please help about my puzzle of my LC/MS system

[meillid]

HI, all,

I have been working on my puzzle for two weeks, I still hasn't got any progress. My system include heap technology autosampler+agilents HPLC(binary pump)+ API 2000 MS. The puzzle problem is that the TIC of the pure standard instrument test solution (reserpine) show peak spliting/mutiple peak/deformed peak shapes. Infusion MS experiment of standard solution (ppg) show that MS is working properly; I tested all the troubleshoot sugested by the HPLC troubleshoot, such as the the one on this website https://www.mn-net.com/web/MN-WEB-mnweb ... WIK-4P5F9L. also I changed new autosample injection port. But the problem still exsit. What is the answer to my puzzle??? I sincerely welcome any opinions and suggestions.

[MG]

That link didn't work for me.

Are you referring to the mass peak for reserpine, or the chromatographic peak?

If it's the chromatography, what solvent is the reserpine std dissolved in, and what is your injection volume, column dimension, flow rate, and mobile phase (or gradient) ?

[JA]

corrected link: https://www.mn-net.com/web/MN-WEB-mnweb ... WIK-4P5F9L

again, it is a shame one cannot attach (small) filesize chromatograms on the forum. A picture says a thousand words and all that

I'll be interested if this problem is MS related, as I've seen some "crazy looking" MS-chromatograms in some recent work which I couldn't personally explain. Naturally, I can't host chromatograms on my work's website!

[meillid]

I mean chromatography. the sovent is compatible, the colume is mini quantitative column, the flow is 200ul/min, mobile phase is not gradient

[MG]

Sorry, I don't have enought information to help you. So far I have

column dimension = 2.1 x something

type of stationary phase = ?

flow rate = 200 uL/min

mobile phase composition = ?

injection solvent = ?

injection volume = ?

[meillid]

the answer is

column dimension = 2.1 x 30

type of stationary phase = c18

flow rate = 200 uL/min

mobile phase composition = water: methanol 1:1

injection solvent = just the standard reserpine solution, it should match the mobile phase, the mobile phase is the one that our contract survice company alway used when they analyze reserpine soultion

injection volume = 10ul

hope this helps and let me know what else should I show you. thanks.

[MG]

Your mobile phase doesn't have any acid or buffer in it? If so, that is at least part of your problem. Reserpine has amine functionality. You need to control the pH of your mobile phase to get any kind of decent chromatography. The easiest solution is to add 0.1% of acetic or formic acid. That will get you well below the pKa of reserpine. You might get some tailing, but it should certainly be an improvement over no pH control. The acid will also improve the ionization of the reserpine for your MS.

Second, since you are doing isocratic analysis, your injection solvent needs to be the same as your mobile phase, or otherwise in a weaker solvent. It sounds like you are not 100% sure of what solvent it is dissolved in. If it is dissolved in a pure organic, or contains more methanol than your mobile phase, you could get misshapen peaks or split peaks.

[Patrik Appelblad]

In addition to previous suggestions, I'd like to stress the importance of keeping a suitable ratio between injection and column volume. This is an essential parameter especially when using isocratic conditions.

A 30x2.1 column has a total column volume of approx. 100 uL and you inject 10% of that, which will indeed have a huge impact on your chromatography. Personally, I never inject more than 1% of the column volume, while persuing analytical separations.

Thus, lower the injection volume to maximum 1uL, and try again.

Good Luck!