Dear colleagues,
I've been working on some lipid extraction analysis with QDa HPLC.
I start with some biological samples that are spiked with a different final concentrations of a lipid mix (consisted of few known lipids), thus to see what is the minimal concentration (and the recovery %) of the lipids that can be detected. In parallel, I use a freshly prepared calibration curve (0;0.5;1;2;4;6;8;10;20).
The problem is that I am not sure of the way I analyze my data, because in some samples I see clear peaks (of up to the ^6-7 intensity), yet, in my excel calculations I get very low or non-existing concentrations, like the extraction was not successful at all. For ex., I have clear peaks and higher calculated concentrations for samples with 50ug/ml final conc; but for samples with 100ug/ml I only have clear peaks with higher intensity, yet the calculations and recovery % show very low/non-existent values.
The way I analyze my data is the following:
1) Use calibration curve (slope&intercept) to calculate the concentration of my samples;
2) I tried to normalize for the blanks (for ex. if I have some very low signal in some of the blank samples)
3) I tried to normalize for the dilution factor (if diluted 10 times, multiple the concentration calculation by 10).
I believe there is a problem with the calibration curve cause a) R= 0.95-8 (approximately)
b) because of the slope or intercept, I get a lot of negative values in my sample conc. calculations.
I already checked the calculations multiple times and I am using a precise automatic pipette, so I really don't understand what the problem might be. Knowing how simple Cal.curve preparation and calculation is, I feel very frustrated that I cannot get proper values every time I do the analysis.
Do you maybe have any suggestions/comment/advice on a proper calculation, on how the slope&intercept affect my concentration and the calibration curve problem?
Thanks in advance,
Margarita
When you are analyse your linear range and calibration range you have to check your column. I guess your column is not correspond for your data!!!
It is difficult to provide an answer without seeing the actual data. However, I will make one observation: you mention that your calibration solutions go up to 20 (ug/mL?) and your samples are 50 and 100 ug/mL. Make sure that you are not trying to calculate concentrations for samples that exceed the range of your calibration.
It's an importat point Robert L. Veazey makes: You have to stay in the calibration range (0.5 - 20 ug/mL in your case). But there are also two other aspects: Check how your calibration line fits with your calibration points. A R² of 0.95 is rather bad. Why? Because of outliners? Then repeat those. Or because either the lowest or the highest concentrations are outside the linear range? In this case, reducing the calibration range might help. The other point is, if you apply relatively high concentrations, the retention time of the signal may shift and possibly the peak is not asigned correctly. This can be the reason for having very low values despite high signals.
When you are analyse your linear range and calibration range you have to check your column. I guess your column is not correspond for your data!!!
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