peak shape problem!!

[jonathonswq]

i am studying on astragaloside iv, a compound from TCM(traditional chinese medicine). The peak shape was very poor, but i have already tried NH4HCO3 2mM, and NH4Cl 10uM. however the peak width of the elute was more than 0.12min, too poor, i think.
the HPLC/MS condition was that:
acetontrile: NH4HCO3 2mM(42:58), 0.2ml/min,40 degree.



can anyone give me some advice to improve the performance of my methods.

[goxy43]

Hello,
why should a peak width of 7.2 seconds be poor? However, could you share details of your method with us: column ID, flow rate, UV cell volume?

[jonathonswq]

in fact, the column is 2.1mm*150mm, 3.5um, ZORBAX XDB C18. flow rate 0.2ml/min , and the moble pahse was acetontrile: 2mM NH4HCO3[PH 6.5], Mass Selective Detector.
and the peakwidth was more than 0.19min, theoretical plate number less than 2000.
the compound i concerned was astragaloside iv.
is it clear?

[jonathonswq]

special thanks to goxy43
thank you for giving me an answer. then give me some advice plz.

[Uwe Neue]

What is the acetonitrile-water composition, and what is the retention time? Are you running a gradient, or is this an isocratic separation?

[jonathonswq]

the composition was acetontrile:NH4HCO3 2mM[41:59], and the retention time of compound was 2.764min. the elute was isocratice, which show better hplc performance than gradient elution.
the peakwidth was 0.1602min, and the performance (theoretical plate number)was 1881.

[Uwe Neue]

your compound is nearly unretained. On most HPLC systems, you will have a rather strong extra-column bandspreading under isocratic conditions. other things are possible as well...

[jonathonswq]

you mean if the performance is better when isocratic than gradient elution, the compound can not stick to the column?
if percentage of acetontrile decreased to 30%, the retention time was more than 10min.so i think the column is ok for this compound.
but i can not understand why the performance of my compound eluted with gradient not better than isocratic elution.

[lmh]

Definitely a good idea to use a lower acetonitrile concentration and elute a bit later. Do you have UV detection on this system? If so, you might compare the peak width with UV and that with MS to check whether the peak width is determined by column etc., or by post-column effects between PDA and MS.

[goxy43]

Well, this forum is not the best place to be sarcastic or cynic. I think that my question about HPLC conditions was justified. Do you really need to elute the substance with ammnonium bicarboante, why not use 0.1%TFA? However, I have eluted that substance on a C18 PepMap column from Dionex (or Acclaim PAII, ditto) by using acetonitrile gradient and0.1% TFA as ion pairing agent.
Success, and please be a little bit more decent when writing comments.
Thank you

[jonathonswq]

i have used 0.1%TFA, but the result was not encouraging.i used negetive ion mode to determine the concentration of my compound.
it is said that TFA can impress the ion response of compounds,
and
can you give me more details about your gradient and detector?

[goxy43]

Well, TFA truely does affect the ion spray. However, it is not the only ion pairing agent that could be used. When using TFA, there is a method for postcolumn addition of pentanoic acid to the mobile phase. This will reduce ion suppression and improve the ionization and all you need is a T-piece after the column or after the UV detector.
The gradient is qquite simple: isocratic run for 5 minutes and increasing acetonitrile concentration up to 100 % B in 45 minutes with a plateau of 100% B for 10 minutes (55 minutes) followed by column equilibration of 15 minutes. Mobiel phase A: 98% water, 2% acetonitrile (0.05%TFA) and B: 80% acetonitrile, 0.04% TFA. What you also need is a good reversed phase column, which is sufficiently endcapped and "base deactivated".

[jonathonswq]

i got it.
TFA was bad for ion response, but good for HPLC peak shape.
i used this method to determine drug concentration in plasma, if the elution lasted more than 60min, i would spend all day with HPLC.
i wonder why there is TFA in A and B, and how it works.
thank you goxy43

[goxy43]

The length of the gradient can be adjusted. You do not have to use the long gradient but can make the change from 0%B to 100%B in e.g. 15 minutes and then equilibrate the column. TFA is in both mobile phase since you need to maintain the ion pairing agent for the analyte, the lower concentration in B is simply for the sake of the UV baseline so that it does not drift.

[jonathonswq]

thanks a lot.
is there another agent can take place of TFA?
because i can not provide a postcolumn setting for TFA, you know, the detect mode was negative ion mode.