Hi,
I have done this few times, not a million though (Respect) and what I have observed is; let me tell you this pointwise, please bear my rambling:
1. when I run a calibration run with a FAME (Fatty acid methyl esters) mix, in known amounts, and the area under the curve or response(the area/height ratio fixed within a fixed limit. The Retention factors of all the fatty acids from c:9 to c:24 fatty acids get established, along with the internal standard for quantification,with these calibrations and with the above mentioned responses we can quantify them.
2. I have seen that the ratio of the the response at 1 microliter/5 microliter are lower with the ratio being 1.9. With, ratio of RT/pA (signal) under the limit.
3. About internal standard peaks; At 5 microliter I get them named (as their RT matches with the fatty acid present in the calibration mix,and with aforementioned ratios under limits) but not at 1 or 2 microliters of injection. I could see the bump at the RT of internal standard on the signal panel during the run but,because of the limitations in the aforementioned ratios i run my at 5 microliter; this made sense to me.
So, theoretically speaking we are heating 5 microliter of the sample so we have to multiply the response at 5 microliters with 0.2 (1/5). But, practically I have see that the ratio is 1.9 (arrived after taking average over 15 samples)
In the case of Internal standard it can not be applied. Because, it would be 0. Anyways, I have see that the ratio of the internal standards response, when see in 1 microliter injection too, is 1.4.
Does it make sense to divide both the ratios and carry on with the regular quantification process using internal standards amounts and multiply with the ratio? Or What should I do?
Also, how reliable the R factors that are obtained from the calibration mix would be, if it is a commercially available mix and the software used in naming the fatty acids and in quantifying them is built around its dependence? Should I be changing the volume of sample injection after running the calibration mix at all?
I am sorry for those who have not read this post
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