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Re: Relating measured retention time to k* in gradients
Posted: Thu Feb 02, 2012 2:32 pm
by Connie Parker
Tom I dont think you did misunderstand my initial question I guess I was just asking the wrong question.
So how do I determine if my analyte is being retained (interacting with the stationary phase efficeintly) on the column when running a single gradient screen? i.e. that k* is between 1 and 10 if we cannot measure k*?
Do I just estimate S as being ~4 (or 5 as I've also seen in the literature) as DJ suggests and use the equation to find K*?
How do you determine if your analyte is being retained? Shoot some uracil on your column. If you compound has the same retention time, it's not being retained. (or did I mess the question
)
DJ I guess the emphasis in my statement should have been on the word "efficiently". Your right of course anything retained after uracil on a normal reversed phase column will be “retained” but k* as I understand it is used as an indication of how efficient the interaction of your analyte with the stationary phase is. k* as with k’ in isocratic runs should be between 2 and 10 although I suspect if you get a nice peak with a half decent retention most folks would run with it regardless of what k* is.
Re: Relating measured retention time to k* in gradients
Posted: Fri Feb 03, 2012 9:12 am
by Peter Skelton
It sounds like you're looking for a way to assign a numerical criterion to something which most chromatographers do by eye.
Re: Relating measured retention time to k* in gradients
Posted: Fri Feb 03, 2012 12:48 pm
by Connie Parker
Dont get me wrong DJ I've been running gradient chromatography for years and measureing the retention by eye but now I've been asked to provide a spreadsheet to measure it its not quite so easy.
However thanks to the responses I've had on here I now know how to work around the problem
Thanks all!
Re: Relating measured retention time to k* in gradients
Posted: Fri Feb 03, 2012 6:56 pm
by DJ
Tom I dont think you did misunderstand my initial question I guess I was just asking the wrong question.
So how do I determine if my analyte is being retained (interacting with the stationary phase efficeintly) on the column when running a single gradient screen? i.e. that k* is between 1 and 10 if we cannot measure k*?
Do I just estimate S as being ~4 (or 5 as I've also seen in the literature) as DJ suggests and use the equation to find K*?
How do you determine if your analyte is being retained? Shoot some uracil on your column. If you compound has the same retention time, it's not being retained. (or did I mess the question
)
DJ I guess the emphasis in my statement should have been on the word "efficiently". Your right of course anything retained after uracil on a normal reversed phase column will be “retained” but k* as I understand it is used as an indication of how efficient the interaction of your analyte with the stationary phase is. k* as with k’ in isocratic runs should be between 2 and 10 although I suspect if you get a nice peak with a half decent retention most folks would run with it regardless of what k* is.
I'm not sure what you mean by "efficient." What is the "goal" of the spreadsheet you're asked to make? It's a difficult question because, tR for two peaks corresponding to analytes that differ in S can actually invert depending on mobile phase program. So in this simplified two component separation, which one is more efficiently retained? Depends on who you ask (which mobile phase program you run).
Re: Relating measured retention time to k* in gradients
Posted: Sat Feb 04, 2012 11:19 pm
by HPLCaddict
If your goal is ONLY to see if your analytes are sufficiently retained, I'd go the pragmatic way: Don't bother with gradient k* and use the "usual" isocratic k. The CDS should be able to present it. Usual criterion is k>2.
Re: Relating measured retention time to k* in gradients
Posted: Mon Feb 06, 2012 6:09 pm
by DJ
Generally speaking, the more hydrophobic, the more well retained the analyte. Therefore, a number related to hydrophobicity may be what the doc ordered for your spreadsheet.
A plot of log Kw (theoretical retention factor in 0% organic) vs octanol/water log P for a series of compounds often yields a linear curve with slope near unity. So, for assigning a number for retention "efficiency," I think log Kw might work. This constant is calculated (numerically, with the equations I outlined earlier in this thread) with retention data from two gradient runs of different tG
Re: Relating measured retention time to k* in gradients
Posted: Sun Mar 17, 2019 2:54 pm
by DavidHPLC
We've actually put together a spreadsheet that does all the calculations. I just set up an auto-reply e-mail address for the download link, so if anyone is interested, send an e-mail to:
gradient-screening-request@lcresources.com
You can put anything you want in the subject or the body. You will get an automated reply with the download link. Obviously, please make sure that your spam filter is set to "whitelist" e-mail from lcresources.com so that it gets through. The "quid pro quo" here is that you will be added to our mail list to receive announcements about upcoming courses (you can unsubscribe from the list at any time).
Is the spreadsheet still available?
Re: Relating measured retention time to k* in gradients
Posted: Mon Mar 18, 2019 12:28 am
by tom jupille
Is the spreadsheet still available?
http://lcresources.com/training/LCR-resources.html
Scroll down; it's near the bottom of the page.