December 04, 2020, 12:17:03 AM
Forum Rules: Read This Before Posting


Topic: HPLC Help  (Read 2489 times)

0 Members and 1 Guest are viewing this topic.

Offline mbckenny

  • Regular Member
  • ***
  • Posts: 20
  • Mole Snacks: +0/-0
  • Gender: Male
  • Undergrad at Leicester uni
HPLC Help
« on: October 06, 2012, 09:50:43 AM »
Hi,

I am running an experiment measuring the rate of the break down of Aspartame to DKP in various buffers (Citrate&Phosphate) at different temperatures (40,50,60 C).


I have run HPLC and have various Area values for different times of the reaction and plotted a graph of Ln(area) vs Time to get a nice linear graph.

In my experiments 'instructions' it states that The area at time t, At, must be corrected for the small reading after the aspartame has completely reacted, A∞ (i.e., [At-A∞]).

It then goes on to say I should plot a graph of Ln[At-A∞] vs time to obtain Kobs.

My Confusion is this... Surely the area when the aspartame is completely reacted (obtaining this data by extrapolating my graph) will be 0 and so At-A∞ will be no different from At on its own?

Offline curiouscat

  • Chemist
  • Sr. Member
  • *
  • Posts: 3005
  • Mole Snacks: +121/-35
Re: HPLC Help
« Reply #1 on: October 06, 2012, 09:57:09 AM »
My Confusion is this... Surely the area when the aspartame is completely reacted (obtaining this data by extrapolating my graph) will be 0 and so At-A∞ will be no different from At on its own?

In theory yes. But many procedures and instruments have a "zero error".  Does your solvent etc. give some tiny background signal? Is there a small amount of Aspartame that remains for ever without break down?

PS. I might be wrong; not an HPLC expert.


Offline mbckenny

  • Regular Member
  • ***
  • Posts: 20
  • Mole Snacks: +0/-0
  • Gender: Male
  • Undergrad at Leicester uni
Re: HPLC Help
« Reply #2 on: October 06, 2012, 10:03:53 AM »

In theory yes. But many procedures and instruments have a "zero error".  Does your solvent etc. give some tiny background signal? Is there a small amount of Aspartame that remains for ever without break down?

PS. I might be wrong; not an HPLC expert.

I've done a bit more research and instead of extrapolating from the graph to obtain A∞, I can obtain A∞ using half lives which should in theory give me a value very close to 0, just working out how to do that now.

Offline Babcock_Hall

  • Chemist
  • Sr. Member
  • *
  • Posts: 4411
  • Mole Snacks: +269/-18
Re: HPLC Help
« Reply #3 on: October 08, 2012, 05:14:45 PM »
In Catalysis in Chemistry and Enzymology, William P. Jencks wrote (P. 561, Chapter 11, Dover edition), "By far the most important experimental quantity which is required for the determination of first-order rate constants is the value of the absorbance, or whatever quantity is being followed, at the end of the reaction, since each experimental reading must be subtracted from the time infinity value [or vice versa]."  Jencks goes on to describe how the plots can look curved near the end of a reaction when the infinity value is not correctly determined.  One way to obtain this quantity is to wait 10 half-times.  One reason why the infinity value might not be zero is if there is a small amount of a second compound with the same retention time as aspartame.

Sponsored Links