You have to control the temperature of derivatization with OPA, including the connecting lines since the reaction rate is temperature dependent.

Dear Dr. Blasko, thank you for your reply.

What I could said is that the auto-sampler comes with chilled thermostat and the mixing of chemical is completed by online derivatization. While the column compartment is thermostated controlled as well.

As for the room environment it is air-conditioned controlled at about 25'c. I'm not sure how can I manipulate the temperature of the connecting lines though.

"Agilent 1200 UPLC system with DAD (G1321B) " ? So that we do not confuse others...

"UPLC" Is a Waters Corp trademark only. The name of the technique you used was HPLC. Let us be good scientists and use the correct terminology. Please refer to this technique as "HPLC".

The Agilent G1321B is a Fluorescence detector, not a Diode Array Detector. Your chromatograms indicate you used a DAD (as you did in fact state earlier). Probably a model G1315 A or B... model. DAD and Fluorescence detectors are commonly employed in AA analysis (separately or together, in-series).

Your method differs from the application note you referenced (that may cause some variations).

Your two example chromatograms (day 1 vs day 2, 1 nmole) do show a significant drop in response (area/height). This may be due to something very simple. For example, are you preparing FRESH mobile phase solutions each day? The derivatizing agents also must be FRESH too. Are you running a separate wash method after each analysis to clean the column? Columns used for AA analysis become quickly fouled (Have poor loading) and show lower and lower responses. This can be corrected with better column washing techniques AND/OR replacement of the column with a new one. AA analysis columns often have very short life-times (100-200 injections is not uncommon for some). Columns are inexpensive and consumable items. They are cheap compared to the cost of solvents, materials, and time. Develop an SOP which can be used to determine when the HPLC column should be replaced (do not base it on number of injections, but the results of an actual analysis).

*I have consulted on several hundred AA analysis methods for clients and most of the problems I see are related to use of old mobile phase / old deriv reagents (not made fresh each day) or use of a fouled column. Those two areas should be checked first when response drops. *I should add that a dirty flow cell and/or failing lamp will also result in similar changes. These are all very simple things to check.

Dear Sir Letter, thank you for your valuable inputs.

First of all, apologize for the terrible mistake for the terminology and module model, I've made edit to the post to avoid confusion.

" Your method differs from the application note you referenced "

By that are you referring to the difference in the system being used by me and the application notes (1260 infinity)? Cause I believe otherwise the setting should be the same, except that I didn't used the extended amino acids stock and internal standard.

" For example, are you preparing FRESH mobile phase solutions each day? "

This could be the cause. I'm still new to HPLC and I used the same buffer and mobile phase for more than 2 to 3 days before replacement (the solvent run out). I will prepare fresh solvent afterward and observe again.

Does ACN:MET:H2O (45:45:10) mobile phase last longer than phosphate buffer, or it should be replaced along with phosphate buffer too?

" Are you running a separate wash method after each analysis to clean the column? "

When injecting different samples, I will inject a blank deionized water samples to rinse the column/system from carry over. I observed small amino acids peak of 1 to 2 unit of mAu intensity during the 1st rinse with deionized water. The baseline become stable with no visible peak in 2nd injection of deionized water.

At the end of the day of analysis, I rinse my column with ACN:H2O (%B: 50) for about 20 to 30 ml at a flow rate of 1.5 ml/min.

Are these the wash method you referring to or should I do something better?

" Columns used for AA analysis become quickly fouled "

It is a new column not older than 1 - 2 months. I hope I have not done any permanent damage to it. I welcome any tips to keep the column healthy and clean.

" old deriv reagents (not made fresh each day) "

OPA is supplied from Agilent in ampoule. I used them as per instruction and replaced the vial everyday. A new ampoule is used every week.

" *I should add that a dirty flow cell and/or failing lamp will also result in similar changes "

This I will have to check with the lab assistant.

Thank you for your helps, I hope my reply answer your question in some way.