Why the negative peak appears? The negative peak maybe refers to the Potassium ion based on the ionic strength between the DI water as a sample and the acidic Mobile phase.

The problem of the negative peak in the sample is back to the citric acid solutions preparation. I recommend you prepare your stock solution in DI Water as mentioned in your reference but make the dilution using your Mobil phase or prepare it in the mobile phase from the beginning.

the positive peak in case you inject the mobile phase also refer to the phosphate ion and methanol

if you can see in both chromatograms, the negative and positive peak cames out at the same retention time of 1.2 min which gives us the argument of the previous explanation, and the comes peaks intensity are small and cand n considered as significant peaks.

I also recommend you to change the column to Agilent Hi-Plex H, 7.7 × 300 mm, 8 µm column

If it is not possible to change the column you can use 100% of 0.1% H3PO4 in the water mobile phase without methanol or you can add 2.5% methanol.

But the acidic mobile phase is necessary for this separation because your sample is a weak acid and the column is C18. So the sample must be in non-ionized (non-charged form) form by dissolving in the same acidic mobile phase in the sample preparation. At least diluted by the mobile phase before injection.

Proper degassed and filter mobile phase. Also prepared injection sample dilution with mobile phase.

Dear Christine Knight,

these peaks are certainly not mysterious :-)

The retention time suggests that the signals occur with the flow time (formerly called dead time). The background is easily explained by the fact that with the injection a change of the eluent composition occurs. In connection with this, the adsorption is also slightly different and there is a minimum signal. This small signal can be positive (increase of adsorption) or negative (decrease of adsorption).

Your sample matrix (water or solvent) is not retarded and elutes together with the eluent according to the flow time.

I also agree with Mahmoud A. M. El-Nouby that it is useful to acidify the mobile phase to force the acids into the molecular form and thus increase the interaction with the stationary phase.

Good luck with your further experiments

Greetings and stay healthy

Christine: A few comments.

• The "peaks" you refer to are associated with the injector (normal) and not any sample. These peaks occur at "Tzero", the column void volume. You should receive some training in HPLC analysis before using the system. No mystery regarding the "peaks" as they are associated with normal Tzero HPLC injection peaks (indeed, as Joachim mentioned, no mystery at all and it is troubling that you are unaware of this). Your question demonstrates that you need to work with someone who has HPLC experience at your school. Such basic knowledge is critical to understanding what you are doing and what the results mean.
• The LC column used is very important and any changes to it may have a dramatic effect on the results obtained (i.e. peaks observed and order). A C18 Poroshell column with 2.7 u particles would be better suited to your isocratic only analysis of organic acids in coffee. Other related compounds such as Caffeine and Acsorbic Acid will also be easy to retain and resolve using such a column with a well developed method. Please get some local help with this project.
• Please keep in mind that at 210nm(4) just about EVERYTHING will absorb, so the method will not be selective for the samples at all. PS, thank you for turning the worthless "Reference Wavelength" feature OFF! Excellent. Never turn that ON.
• Make sure you develop a method which has excellent K primes for all samples (and once obtained, verify the Rt using standards). Achieving proper K primes is very important for all methods, but especially so for a method with such poor selectivity as retention alone is the only selective element you will rely on.
• Always dissolve ALL samples in the mobile phase before injecting.

Two free articles which you should become familiar with before starting any HPLC sample analysis work.

• Determination of HPLC Column Void Volume / Dead Volume, Dead Time (T zero); https://hplctips.blogspot.com/2011/05/determination-of-hplc-column-dead-time.html
• K Prime (also known as: Capacity Factor, Ratio or Retention Factor): One of the Single Most Important HPLC Parameters of All; https://hplctips.blogspot.com/2015/06/k-prime-also-known-as-capacity-factor.html

Kindly check..

https://www.chromforum.org/viewtopic.php?t=1887