Reverse horizontal integration parameters

[gelcell]

Hi

We have a bit of a problem understanding the difference between "reverse horizontal integration by time" and "reverse horizontal integration by peak". We do not understand what the parameters and what they mean. We also need to know if this form of integration is GMP compliant. If anyone can help we would really appreciate it.

Thank you

[danko]

I have never used it, so the following is a qualified presumption/guess:

The most often used (by me anyway) parameter is Forward Horizontal by time. The rationale is as follows: If the baseline is horizontal and it typically is – in well functioning isocratic chromatography – then its best drown by the software and if you define the time range in which the peaks of interests are to be found, then you are well on the way to a useful integration. The procedure goes like this: Define start point (f .ex. 5 min.) and end point (f. ex. 20 min.) The software (Millennium/Emower anyway) starts the baseline at 5 min. and draws a horizontal baseline until it reaches the 20 min. - end point.
In the case of Reversed Horizontal by time, I guess the baseline starts at 20 min. and is drawn backwards to the 5 min. point.
If the attribute “by peakâ€

[mbicking]

Although I agree that isocratic separations "should" produce flat baselines, that does not always happen. I would be very careful with this technique, as a very small shift in the baseline will not be corrected, and your peak size will be in error. If these are very small peaks, then this is a dangerous option, unless you look at the baseline very closely. Try to learn more about your integration settings, and maybe you don't need this option.

As for GMP compliance, from my limited reading, there is really nothing in GMP at this level of detail. Others may want to comment in more detail, but my understanding of GMP is: "You must set up reasonable rules and you must follow them!" Most auditors know little, if any, chromatography, and would be unable to tell if you were doing things correctly or not. They are more concerned about whether everything is documented and signed.