I am trying to measure extracellular sugar concentrations in media via HPLC. The peak of the sugar that I get is fused to the phosphate peak, which is also present in media. I have tried different flow rates, but the two peaks never separate. So, I wanted to know whether the concentrations of my sugar determined with this chromatogram are still acceptable. I am attaching a pdf file for reference. The peak at 26.3 RT is that of suagr and at 28.6 RT is that of phosphate. Thanks.
This is acceptable as the both peaks are integrated via perpendicular adjustment.
Additionally, for more precise calculations, you can measure area of phosphate peak in a Blank Injection, then minus the area with sample injection.
It is always preferable to quantify without correction. To achieve this you need to improve your chromatography, however, this is a little difficult as we have no information regarding the column, eluent or flow rate. What do you mean by "sugar" - sucrose, glucose, fructose, . . . ? How did you identify this "sugar"? What else is in the "media"?
Shilpi Kumari: That is NOT an acceptable chromatogram. You need to baseline resolve the peaks. You have co-elution which is not acceptable and invalidates the analysis data. BTW: Never use peak subtraction to fix an unacceptable method. The method must be selective for the sample and must follow good chromatography fundamentals. As you did not share any of the chromatography method details (all we really know is that you are using RID), no additional comments can be made. Please contact an experienced chromatographer for assistance in developing your sample method. This will insure that valid HPLC data is collected and the conclusions made are also scientifically correct.
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