I don't exactly understand your question. For quantifying a compound against standard using HPLC you need to obtain a standard curve. That is made by preparing solutions of the standard at different concentrations from a stock solution (high concentrated). After that you graph the area or height of the peak from the chromatogram against each concentration value. I hope this is helpful for you.
there is no formula; we call it a "method"; adding to what Karla said: there are different approaches to compound=unknown determination; find below a good discussion
http://webcache.googleusercontent.com/search?q=cache:XCc1d29iYBgJ:bionmr.unl.edu/courses/chem116/lectures/chapter-05.ppt+&cd=4&hl=en&ct=clnk&gl=us
Dear Khushboo Nawal,
if I understand your question you need to determine bisphenol A in water samples by using an external standard.
If it is the case, you have to construct a calibration curve with the reference standard, covering the concentration you believe bisphenol is in water samples.
After, you have to prepare the water sample and analyze it using the developed method. According to the peak area obtained you can determine the concentration of bisphenol A.
Assuming, your concentration is on the standard curve;
Result = (rU/rS) × (CS/CU)
rU = Peak response of Bisphenol from the Sample solution
rS = Peak response of Bisphenol from the Standard solution
CS = Concentration of in the Bisphenol Standard solution (ug/mL)
CU = Concentration of Bisphenol in the Sample solution (ug/mL)
In order to precisely determine the compound or molecule, molecules in standard and sample solution should be dissolved well. Thus, we have made biotin standard solution by dissolving in methanol (please see file; Netherlands biotin).
Bisphenol A also seems to be very hydrophobic molecule, and should be dissolved into methanol, ethanol, or 2-propanol for both sample and standard (both should have the same ratio of water-content).
I must add that an LC-MS/MS machine is a high price machine, and yet is a non-quantitative machine at all.
Therefore, I would like to recommend the use of the quantitative and reliable RP-HPLC-photometric method via gradient elution technique. Gradient elution method can elute and/or wash out the contaminant molecules with higher hydrophobicity remained in the ODS column.
I have carried out same research project as you questioned....but using LC-MS/MS.
You will want to see this;
http://www.restek.com/chromatogram/view/LC_GN0559
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