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## hplc retention question

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### hplc retention question

Hi guys,

I'm having trouble answering this question after a practical i did on reversed phase HPLC and was wondering if perhaps any of you could help me.

Qs; "The retention in this form of chromatography can be approximated by the equation:
log k = log k(w) - S theta
where theta is the volume fraction of organic solvent in mobile phase
k = retention factor
k(w) = a constant related to the retention time of the substance if pure water was used as mobile phase
S is a constant for the compound on a given column with a given binary aqueous/organic mobile phase

Explain whether this eq holds for your results by plotting an appropriate graph for toluene? If so, use the graph to estimate the retention time of toluene in 40% methanol/ 60% water."

No values for the constant have been given and have one chromatogram of Toluene with mobile phase of 80% methanol/20% water and came out with a retention of 3.323
70%3.358
90%2.379

uracil unretained 1.484

If anyone can give me any pointers on how to tackle this I would be very grateful as I really have no idea at the moment

### Re: hplc retention question

have one chromatogram of Toluene with mobile phase of 80% methanol/20% water and came out with a retention of 3.323
70%3.358
90%2.379

uracil unretained 1.484
Then you have *three* chromatograms for toluene, not one.

In a nutshell:
1. You know how to calculate k', right? So calculate the k' values for toluene at your known %B values (70%, 80%, 90%).
2. Plot log(k') vs %B and draw the best straight-line fit to your three data points.
3. You can then visually estimate k' for any other %B from the graph.
-- Tom Jupille
LC Resources / Separation Science Associates
tjupille@lcresources.com
+ 1 (925) 297-5374

### Re: hplc retention question

think a little bit harder...

What information do you actually have about your compound and what can you calculate with them...?
Then it's just straight-forward to draw a scatter plot and apply a linear fit and estimate the new retention time.

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