Fluorescence and chemiluminescence responses of thiols derivatized with o-phthalaldehyde (OPA) and primary amines. It is suitable to make a pre-column derivatization  Among thiols tested, those having an alpha-ketothiol structure [R-CH(SH)-CO-]  give extremely low fluorescence intensities when derivatized with OPA and L-valine. The relative fluorescence intensities of alpha-ketothiols, such as mercaptoacetic acid (MAA), 2-mercaptopropionic acid (2-MPA), mercaptosuccinic acid (MSA) and N-(2-mercaptopropionyl)glycine (MPG), provide below 3% of that of N-acetyl-L-cysteine (NAC), having no alpha-ketothiol structure. The addition of detergents such as sodium dodecylsulphate shows no effect on the fluorescence and an aprotic solvent (dimethylsulphoxide) showed little effect, whereas these thiols chemiluminesced intensely when derivatized with OPA and N-(4-aminobutyl)-N-ethylisoluminol as a primary amine and successively detected by high-performance liquid chromatography (HPLC) with postcolumn chemiluminescence reactions using hydrogen peroxide and haematin. Due to the low fluorescence quantum yields of the derivatives, and HPLC with a chemiluminescence detection system is useful for the sensitive detection of thiols. OPA derivatization also allows detection of the rare amino acids.HPLC analysis of amino acids derivatized with OPA/MCE requires exact control of reaction times.  9-fluorenylmethyl chloroformate

(FMOC) for secondary amino acids; the automated derivatization is then integrated

with rugged HPLC analysis. The complete procedure is rapid, accurate, sensitive, and reproducible using the Agilent 1100 HPLC. Combining OPA and FMOC chemistries enables fast pre-column derivatization of amino acids (AA)

for chromatographic analysis. The reaction mixture is buffered at a

pH of 10.2, which allows direct derivatization of acid hydrolyzed protein/peptide samples. The primary AAs are reacted first with OPA using 3-mercaptopropionic

acid (3-MPA). The secondary AAs do not react with the OPA; but are then derivatized using FMOC. The incorporation of 3-MPA into the indoles decreases their hydrophobicity, and as a result, the OPAderivatives elute chromatographically

before the FMOC derivatives. Addition of acetic acid will stop derivatization and form racimic mixtures or false intermediates which will put on changing the energy absorption and elution and operation of HPLC become diffult.  It will create huge back pressure on the column containing the smaller, 3.5 µm, particles is 240-300

bar (3530-4410 psi), while the backpressure of the column having 5 µm particles is 160-210 bar (2350- 3090 psi). 

@Ravi Kant Upadhyay:

You are describing the general procedure but your answer has nothing to do with my question.

Further ideas concerning my question? Anyone?

@Krzysztof Bielikowicz:

As eluents I use methanol and an aqueous acetate buffer which keeps the pH of the eluent at 6,5.

So, this can't be the reason.

Maybe the proceduro requires further explanation: The OPA reagent is injected into the sample. The incubation time is 2 min. Afterwards, acetic acid is injected into the sample. Directly after that the HPLC-run is started by injecting the sample into the column.

Any further ideas?

@ Krzysztof Bielikowicz: assuming you are right....

My questions in the 1st post are still not answered. Please help.

Hi, I agree that it is not necessary to quench that reaction.