Basic HPLC Terminology

[JHC1953]

Hello all -

I am a biologist by trade who has been thrust into the world of analytical chemistry, specifically HPLC and HPLC/radiometric detection. I keep hearing methods and equipment being discussed in terms of things like “sensitivity” and “resolution”; for example, if we do “A”, we will increase sensitivity, but sacrifice resolution, or vice versa. This usually takes place in relation to injection volume, sample loop size, or in the case of radiometric detection, sample cell size. In a discussion of another lab’s procedures, they use a 500 uL cell, and we use a 1 mL. It was said that they get better resolution with a smaller cell, but less sensitivity. I’m not clear on what the difference is between sensitivity and resolution, and how each relates to sample size, injection volume, etc. When I ask my colleagues, I get vague, non-specific answers that are not very helpful. Can someone concisely explain this terminology, and how they are related to each other? Alternatively, if you can direct me to a reference that will explain it, that would be greatly appreciated also. Thanks much.

[paulw]

Sensitivity relates to the amount needed to generate a response from the detector. Resolution is the degree by which two analytes are separated. If you have a flow cell that is 1 mL compared to 500uL, it is twice as big. That means that you should roughly be able to use a solution half as concentrated and get the same response from the detector. What you sacrifice is that because the cell is twice as big, twice as much liquid is present at any given time. This means that two closely eluting analytes could both be present in the cell at the same time (due to the extra 500uL) or even just delaying the complete transition of the earlier eluting compound through the flow cell, thus not giving you resolution between the detection of each analyte.

You would need a more sensitive setup to accurately and precisely detect lower concentrations. A more sensitive setup would also allow you to use smaller injection volumes.

Hope this helps clarify the terminology some.

Paul

[Peter Apps]

Hello all -

When I ask my colleagues, I get vague, non-specific answers that are not very helpful.

This probably means that they don't know either

Peter

[mattmullaney]

Overall, Agreed with Paul, Peter gives a nice explanation of sensitivity.

If you have some funding or a good library near you, try these texts, as well:

Practical Problem Solving in HPLC, Stavros Kromidas, ISBN: 978-3-527-29842-6, Paperback,193 pages, June 2000, US $105.00.

More Practical Problem Solving in HPLC, Stavros Kromidas, ISBN: 978-3-527-31113-2, Paperback, 309 pages, January 2005, US $105.00.

These Will Answer Your Questions and Provoke Others. Not always a bad thing.

This Web Site:

http://www.waters.com/waters/en_US/HPLC ... d=10048919

may be a bit too basic, but there are some nice illustrations of other concepts that may be of interest to you down the road.

Best Wishes!

[JHC1953]

"If you have some funding...."

Funding. Ha ha...that's a good one!

Thanks, guys. Good helpful information. I will check out the Waters site.

[mattmullaney]

JHC1953--I hear you. I've not much "funding" myself.

Barely enough for a library card.

Fortunately just enough for the children.

At Least, We'll Not Starve to Death Today...

If there remains "dazedness and confusedness" after your visit to the Waters site, I'm open to respond to new questions on your part should they arise.

[HippyLabRat]

Try this?

http://www.mac-mod.com/resources-chroma ... -terms.php