Chromatography Forum

Hello all,

I'm analyzing samples to detect monosaccharides, organic acids and alcohols in HPLC. Unfortunately, the peaks for two primary products, ethanol (Eth) and butyric acid (HBu) tend to overlap while the retention time of other organic acids (e.g lactic acid) are bit too close to the RT of HBu and Eth for my comfort (1.5 min before HBu).

Can someone please tell me how I can separate the Eth and HBu peaks? Additionally, if there's something I can do to distance the lactic acid peak away from Eth/HBu that would be wonderful.

For reference, please find the specifications of the HPLC machine and the restrictions below:

1. HPLC: Shimadzu LC10A
2. Column: Rezex™ RHM-Monosaccharide H+ Ion Exclusion (300 x 7.8 mm)
3. Detector: RID (UV-Vis not possible)

4. Mobile Phase: Water (Reverse Osmosis)
5. Flow Rate: 0.5 ml/min [Isocratic Flow]
6. Column Temperature: 75 degree C (max. 85 C)
7. (Detector) Cell Temperature: 40 degree C
8. Injection Volume: 20 uL (Direct Injection)
9. (Detector) Response: 1.5 seconds

Sample Preparation
- 400uL of 0.02N H2SO4
- 300uL of sample (passed through 0.22um filter)
- 100uL of ISTD for organic acids (crotonic acid)
- 100uL of ISTD for alcohols (TBD)

Try adding some H2SO4 to your mobile phase (maybe 10 mM for starters?). That will hopefully increase the retention of the acids while leaving alcohols alone.
It may also help minimize the possibility of displacement of H+ on your column by other cations in your sample

Thank you so much for the suggestion. It helped solve the issues with the overlapping peaks.

I'm still facing issues with interfering peaks for other analytes (monosaccharides), namely glucose and malic acid. I don't think I can increase the concentration of mobile phase any further without causing damage to the instrument. Do you have any suggestions regarding the sampling (acquisition) rate and response time of the detector maybe? I've set them to 10 Hz and 0.05 seconds at the moment.

mixed-mode chromatography is your answer. Your acid will retain by combination of RP and anion-exchange, and your alcohols will retain based on pure RP mechanism. You can pull them apart with almost any conditions.

Contact me if you have questions.