Chromatography Forum

Hi,

I'm quite new in the chromatography field, and so it could be a "silly" discussion, but I have a lot of doubts about it....

If I only want to quantitate one analyte (and also the corresponding IS-deuterated).... is always the ISOCRATIC way the best to fit?
In other words; although the isocratic methods works good, is it better to work with a GRADIENT one???

Please, all the comments will really great wellcome!!!

Thanks,

Another opportunity to throw in my standard answer: It depends .

Generally I'd say yes. If you have to quantify only one analyte, isocratic is usually the way to go. Less practical work involved, less demanding on the HPLC machine, less opportunities for errors, less hassle when transferring to different HPLCs, no reequillibration needed, . . .

There CAN be situations, though, where even for such a "simple" analysis gradients are better:
- Ugly matrix components in the sample. With a (step-)gradient you'll be able to clean the column after every injection and prevent stuff to accumulate over the sequence, possibly increasing column lifetime.
- Difficult separations, where a separation of the analyte from matrix components and/or degradation products can be easier via a gradient.
- Gradients might yield better signal/noise ratios than isocratic because of peak compression.
- Depending on the field you're working in, it might be possible to use a generic gradient for quite different analyses.

Thqnk you!!

I thought the general answer will be "it depends"

I'll think about it...
Regarding my working field it's on ARVs drugs....

Another time...

If I have some bad resutls regarding precision... using an isocratic method....
I could:
1. change to a gradient one?
2. check the preparation of the samples (the quantity of the analyte added)
3................

It depends .

First of all, what do you mean by bad precision? I suppose too big a variance in peak area? Is retention time precision also bad? Several injections from the same vial or several distinctive samples?

If
- retention time precision is good
- and you get decent precision when injecting several times (6-times is usually used) from the SAME vial (decent meaning %RSD <1.5% for a reasonably big peak)
then the method itself is most likely NOT to blame.

On the other hand, if you get too much variance with retention time and/or peak area with this 6-fold injection, either the system (the HPLC itself) or the method is the likely culprit. Is the system OQ'ed regularily?

If the bad precision refers to several distinctive sample preparations, then I'd had a closer look on the sample preparation procedure...but first make sure the HPLC and chromatography are OK.

Thank you!!

I refered to bad variance on peak areas from DIFFERENT samples with the same theorical concentration. The retention time is quite good.
I'll try to inject 6-times the SAME vial to see what is happening...

Regarding OQ, what do you refer to, exactly? Is it the maintenance of the HPLC and/or MS/MS systems?

Thank you!!!