I want to measure enzymatic reaction with nucleoside phosphates, stop the reaction with methanol and then measure the NTPs on C18.
For the separation I need 1% or lower methanol concentration (I'm determining that right now). But if I diluted the samples to obtain 1% concentration of methanol, the concentration of substrate and product would be too low.
Thus the question is, is it better to load e.g. 10ul of sample with 50% methanol or 50ul of sample with 5% methanol? In both cases the amount of NTPs and methanol will be the same, but it will take different time to flush the methanol away and thus until the nucleotides bind to the column and thus will separate.
I have a simple question. What was the equilibrium concentration (A) of methanol to C18 column? Are using same solvent or do you want to modify? If concentration of solvent used more than A elution will take less time if concentraion used less than A the elution time will be more. If further make to hydrophobic then binding will be more to column, so it take more of time to elute. Area shall be used on HPLC or fractions collected may be tested using spectrophotometer as said in nucleic acid volumes for various bases, nucleotides at different solvents. NTP->NMP or NDP or PPi or Pi.
Dear Thyaga Raju Kedam, I cannot see your question here, from what I read on the front page of RG, you're asking for the starting conditions? It seems I will have about 1% MeOH, maybe less. I'm still trying to figure out what conditions will be the best for the nucleoside phosphates.
I tried H3PO4 with KOH in 1:1.5 ratio (pH ~4.25), which should be OK for the phosphates, but it seems that adenine has pKa somewhere around 3.9-4.2, so I switched to 0.1% formic acid (pH ~2.5), but I think at that pH the phosphate should be about half protonated and half deprotonated. The peaks are visibly highly tailing. But I cannot go much lower with the pH, as the column can withstand range 2-9.
Tomas -
Is the initial sample extract in methanol? If possible you can concentrate methanolic extract to dryness and re-dissolve. The way I do this is concentrate extract to dryness, add 100 uL MeOH to re-dissolve sample, transfer the 100 uL to autosampler vial then rinse the extract vial again using buffer say 900 uL. Now your final sample will be 10% MeOH and 90% Buffer. This should be sufficient for injection.
Regarding C18 column, the nucleoside (NTPs) are strongly hydrophilic and will not be separated using C18 column. Try anion exchange or HILIC columns.
Good Luck
Incorporating an evaporation step offers some important benefits: to concentrate the analytes and lower solvent strength, but this takes time and adds another source of variability.
I would say a larger injection volume with lower % of methanol may have a better chance to work in this case.
Ideally, you can figure out better conditions (column, mobile phase, detection) which allows (1) better retention of the analytes and (2) higher detection sensitivity (so the injection volume can be reduced).
No, it's not extract. It's enzymatic reaction stopped with twice volume of methanol, thus ~66% MeOH. I'd rather avoid evaporation, mainly for what Hanghui Liu said.
Generally it is better to have a larger volume of sample and sample similar to the initial conditions. in your case 50 uL volume is not that much, especially if your column uses flow closer to 1 mL/min. Still there is an alternative solution, that you should keep in mind: Depending on the kinetics of the reaction you are monitoring, the best approach is to monitor the sample automatically right from the reaction mixture. This typically means that the reaction has to be relatively slow. In any case, adjustments need to be made regarding concentrations, etc. In my experience monitoring reactions directly by HPLC or CE is the best approach. No quenching necessary and no doubts whether the quenching worked or not. The enzyme will elute quite early, and it will be a small peak, and the NTPs later and they will be large peaks at 260 nm. C18 is probably not a good chromatography to use. For reactions with NTPs I always prefer CE.
Best of luck
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