Hello, I am running fraction collection on an Agilent 1100 stack with a 1200 series fraction collector and a C18 Zorbax Eclipse column, and we are running into purity issues when isolating the compound for NMR analysis.
The sample is green vegetable sprouts processed using the EN15662 QuEChERS method, and the goal of the fraction collection is to collect a novel unknown plant secondary metabolite, so that we can elucidate the compound via NMR. Because the metabolite is unknown, we are unable to exploit its physical and chemical properties in order to aid isolation of the compound, so we have been relying solely on the HPLC for isolation.
Our process has been as follows: Run about 30 fraction collection runs for the peak of interest (mobile phase is water + methanol with 0.1% formic acid), lyophilize (freeze dry) the collected eluent to concentrate it, reconstitute with mobile phase, and Fraction collect (also ~30 runs) the sample again with slightly higher %organic to improve separation and purity.. The collected eluent is freeze dried again and then reconstituted with deuterated water and sent for NMR analysis. However, the past three times we have attempted to elucidate NMR samples the NMR technician has told us that our samples have too many impurities. I have run lab diagnostics tests on the DAD and on the fraction collector and everything appears to be working fine.
I understand this is a vague issue, but was hoping someone could point me in the right direction for troubleshooting this issue, let me know if there is more information I could provide on my end to help.
Thanks in advance!