I need to detect HMF in 2-secbutyl phenol. Ours is a single pump HPLC so we cannot use gradient flow. Presently I have been running it with mobile phase (ACN:Water : 70:30 at 35 C, isocratic). The 2-secbutyl phenol is not eluting out fast and is giving broadened peaks which cover even the HMF peaks. Please suggest isocratic conditions to tackle this problem.
Adapt the ICUMSA (International Commission of Uniform Methods of Sugar Analysis) HPLC method. It might work for your sample.
For better elution use IPA (a stronger solvent) than ACN.
What are the column dimensions? What is the flow rate you are using? Is HMF diluted or not with respect to the phenol?
Prof. Omar, The column dimension is 150 x 4.6 mm, its a 5um EVO c-18 100 A column. I am using 0.8 mL/min as flow rate and HMF is diluted with respect to 2-secbutyl phenol.
Can you attach a chromatogram please. Detection wavelength? Sample volume? Is the sample diluted in the mobile phase?
Prof. Omar, Sorry for really late reply. Please find the attached chromotographs. I use 284 to detect the HMF. I now found a technique to separate HMF and Organic phase but I struggle to find a way to separate HMF from Levulinic acid. Can you give me directs on that. Thanks
According to the attached chromatogram I understand that 2-secbutyl phenol has a retention time similar to HMF, nonetheless levulinic acid leaves the column in what seems to be your void volume (3 min). To increase the retention of levulinic acid use phosphate buffer instead of water. For the original separation (2-secbutyl phenol from HMF) it seems to me that further dilution will end up in separated peaks. If you want to improve resolution, you will need to lower the proportion of ACN in the mobile phase or increase the temperature or decrease the flow rate.
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