• Without any detailed chromatography conditions (column, flow, mobile phase, detection, sample type, inj vol...), it is not possible to provide any useful suggestions.

Please provide your chromatography conditions (DETAILS) so we can try and understand what you are doing. The chromatogram shows several peaks, including peaks which elute out after your Stop Time of 18 minutes. That implies your Stop Time is too early (Iso) and the sample is still eluting off the column (*change your Run Time to 35 minutes so you can capture the data). Maybe you need a better method (you did not share it with us)? Maybe you have a leak (Besides failure to wash and equilibrate the column, a leak is the #1 cause of sudden change, as in a delay, in Rt) or your HPLC requires servicing. Please verify correct operation of your entire HPLC system before running samples.

Air bubbles: Bubbles are often the result of a poorly functioning HPLC Pump (Due to leaks, poor maintenance, bad connections, wrong settings etc) or lack of any mobile phase degassing. Are you degassing your mobile phase? With a RID, you should be. RI is very sensitive to bubbles, pressure, flow rate, temperature, and just about everything in general. Bubbles can result in a check valve floating and reduced flow. It is not acceptable to have bubbles during an HPLC analysis, so this must be solved before you begin to look at samples.

Shifting Retention Times; Just like bubbles, any change in peak retention times indicates that: (1) the method may be very poor in quality (e.g. Poor choice of mobile phase; insufficient equilibration time between runs; failure to wash column off with stronger solvent than mobile phase after each run) AND/OR, (2) that you have a mechanical problem (e.g. such as one of more leaks, bubbles, inaccurate mobile phase composition...).

I have linked a few HPLC troubleshooting articles below which may help you.

• "The Three Most Common HPLC Questions and How To Solve Them";
• "HPLC Retention Time Drift, Change, Variability or Poor Reproducibility. Common Reasons for it."
• "Common Causes of Baseline Noise in HPLC, UHPLC."

https://hplctips.blogspot.com/2017/03/the-three-most-common-hplc-analysis.html

https://hplctips.blogspot.com/2015/11/hplc-retention-time-drift-change.html

https://hplctips.blogspot.com/2014/09/common-causes-of-baseline-noise.html

In case of solvent i.e. in HPLC as mobile phase, it should be unreactive (do not react with your compound), which totally do not affect your results.

De-Gassing of solvents is more important before using for analysis. It could be achieved by either proper filtering or sonication. Doing both would be more beneficial. Improper removal of gas does not only interfere with results and also reduce the life time of instrument, in particular column and detector.  

Dear searcher 

the problem is related to the detection system, so you can use a non-absorbing solvent at using wavelength,  you can use a solvent HPLC grade or higher

Hello Mohamed,

Bill has provided a very detailed response to your question and I agree with all of his recommendation. In most cases, solvent peaks are unavoidable and if they happen to interfer with a peak if interest, my suggestion will be to try another column and method. If your method permits, try another mobile phase or solvent medium for your sample.

Best wishes,

Chigozie

Hi! William Sir has given a proper suggestion regarding your problem. I have few things to say,

1. Maybe in the HPLC program, the runtime is 18 minutes, but the acquisition time of the detector is more than that. Please check that.

2. In the isocratic system, I used to get solvent peaks within 2 minutes. It is very surprising to me that solvent peaks are coming after 5 minutes in 1 ml/min flow rate. But in reverse phase gradient system, I never get solvent peaks in my system. Maybe the bubbles are creating problems. Personally, I don't think the peak at RT 8 minute is for the solvent.

3. Irrespective of columns, 5000 ppm of chemical dilutions is excessively high for HPLC system. I never use more than 1 ppm in the system for any analysis.

4. You can decrease the oven temperature of the order of 25-28 degree C.

5. As per my experience, you can go for gradient flow with methanol and water at 1 ml/min flow rate with a more comprehensive method and I believe, you will able to get all the peaks within 20 minutes.

Thanks and best wishes.

Dear Yavuz,

I presume you are using RI detector for this analysis, as many sugar analysis are carried out by RID. In this, it is very important to have the mobile phase solvent degassed, as well as the final sample dilution solvent system should match your eluent mobile phase. Also for RID, a constant temperature is very essential, as detector takes time to stabilize, otherwise fluctuations will lead to baseline issues. The stationary phase column should also be at constant temperature (use heater jacket) to avoid retention changes and stability of the system. Normally sugar analysis is carried out at column temp. bet. 50-70 deg. Celsius. The columns are also specific for sugars with mostly water as eluent.

Best regards.