By Anonymous on Monday, January 13, 2003 - 01:00 pm:
I am seeking information regarding any problems or bias that may be encountered with the accuracy of recovery of spiked low level (0.1% - 0.5%)impurities into a drug substance with a method that uses a flow rate gradient (1.0 mL/minute - 2.0 mL/minute in 16 minutes)when compared to the same method with a constant flow rate. The method is a reverse phase system with UV detection. Quantitation of chromatographic impurities is performed using a low level external standard of the main active compound. Is there any theoretical explanation that would relate to accuracy differences when using a flow rate gradient? (not mobile phase gradient)
All responses are appreciated.
By Uwe Neue on Monday, January 13, 2003 - 03:39 pm:
Yes, there is a problem with quantitation in a flow gradient with UV detection. The UV detector gives you a signal that is proportional to concentration. If you integrate this signal (= peak area), you get concentration x time. At constant flow rate, you multiply this signal with the flow rate (= volume/time), and you get concentration x volume = mass. In other words, peak integration is proportional to the mass injected. At constant flow, the integrated signal is always the same, independent of the retention of the peak. Not so in a flow gradient! If the retention time of your peak shifts, it will elute at different flow rates, and the integration will give you a result that depends on the retention time. This will affect the reproducibility of your determination.
For a rather slow increase in flow as described by you, you may get away with it, and the problem may be small. As long as you know what is going on...
By Anonymous on Tuesday, January 14, 2003 - 07:06 am:
Thanks for the quick response. Can you give me a data reference for this information?
By Uwe Neue on Tuesday, January 14, 2003 - 04:32 pm:
Nothing in my book. The closest that I could find is a table on page 653 in Snyder/Kirkland "Practical HPLC Method Development".
By Uwe Neue on Tuesday, January 14, 2003 - 04:38 pm:
Here is another one: there is a section in Veronika Meyer's "Tips and Tricks in Pictures", or whatever the book is called in English. I only have the German version. Check at amazon.com for the real name. Chapter 2.20, "Influence of the Flow Rate on the Peak Area".