Report on the results of process development using Ultisil Cellu-D-SLL
Test Report
Figure 1 Customer Analysis Spectrum
Chromatography conditions:
Instrument |
Welch WelPrep2000 |
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Column |
Ultisil Cellu-D（4.6×250mm,5μm） |
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Mobile Phase A |
Chromatography-grade n-Hexane |
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Mobile Phase B |
Chromatography-grade Ethanol (containing 0.4% Diethylamine) |
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Injection Volume |
10 μL |
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Flow Rate |
1.0 ml/min |
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Detect wavelength |
260 nm |
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Elution procedure |
|
The analysis spectrum is shown in Figure 2
Serial number |
Retention time (min) |
Peak Area(mAU*min) |
Area percentage(%) |
Resolution(EP) |
Tailing factor |
Number of plates(EP) |
1 |
7.933 |
21217.985 |
66.048 |
2.3 |
1.206 |
4574 |
2 |
9.107 |
10907.200 |
33.952 |
0.0 |
1.278 |
4530 |
Figure 2 Method development map
Conclusion:
After communicating with the customer and learning about the process optimization, we determined that the peak with RT 8min accounting for 70% was the target.
1.2. Separation and purification process
1.2.1. Sample preparation 1
Sample solution: Dissolve 75 mg of sample solution in 750 μl of ethanol and dilute to 3 mL with n-hexane. Analyze according to the following chromatographic analysis method
Instrument |
Welch Sail1000 |
Column |
Ultisil Cellu-D(21.2×250mm,10μm) |
Mobile Phase |
n-Hexane: ethanol (containing 0.4% ammonia water) = 93:7 |
Injection Volume |
75mg |
Flow Rate |
20mL/min |
Detect wavelength |
260 nm |
Run time |
15min |
The preparation spectrum is shown in Figure 3 below:
Serial number |
Retention time (min) |
Peak Area(mAU*min) |
Area percentage(%) |
Resolution(EP) |
Tailing factor(EP) |
Number of plates(EP) |
1 |
8.883 |
189488.328 |
43.573 |
1.7 |
0.111 |
70302 |
2 |
10.897 |
245385.311 |
56.427 |
0.0 |
0.123 |
370 |
Figure 3 Sample preparation diagram
Preparation and Collection of Target Fraction 1 and Detection.
1.2.2. Fraction Analysis 1
Perform liquid chromatography analysis on the fraction collected in step 1.2.1. The specific analysis conditions are the same as those in "Step 1.1." The chromatogram is shown in the following figure:
Serial number |
Retention time (min) |
Peak Area(mAU*min) |
Area percentage(%) |
Resolution(EP) |
Tailing factor(EP) |
Number of plates(EP) |
1 |
8.410 |
21255.819 |
99.536 |
2.6 |
1.234 |
4553 |
2 |
9.787 |
99.169 |
0.464 |
0.0 |
1.143 |
4906 |
Figure 5 Fraction Analysis Chart
Conclusion: The photochemical purity of the target compound is 99.08%, meeting the client's requirements. The fraction was evaporated to dryness at 40℃, yielding 49 mg of product with a recovery rate of 65%.
Conclusion:
Using Welch Ultisil Cellu-D column under the corresponding chromatographic conditions, the target peak was prepared and collected. The sample load was 1.5‰ of the packing material's capacity, with a recovery rate of 65%. The photochemical purity of the target compound was 99%, and the preparation results met the client's requirements.