Broad peaks and tailing for drugs

Discussions about HPLC, CE, TLC, SFC, and other "liquid phase" separation techniques.

7 posts Page 1 of 1
Hi all, I am running Quercetin and Cyclosporine drugs on RP-HPLC, on a C18 column with Mobile phase ACN:H2O with 0.1% Formic acid (90:10) at flow rate 0.5ml/min and 60°C. Earlier I used to get acceptable peaks with this method. I added Formic acid since Quercetin showed high tailing. In between my column had possibly got contaminated due to insufficient washing, so I flushed the column as per the methods given in chromacademy. The column was working fine after that. But now after runs, the peaks for the drugs appear broader than earlier and Quercetin shows tailing even now, even with 0.1% Formic acid. Can someone please help?
Install a brand-new column and report whether stuff got better, worse, or stay the same.

If you don't have a brand-new column on site, then your pointy-haired boss needs to better understand cGMP.
Is this a method that you developed, or one that has proven its robustness before? I can imagine quercetin eluting very (too?) early under these conditions.

The column temperature is quite high, can you check your column specifications if it's useable at 60°C? I'm also wondering why you think formic acid would solve the tailing problem.
Yes, I developed this method for both drugs. I'm using 60°C as in most papers, high temperature is used for Cyclosporine. But the column I'm using can only be used till 60°C and not more. And I'm using Formic acid as a peak modifier for Quercetin tailing which my other colleagues also use for the same drug and they get good results. Quercetin is eluting at 4.8-5.0 min. It was working absolutely fine before, till the column was flushed and the frit in the system was replaced. Is there any possibility that column flushing is leading to broadening and tailing?
What are the column dimensions? 250x4.6mm by chance? Then a retention time of 4.8-5.0 minutes corresponds to the column dead time...
Can I ask if you have tried reversing the column? This is something we don't like doing, but if all else fails we give this a try before buy a new column. If it works for you, keep the flow permanent in the reversed direction (ie. don't swap back).
Ruchita wrote:
Yes, I developed this method for both drugs. I'm using 60°C as in most papers, high temperature is used for Cyclosporine. But the column I'm using can only be used till 60°C and not more. And I'm using Formic acid as a peak modifier for Quercetin tailing which my other colleagues also use for the same drug and they get good results. Quercetin is eluting at 4.8-5.0 min. It was working absolutely fine before, till the column was flushed and the frit in the system was replaced. Is there any possibility that column flushing is leading to broadening and tailing?


Posting a picture of the before and after chromatogram might help. I would be very surprised if replacing the frit is causing column broadening. Have you tried running the same method with the same solutions on a different instrument with a different (but identical) column? If you want to rule out the column flushing or the frit, then using an identical but separate column on a different instrument might help with figuring that out.

How long are your run times? I'm not familiar with the analysis of these drugs on a C18 column but if you are operating at the maximum operating temperature of the column for prolonged periods of time, that may or may not have an effect on the stationary phase - which may or may not lead to broadening.
7 posts Page 1 of 1

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