Hello,
I try to develop a method for determination of PITC derivatized amino acids in cockroach but immediately i established my conditions for the separation (amino acids standard) which gives the perfect separation as evident in the chromatogram i have, the next step (qualitative analysis; identifying each peaks) is not clear to me.
I used Agilent 1260 Infinity Binary LC system equipped with a degassing unit, a quaternary pump, and infinity diode array detector (G4212B). The column temperature is 33 oC, wavelenght 254 nm, flow rate 0.5 mL/min, column is Agilent extend- C18 columns (25 cm × 4.6 mm i.d., 5 μm), injection volume is 20 μL, Standard concentration is 1.25 μmol/mL . The system was linked to a Lenovo desktop for recording chromatograms, using a ChemStation edition software. The mobile phase is Acetonitrile : water (60:40) and 0.14M sodium acetate containing 0.05 percent TEA adjusted to pH 6.5 using glacial acetic acid.
My question is do i need to separately collect each peak and go for GC - MS for qualitative analysis? N.B we have no LC-MS.
if yes, remember each will contain water because of the mobile phase and can that work for GC- MS?
Any other way to carry out the qualitative and quantitative analysis?
Thanks
Given the proximity of the peaks you will need to collect small volumes in each fractionation tube. If you are worried abut water, collect your samples and evaporate them. Redissolve the residue in a volatile solvent and run GC-MS.
Why are you re-inventing the wheel when Waters already has PITC methods for amino acid analysis called PICO-tag and Accu-tag?
@ Bruce Neagle........ Thanks for the contributions, really appreciate it. We don't have the PicoTag Reagent Kit in our Lab and not ready to buy one (lolz)
@Omar..... very grateful for the hint. i will do that but wouldnt my mobile phase (salt) has effect on the amino acids (i.e MS )?
Acetate will not since its concentration can be 'blanked out', however TEA cannot be completely washed from the column, so the column will have to be dedicated. In addition TEA will permanently change the column chemistry/chromatography.
seriously , i have experieced that now. After several days of checkmating the procedure if truely reproducible, today i faced another shock from this column. The peaks totally disappear even though i had used newly prepared solutions/samples. I just got confused now if to regenerate the column or just flush with ACN:H2O
As Bruce stated, perhaps I can elaborate. It sounds like you have a nice hplc, please take good care of it. Please do not ruin a gcms with any water. You do not need a gcms. There is already tons of literature from Waters, Agilent, and many others on ID and quant by hplc of derivatized amino acids. Standards are available. You could start with the 20 most common aa's. This could be an interesting contribution to cockroach biochem if you proceed carefully and skilfully.
Backflush the column with isopropanol, etanol, and water. Do you have a guard column?
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