My colleague doing the organic acid detection using pelkin almer HPLC unit, RP-HPLC, UV detector. But she got difficulties because some of the organic acid peak were existed at similar RT especially gluconic and oxalic acid. She already play around with the percentage of solvent used. For now, she use KH2PO4, 93% and methanol 7% as a mobile phase.
Mrs/Miss Talib,
Let us start with the experimental fact that the determination and quantification by means of UV-detection; in addition to, quantification based only on the chromatographic data do not provide highly accurate, precise, selective and sensitive analytical informations in quantitative terms.
There are a set of major drawbacks to both these methods (UV- and chromatography,) which prevent currently their employment for abolute or highly precise analytical determination of analytes.
Furthermore, the quantification and even the qualitative analysis are significantly complicated with increasing in the complexity of the molecular structure of the analytes.
(In your case, there are organic acids.)
Currently, there is only one hyphenated method which is only capable of obtaining the most accurate and precise as well as selective and sensitive analytical information of the analytes despite their molecular structure, chemical classes, and number of the components in the mixture, respectively. This apporach is chromatography coupled with mass spectrometry. However, talking about the mass spectrometric methods for quantification, there is important to underline that only our stochastic dynamic method developed more recently as well as our model formulas, which we have introduced provide exact of absolute quantitative, qualitative and even 3D structural information of the analytes.
(Of course, there is a remark, that depending on the complexity of the analyte mixture our mass spectrometric based method provides absolute quantitative information even without employment of chromatographic analysis.)
In this context, consider the content of my answers to the following discussions [A,B] as well as the corresponding references cited, therein with the references cited in the publications.
[A] [https://www.researchgate.net/post/Sensitivity_Coefficient_in_Uncertainty_Calculations_for_HPLC_assay_method_UV_assay_methods_and_pH_measurement]
[B] [https://www.researchgate.net/post/How_can_we_detect_VOCs_in_exhaled_breath_sample_using_ISQ_7000_single_quadrupole_GC-MS_system]
In particular, the capability of our innovative method of exact determination of the 3D structures of the analytes, have been highlighted in our recent review-article [1*].
[1*] Reviews in Analytical Chemistry, Band 38, Heft 2 (2019)
A stochastic dynamic mass spectrometric diffusion method and its application to 3D structural analysis of the analytes; Bojidarka Ivanova, Michael Spiteller
Also, reference [2] in discussion [B] contain chemometric treatment of chromatographic data, because of our new approach has been validated by means of independent chromatographic analysis. Therein, you are able to evaluate the significance of the error contribution of the mathematical data processing of the chromatographic outcome to the analytical quantities.
Further, discussion [B] details the problems with the error contribution of the chemometric treatment of some optical spectroscopic methods, which are valid to the mathematical data processing of the UV-VIS experimental patterns, as well. Therein, you are able to evaluate the data on few experimental spectra, as well.
1- you have to select the right wavelength.
2- try to change the gradient program at the overlap area.
3- try to change the column that used.
4- try to used UHPLC instead HPLC.
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