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- Posts: 8
- Joined: Tue Feb 17, 2015 11:32 am
Recently my supervisor assigned me a project, which to run diphenylguanidine (DPG) compound using HPLC.
The model of HPLC that using is Agilent 1100 auto-injection, DAD detector, and Zorbax c18 reversed phase column (150 mm)
I used 95:5 Acetonitrile: Deionized water to run the elution. Some website suggested to add 0.1% trifluoroacetic acid to mobile phase. However, I dont have the TFA, so I used 0.1% acetic acid to replace it.
Here is the problem. I tried to run standard solution for calibration curve. For standard preparation, I dissolved DPG powder into DCM. However, I noticed that the retention time of my DPG compound inconsistent. The retention time is decreasing when the concentration of standard is increase. Here is my data:
Mobile phase: A - 1 L (0.1% acetic acid), B - 1 L Deionised water (0.1% acetic acid)
Flow rate: 3.0 mL/min
Thermostat: 35 celcius
80 ppm - 1.655 min
60 ppm - 1.678 min
40 ppm - 1.704 min
20 ppm - 1.735 min
(Solvent peak 0.498min ~ 0.500min)
R-square for my calibration curve = 0.99979
I also tried another set with no adding acid into mobile phase, the outcome still the same, but with longer elution time (approx. 15 min). Besides that, I changed the mobile phase ratio, change mobile phase to methanol ... it also don't give a consistent retention time. I even try to dissolve DPG in acetone, but the outcome still the same.
So, may I know what is the problem with this HPLC elution?
The pressure is almost the same, which means the pump is good in condition.
Is that possible the problem is with the column?
Hope HPLC expert can give me a solution ASAP, it is very urgent. Thank You.