Chromatography Forum

I and a lab partner are new to HPLC. We are using it preparatively to separate steroid metabolites from plasma, blood serum and urine (she with marine lizards and I with dolphins) from male and female sample animals. These have not been characterized in either spp, although we presume that they're very similar to most other animals. We are working our way through learning the configuration (a 7-yr old Hitachi Elite LaChrom with a 4.66 diam. Waters column) we need, and have been able to run a general sample with unknown estrogen metabolite concentrations through, and generate a very noisy chromatograph including the baseline. We ran it with the equilibration set in the first 10". We do know how we want to prepare our standards. Our problem is, we really don't know where to start in adjusting the peak width, shoulders, etc. to clean out the chromatogram before running some very expensive steroid preps through. I can email the screen shots of what we've obtained so far if it will help in giving us advice.
shomerd@fau.edu.

One of the general problems is that different data systems use different terms for equivalent functions (and all too often have different functions for apparently equivalent terms!). You can show chromatograms on the Forum by linking to images (how to do it is explained here: viewtopic.php?t=2617 ) that may spark some suggestions.

My guess is that you need someone who has experience with that specific software to guide you through. You might try contacting your friendly local Hitachi sales rep to try to find another customer who can advise.

This was after playing with peak width, etc. a little, and zooming in on a part of the graph.[img][img]http://i52.tinypic.com/34zkhg6.jpg[/img][/img].
Blue is the baseline, green is pressure and black is the UV.

Hi, you are using Lachrom Elite, which is the same software as EZChrom Elite. First thing I can recommend is to lower the acquisition rate, it seems quite high. For a good integration you need roughly 30-40 data points for the narrowest peak. So select your narrowest peak (not smallest) in the chromatogram. When in the chromatogram review window, you will find a small button with 5 Hz displayed, most likely at the bottom of the screen. This will give you a calculation of the advised sample rate for the narrowest peak. By accepting this the sampling rate in the UV detector will be adjusted accordingly.
In the time event table you can adjust the peak width so that you will have an optimal width throughout the chromatogram. Do this also for the narrowest peak and adjust it for those peaks that are broader (possibly later in the chromatogram).