5-HMF is the compound you wish to detect? RID is assumed to be refractive index detection. In which way is the baseline unstable? Noisy? Drifting?

Without more detail, I can only make barely educated guesses and suggestions.

It would be helpful if you described you method in more detail, and maybe post a chromatogram. A negative peak is from something with a lower refractive index than the mobile phase. Depending on how you dissolved your sample, it is possible it is the solution used to inject your sample.

Note that refractive index detectors can't be used with gradients since the mobile phase refractive index changes.

Jack Silver Many thanks for your help. and please excuse my ignorance in some issue related to HPLC.

Actually noise and drift are exist

my method : 0.5 mL/min H2SO4 0.005 M, 30 °C, 20 μL, 975 psi

my column: Bio-Rad Amniex HPX-87H

I attached one screenshot but I will provide another one once I perform the experiment again.

I have experience with that specific column. I used it for oxalate, but it is also intended for alcohols and sugars. If you dig deeply into the guides on BioRad's website you'll see that 60 degrees Celsius can give you better resolution than lower temperatures. Regarding your problem, how do you know that peak is not your HMF? I have no experience with RID, but a negative peak might not necessarily be bad. More importantly, are you finding any peaks that grow in proportion to your analyte?

Also, this column chemistry is just like water softener, so it is polystyrene cross linked with divinylbenzene, and somewhat sulfonated. The aromatic groups might retain your analyte significantly. Perhaps try a small amount of organic modifier to pull your compound off. I believe that recommendation can be found in their product literature. Certain alcohols are forbidden as mobile phase for this column, so be careful in reading.

Your mobile phase Strength must be know any author even you just wite its not scientific 0.005 H2SO4 other thing agree with Dr. Adam L Vanwert

Harish, that column is normally used with 100% aqueous and 5 mM sulfuric acid to modify the sulfonate charges. However organic modifier is sometimes recommended with this column in circumstances where the analyte is not eluting normally.

Adam L Vanwert Many thanks for your very supportive and helpful advice. Highly appreciated

@Noor Aljammal, you're welcome. Sometimes the simplest protocols still give confusing results. I believe persistence will reward you.

Noor Aljammal Thank you for posting the image.

First, lets start with the basics:

• Is the system running correctly? Has this configuration been run before? I see the negative peaks, and it appears the detector is using an analog/digital converter (an interface box) for the signal. Make sure the polarity of the input is correct. This looks very much like an Agilent system.
• Verify the flow rate is correct (pumps working correctly)
• Do you have a reference compound you can run? 5-HMF isn't expensive, and is available from Sigma Aldrich, and probably others. https://www.sigmaaldrich.com/catalog/substance/5hydroxymethylfurfural126116747011?lang=en®ion=US Make sure a reference is running correctly before running you sample; a blank is often useful, too. I see several inverted peaks eluting after 15 minutes- how do you know that one of those isn't your compound?
• Remember that RI detectors are affected by temperature. Does your system have a column heater? The baseline drift might be simply a change in room temperature, especially if the detector is outside the HPLC system.

May I ask why you are using sulfuric acid in your mobile phase?

Noor, i wanted to know why you are using sulfuric acid. Is there a particular reason? Also can you state your gradient? Did you check if the column is working in fine condition?

Adam L Vanwert thank you so much.

Jack Silver many thanks for your detailed answer. yes it is an Agilent system.

Actually , I prepared several solution of HMF (Sigma) to obtain my calibration curve.

• No, it is the first time I run this column with RID, my colleague used C18 and C8 with DAD detector
• pump is working properly
• I will check the heater ( many thanks for this remark)
• The baseline drift might be simply a change in room temperature, especially if the detector is outside the HPLC system. ( many thanks for this remark).

Many thanks for flagging these points, I will check and revert back to you.

Navid J. Ayon I am just repeating standard method to detect HMF using Amniex column, and this mobile phase recommended for this compound and column

Injection volume? - When using instruments for multiple projects, multiple chemists - sometimes you will need to change the sample loop to accommodate large volume injections. Make sure your sample loop and syringe is sized right for your injection volume. (just a thought) - good luck!

Peter Philbrook thank you so much for flagging this issue

i am also experience same pattern.(not problem). RID having much sensitivity towards temperature, solvent change, column equilibrium.

Whenever my mobile phase RI is difference between solute and mobile phase, (with me) usually i found that.

rest base line...i have to saturate it at-least for 2-3 hrs after purging 1 hrs before each analysis.