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Injection Volume |
20μL |
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Flow Rate |
1.0 ml/min |
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Detect wavelength |
214 nm |
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Elution procedure |
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Note |
After the analysis of the above batch of chromatographic columns, use the initial proportion of mobile phase 0.2ml/min flow rate to flush for 24 hours and then analyze again. |
Chromatogram and Data:
1) Chromatographic column 1 (SN: 60220601601) analyzes the test solution chromatogram:
Serial number |
Peak name |
Retention time (min) |
Peak Area(mAU*min) |
S/N |
Relative peak area(%) |
Resolution(EP) |
Number of plates(EP) |
Asymmetry(EP) |
1 |
Main materials |
27.242 |
70.234 |
196.8 |
98.05 |
19.02 |
397233 |
1.21 |
2 |
Abnormal peak |
33.765 |
1.399 |
1.0 |
1.95 |
N.a. |
68855 |
0.81 |
Sum |
|
71.633 |
|
100.00 |
19.02 |
|
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2) Chromatographic column 2 (SN: 60230403506) and chromatographic column 3 (SN: 60230403507) analyze the test solution chromatogram:
Serial number |
Retention time (min) |
Peak Area(mAU*min) |
S/N |
Relative peak area(%) |
Resolution(EP) |
Number of plates(EP) |
Asymmetry(EP) |
1 |
27.555 |
73.030 |
207.6 |
98.49 |
10.48 |
353181 |
1.08 |
2 |
29.838 |
1.121 |
2.9 |
1.51 |
N.a. |
225042 |
0.75 |
Sum |
74.151 |
|
100.00 |
10.48 |
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- 3) Analyze the test solution chromatogram after low-speed flushing of the mobile phase with the above initial ratio of the chromatographic column.
Serial number |
Peak name |
Retention time (min) |
Peak Area(mAU*min) |
S/N |
Relative peak area(%) |
Resolution (EP) |
Number of plates(EP) |
Asymmetry(EP) |
1 |
Main materials |
27.158 |
71.999 |
203.2 |
100.00 |
N.a. |
392779 |
1.15 |
Sum |
71.999 |
|
100.00 |
0.00 |
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Conclusion:
Using different batches of Welch hpcl column Xtimate XB-C4 (4.6×250mm, 5μm300A), under this chromatographic condition, analyze the solution of the test sample. An abnormal chromatographic peak appears at a retention time of 29-34 minutes. After flushing with the initial proportion of mobile phase at a flow rate of 0.2ml/min for 24 hours, reanalyze the solution of the test sample. No abnormal chromatographic peaks appear during the corresponding time period.