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denise.pattavina@gmail.co
science forum beginner

Joined: 06 Jul 2006
Posts: 2

Posted: Thu Jul 06, 2006 3:43 pm    Post subject: HPLC/UV Effect of Flow rate on Area Count

Clarification on earlier post:

Can someone please explain why and how flow rate has an effect on peak
areas? A coworker is in the middle of validating a new method for the
assay of one of our products. We have a method that works but uses an
unbuffered mobile phase which causes problems from time to time.
Basically the only change to the method is using a buffered mobile
phase and changing the flow rate. She is getting the following flow
rate:area ratios regardless of which mobile phase is used: 1.0 mL/min
: 295,000; 1.5 mL/min : 233,000. I have noticed that this is
typically the case with HPLC. Is there a standard explanation for why
this occurs?

Thanks!
David Stone
science forum beginner

Joined: 27 Mar 2006
Posts: 14

Posted: Mon Jul 10, 2006 2:50 pm    Post subject: Re: HPLC/UV Effect of Flow rate on Area Count

denise.pattavina@gmail.com wrote:

 Quote: Clarification on earlier post: Can someone please explain why and how flow rate has an effect on peak areas? A coworker is in the middle of validating a new method for the assay of one of our products. We have a method that works but uses an unbuffered mobile phase which causes problems from time to time. Basically the only change to the method is using a buffered mobile phase and changing the flow rate. She is getting the following flow rate:area ratios regardless of which mobile phase is used: 1.0 mL/min : 295,000; 1.5 mL/min : 233,000. I have noticed that this is typically the case with HPLC. Is there a standard explanation for why this occurs? Thanks!

Do the results you cite reflect unbuffered vs buffered, or are they
both for buffered? If you are changing the pH and the compound has
an acidic or basic group, you may be changing the fraction ionised
and, therefore, the fraction which absorbs at your monitoring
wavelength. If both results used the same pH mobile phase, see my

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