Running a Sequence...

[Student]

Hello, I'm new to HPLCs and I was wondering if you could help me with what most labs do during a typical autosampler run. For a non-regulated run and you wanted to analyze 5 samples; what would the order of the sequence look like. Do you run blanks first- how many, run samples - all in a row, run standard - how many? Also for system suitability - minimum accetable parameters to look at and values. My last question is minimum levels for a calibration curve- how many calibration points would be considered acceptable ?All these questions are for a non-regulated sample, and I just wanted to get a feel as to what most labs would do for setting up a sequence given good lab practices. Thanks for your help.

[DR]

Typically, you want to run the system until a flat, level baseline is seen for several minutes, then inject as many blanks (1-3) as it takes to see a fairly flat, level, repeatable baseline in the blank. Then inject at least 3 standards so that you can calculate %RSD and see that this is low. Inject samples, inject another standard and make sure that the %RSD for all standards is low.

For a cal. curve, inject a series of standards (at least 3, I usually use at least 5) that brackets the expecteced concentration range of your samples. Calculate r². If r² is at least 0.99, inject your samples and follow them with a couple of standards from each end of the curve so you can make sure that nothing has changed over the course of the run.