Chromatography Forum

Hello, I am trying to measure pyrrolidine by HPLC-DAD. I tried different columns (ODS and C18) and different ratio of mobile phases (water and acetonitrile) but all in vain. The UV-VIS spectrum shows there is a adsorption at 190-200 nm. Would somebody give me a hint...Thank you and I appreciate your help.

Try hydrophilic interaction chromatography (HILIC). Start with a well-coated material such as our PolyHYDROXYETHYL A, Tosoh's TSK amide-80, etc.; do not use uncoated silica, which would manifest cation-exchange in addition to the hydrophilic interaction at pH's above 3. Run isocratically in a mobile phase containing, say, 60% ACN and note the retention time with a pyrrolidine standard. Increase the ACN concentration to 65%, allow the column to reequilibrate for 45 minutes, and repeat your run. Continue increasing the ACN concentration in 5% increments until you reach a concentration that affords satisfactory retention (say, between 6 and 20 minutes). The sample solvent should contain the same concentration of ACN as the mobile phase, and both should contain a buffer that's transparent at 200 nm. I recommend 15 mM triethylamine phosphate (TEAP), with a stock solution prepared by dissolving H3PO4 in water and adding triethylamine until you reach the pH you want (which is itself a variable to be assessed). Once you have the retention you want, then your next problem is going to be whether or not anything else in your sample which absorbs light at 200 nm coelutes with pyrrolidine. If your sample is complex to the point that there is no prospect of getting a unique elution window for pyrrolidine, then you have two alternatives:
1) Use a detection method that can measure pyrrolidine in the presence of other compounds: mass spectrometry.
2) Collect the fraction containing pyrrolidine (plus the interfering substances) and rerun it on some complementary mode of chromatography. The chances that two compounds will coelute in two different modes of chromatography is low enough that you stand a good chance of success, at the cost of more time and trouble.