Chromatography Forum

With 70% aqueous as the sample solvent and a starting gradient of 3% aqueous I would expect that the chromatography would be poor, ie, the sample will mostly be carried with the injection solvent through the column with a good portion of the sample being unretained followed by a smear of the sample across the column resulting in a large hump of a peak.

What is the concentration of the sample?
Is this a prepatory method? It seems like it with 200 uL injected at 3.5 mL/min.
How do you already know that HILIC is the best method for separation?

EK8: I agree with "itspip" and had the very same thought about using dialysis. It does sound like you may have a very unusual (and inappropriate) method or do not have the required training to set up this method. I would encourage you to consult with a professional full-time chromatographer, not someone in academia for advice on how to proceed. Using any type of HPLC method where the sample is not fully soluble in the mobile phase will always lead to the creation of a scientifically invalid method of analysis (and invalid results). These types if general questions are not appropriate for a web forum (too much info is missing and you need local assistance for these types of issues).

EK8 wrote: " leaving me basically to figure everything out on my own."

Unfortunately, even learning the basics of HPLC is not something that one can ":figure out on their own". Not ever. It takes many years of professional training just to develop a 'basic' level of skills, BASIC, not enough to develop methods. The technique is far more complex than most realize and each instrument (and CDS) requires a great deal of new knowledge to be acquired. If your school/business wants to use HPLC, then they need to understand they will need to invest in hiring a full time professional chromatographer from nidustry OR send the work out to a professional analysis lab. They will waste what little money they have (and of course TIME) leaving someone to "figure it out". They clearly do not understand what they are asking you to do (and you do not have the experience to help them). We see many businesses attempt this poor route thinking they are saving money. In all cases, they loose money and time, with no scientifically correct results. It is a lesson best learned early. Delay, and continue to waste time and money, not learning from others.

I know the target analyte is soluble in methanol. Would it be worth trying to use Methanol/H2O r as solvents as opposed to MeCn/H2O?

Hi,

We were in a very similar situation a few years ago. A bioactive unknown compound (for which stucture AND concentration were unknown) which we could only track by bioactivity. It would elute in the solvent peak in reverse-phase and could only be satisfactorily retained with HILIC. It was poorly soluble in ACN so we tested different things and even published a (quite cool in my opinion) paper on at-column dilution for HILIC-based prep HPLC (https://doi.org/10.1016/j.chroma.2017.11.071). We did all kinds of tests on solubility, molecular cut-off filters, SPE sorbents, buffers, and used different HILIC and ion-exchange columns. We purified several fractions and fractions of fractions, performed NMR on active fractions, identified certain compounds, retested these compounds which proved inactive, restarted the entire process with different chemistries etc. But despite years of efforts we never found what the compound actually was.

I agree with previous comments that this is not a job for beginners nor for labs which have few resources. I even believe that you may need several experts from different fields (e.g. analytical chemist, natural product chemist, organic chemist, biologist). Have a clear discussion with your supervisors so they really realize what it takes to identify an unknown bioactive susbtance from a complex biological extract. Sometimes you can be very lucky and get your molecule almost by serendipity, but in most cases you'll struggle a lot, especially if only HILIC works since there are lots of highly concentrated primary metabolites able to interfere.

Good luck!