I have HPLC data for my samples and due to bad separation of the compounds, we made an integration of the peaks for Rt 15 to 26.
I really do not understand your question? If your separation was poor, then you need to improve your method first. Develop a proper HPLC method of separation, run appropriate standards and perform calibration. If you integrated the entire area from 15 to 26 minutes, then the value obtained from this is not useful during quantification. Re-run the integration and adjust the settings to properly integrate all of the peaks. If the peaks were not baseline resolved or separated (IOW: if they co-eluted, then your method is not acceptable so you will need to try again).
If you are unable to run the sample again AND have a printed chromatogram showing the peaks of interest, then their is one other way of determining the actual area % of each peak using an analytical balance. To do so, the peaks should be baseline resolved (accurate results depend on this). Cut out each resolved peak from the chromatogram, containing all of the individual peaks (very carefully), and then individually weigh them. Determine the total mass of all of the individual paper "peaks" obtained. You can use the mass of the peak(s) of interest vs (divided by) the total mass of all peaks to calculate the % area for each one (simple division).
*This technique may sound a bit crazy to some of the younger scientists out there, but this is a real scientific technique that we used before integrators and computers were available (when we only had strip chart recorders) to calculate peak areas. No calculus was required, just a good pair of scissors and a balance.
Hi, What did you mean bad separation? What was the value of Rs? I think you cannot do integration of all peaks of Rt 15 to 26;p You should optimize your HPLC conditions to have good separation first, I think for doing quantification by HPLC, you have to run first calibration standards of your compound(s) . From the regression line of the calibration standards you can do quantification of your target peak(s) by interpolation. For quantification you can do both external or internal standard calibration.
Hello,
Quantitative estimation of compounds by HPLC can be done by two ways:
1. you can run a series of standard with various concentrations. then from calibration equation you can get the quantification of your compound.
2. In another method, you can run single standard compound with known concentration followed by sample analysis. Later you have to measure area under the curve for standard as well as sample and then find out concentration of sample just by cross multiplication.
Second method is easy to perform but first one is more accurate.
Regards,
At the onset, it is highly suggested to optimize your HPLC separation conditions.
Peak integration in bad chromatographic separation will leads to faulty quantification.
You don't want your hard work to go in vain, therefore, you must repeat the experiments and optimize the HPLC conditions to get better separation.
Quantification can be done via two ways.
1. Run series of standard compound dilutions to obtain rugged calibration. Quantify unknown concentration via regression analysis mediated interpolation.
2. Follow area under the peak method.
I really do not understand your question? If your separation was poor, then you need to improve your method first. Develop a proper HPLC method of separation, run appropriate standards and perform calibration. If you integrated the entire area from 15 to 26 minutes, then the value obtained from this is not useful during quantification. Re-run the integration and adjust the settings to properly integrate all of the peaks. If the peaks were not baseline resolved or separated (IOW: if they co-eluted, then your method is not acceptable so you will need to try again).
If you are unable to run the sample again AND have a printed chromatogram showing the peaks of interest, then their is one other way of determining the actual area % of each peak using an analytical balance. To do so, the peaks should be baseline resolved (accurate results depend on this). Cut out each resolved peak from the chromatogram, containing all of the individual peaks (very carefully), and then individually weigh them. Determine the total mass of all of the individual paper "peaks" obtained. You can use the mass of the peak(s) of interest vs (divided by) the total mass of all peaks to calculate the % area for each one (simple division).
*This technique may sound a bit crazy to some of the younger scientists out there, but this is a real scientific technique that we used before integrators and computers were available (when we only had strip chart recorders) to calculate peak areas. No calculus was required, just a good pair of scissors and a balance.
I think your method needs to be improved. Play around with the chromatographic conditions and optimize it in such a way you get discrete peak of the compound of your interest. As an external quality control, you can confirm by LCMS or LCMS/MS.
@ Bill Letter,
That's the first time I heard of that technique and it seems very sound (and it is in your experience). That's a new thing I learned so I would like to thank you, knowing a low-tech backup technique is always useful.
I agree with Bill Letter. Many years ago, using a room temperature GC system (state of the art back then), I did a supervised research project under a Professor at a local university for a high school science fair project as to which of three methods gave the most accurate results. Using this "cut out the resolved peaks and weigh method" came in as third from the top with a reasonable error (back in the late 50' and 60,s) for the technology. However, with a slightly larger error, coping the peaks on a quality scanner and cutting out the partially resolved peaks having a reasonably close to baseline resolution estimating the slope to baseline (similar to the algorithms used for computer assisted integration) can be used to get additional weight ratios for these partially resolved peaks. The further from baseline resolution, the greater the error of integration.
I too agree with answer of Rohit Upadhyay........first & foremost thing is that without data of standards with same analytical conditions no one can do quantitative analysis with any of technique either chromatography , spectroscopy or others.........................
This is also an important factor in chromatography either GC or LC for accurate quantitative analysis you should have proper resolution between peaks. for isomeric analytes of one group of compound you can use group quantitation if proper peak to peak separation can not be achieved or standards of all peak not available.
in case of Novel molecule whre standards not available you can prepare standard by own by purifying compound either with Column chromatography or Preparative HPLC. Once compound purified you can check purity of compound using LC/LCMS or GCX/GCMS along with NMR.
i also want to know if there is any software which can provide me information of the peaks in HPLC chromatogram so that i may be able to calculate the concenteration of my product. Thanks
No, software is not the solution to fixing problems generated by the use of poor quality methods. Poor quality methods and/or data are just that. Developing scientifically correct HPLC methods which properly retains and baseline resolves the peaks apart is required for accurate results. Only after developing reproducible, scientifically correct methods can you begin to analyze a series of high purity calibration standards of your sample to determine concentration by the same method (through interpolation, not extrapolation).
If you do not understand the importance of this fundamental scientific requirement ("collecting data in a scientifically acceptable manner"), then please take the time to familiarize yourself with the analytical technique used before trying to find short-cuts to utilize poor quality information.
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