Annisa Mutiara: (1) Following good chromatography practices, your sample should be fully dissolved in the mobile phase. Dissolving samples in stronger or weaker strength solutions can lead to unwanted diffusion, peak splitting/distortion, precipitation, retention time variation, poor reproducibility etc. In your example, 8% water/92% ACN would normally be used to dissolve the samples in, HOWEVER...

(2) Your proposed method may be invalid and have poor to no retention on the column using the mobile phase containing 92% ACN (*Basic details about your LC method are needed to be sure). With such a high percentage of ACN, just about everything in the sample would be expected to elute off the column quickly with minimal interaction with the support. While you did not provide us with your HPLC method details, proper retention of the sample on the column is needed before any discussion can start. For most RP modes, please target a sample K prime of greater than 1.5 (a minimum of 2.0 is preferred) to demonstrate proper selectivity. Here is a link to an basic article on the topic:

• "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

Please contact someone at your school with professional experience in HPLC operation and method development. The technique of HPLC can NOT be learned from taking a class or viewing web forums. Working with an experienced professional will allow you to acquire practical knowledge in a shorter period of time so you can complete your analysis goals.

Hello Annisa Mutiara,

Do you alternatively dissolve your sample in acetonitrile instead of methanol or do you just dilute the sample with some ACN?

I can imagine that hydrogen bonds are formed with methanol, which then shift the retention time to longer times. By changing the solvent or diluting with acetonitrile, this interaction is cancelled or reduced, which then leads to faster retention.

Best regards

Joachim Horst