Can you please mention what buffers are you using and at what pH? Also, is the separation achieved using a binary gradient or involves more than two buffers?

I am planing to use Acetate buffer (mid ph) and the Phosphate buffer (high ph). i just wondering is there any relation between mobile phase ph and the ph of reagent solution?

They are different since they have different functions;

1.  The mobile phase pH is used to optimize both the separation between the peaks and sharpen the peaks.

2.  The reagent pH is used to maximize the derivatization of the amino acids with OPA.

Thanks Bruce Neagle.

We perform pre column derivatization using OPA reagent and separation with acetate and phosphate buffers.

We have experienced that too acidic samples do not separate well and hence adjust the pH of samples to 5-6.5. Our buffers are in the mid-range pH or near neutral.

Please find attached article. Might be helpful.

Thanks Mihika Dave.

Basically, I am going to do same separation what you have performed, the only difference is that I am trying derivatization after the column.

I have performed Gly and Lys without column just to see interactions with my OPA solution and the response from DAD and got peak DAD 338nm. However, when I try aspartic acid got negative peaks. I have just injected the solution of water with few drops HCl which  I dissolved aspartic acid with it because of low solubility in water, and got same negative peaks.

Do you have any suggestion for that?

Negative peaks would generally relate to the elution of a compound with less detector response than the mobile-phase baseline.  

If you have injected only water with little amount HCl, possibly the response isn't enough and hence a negative peak. You could try injecting blank first and then all sample preparation reagents one by one to rule of the cause of negative peak. 

We use FLD detector for Amino acid analysis and did not have the issue of negative peaks. Hope this helps!

Thanks Mihika.

I have injected mobile phase only then injected my samples and got the peak of them except aspartic acid. it seems that there is no interaction between aspartic acid and the OPA solution however I am going to try to mix them manually and inject to the instrument. Will see DAD response or not.

Again thanks for your help.