Late negative peaks found by using HPLC with RI detector

[sisay]

Hi all

Currently i am testing USP monograph, Mannitol, related substance:

USP parameters are as below:
Mobile phase: Degassed water
Mode: LC
Detector: Refractive index
Column: 7.8-mm × 30-cm; packing L19
Temperatures
Column: 85 ± 2
Detector: 40 (maintain at a constant temperature)
Flow rate: 0.5 mL/min
Injection volume: 20 µL
Run time: NLT 1.5 times the retention time of the Mannitol peak. [Note—The retention time for Mannitol is about 20 min. ]

The HPLC I used is Waters e2695 with 2414 RI detector (corrected). The column I used is the same type L19 and the same length (Phenomenex RCM). Rest of parameters follow USP monograph. MP and needle wash is water (Merck, for chromatography). Diluent is the same solvent, which means that the refractive index should be the same with m.p. However, after the injection of diluent, 3 negative peaks appeared in the region where the Known Impurities elute at about 18 min. Even I inject 0mL of diluent, 3 smaller negative peaks still appeared in the same region.

At first, I thought the different external volume might cause those peaks shift because those negative peaks can be separated from Impurities by using the same Instrument and column about one year ago. So I changed the different tubing length between HPLC and column. However, the retention time didn’t change at all.

Therefore, I assume that there might be something wrong with the water to cause negative peaks (Merck, for chromatography. Same situation occurred for in-house purified water) regarding the result of 0mL of diluent (I guess that the residual water adhered to the outside of syringe was brought into the system while injecting). But I could not explain why there are “late” and “negative” peaks as diluent is the same solvent.

Hope you could provide me any valuable suggestion to solve my problem. Thank you in advance!

[scottythree]

The HPLC I used is Waters 2489.

Waters 2489 is a UV/Vis detector. You probably meant to say something else. Could you perhaps double check your model number?

[yazmer]

It's pretty common to get negative peaks with an RI detector. If you have compounds in your sample that have a lower refractive index than your mobile phase - you will see a negative peak. did you try a blank injection and a null injection to troubleshoot? You need to determine if those negative peaks are an artifact of your sample or something else going on with your system.

[sisay]

It's pretty common to get negative peaks with an RI detector. If you have compounds in your sample that have a lower refractive index than your mobile phase - you will see a negative peak. did you try a blank injection and a null injection to troubleshoot? You need to determine if those negative peaks are an artifact of your sample or something else going on with your system.

Hi yazmer

Thanks for your comment.
Mobile phase is 100% purified water, a blank injection (purified water) showed 3 negative peaks around 18 to 20 minutes and 0 uL (null) of blank injection showed reduced 3 negative peaks. However, it was found that one negative peak might vary its intensity inter-day. So i am not sure that if these three peaks are artifiacts or interference from solvent or somewhere else.

Is there any suggested procedure, inspection, or troubleshooting reference for me to investigate the unwanted negstive peaks? e.g. check tubing installation, RI purge procedure, injector cleanness, etc. Thanks in advance.

sisay

[Gerhard Kratz]

Why do you use different temperature for column and detector???
Flow rate is 0,5ml/min.
Your back pressure is constant???

[sisay]

Why do you use different temperature for column and detector???
Flow rate is 0,5ml/min.
Your back pressure is constant???

As it is a USP37 S2 monograph method, i can't modify the chromatographic parameters. Back pressure is about 170 psi, delta psi is 7.

Here is an update for the investigation, negative peaks appeared around 0.5 min while connecting pump system with RI detector directly (linked with tubing only). So i assumed that the issue regarding column might be excluded. Therefore, the pump, injection and detector system might be the interference sources.

We used another Waters pump system with the same RI detector, it showed similar retention time but different peak shape and size. the injection system might be questioned because no interference appeared during real-time monitoring. The interference will appear only after an injection action was taken (even 0 uL injection).

thanks,
sisay

[saikorem]

Dear

Can you please tell us how you have resolved the above discussed issue?.

the same problem is facing at our end.

your quick response is appreciated.

[KM-USA]

Currently I am testing USP monograph....

Unfortunately, many "compendial" methods are lacking or just obsolete technically, whether USP, EP, etc.

As it is a USP37 S2 monograph method, i can't modify the chromatographic parameters.

Says who? Even <621> details what parameters can be changed WITHOUT validation studies.

[sisay]

Dear

Can you please tell us how you have resolved the above discussed issue?.

the same problem is facing at our end.

your quick response is appreciated.

Hi, i remembered that we can only reduce the negative peaks by replacing the sampling o-ring (don't know exact name) or something in the sampling region where a residual solution may adhere to.

If first sample solution injected contains organic solvent or other solutions than your mobile phase, the solvent may adhere to the sampling o-ring which is used to clear the sampling needle. Once the 2nd sample is sampled by the same sampling needle, the residual solution may be introduced to the HPLC vial via the needle and that is how our negative peak was observed.

As we only reduced the negative peak instead of removing it, part of our test results were justified accordingly.

This may be a cleanness issue regarding auto-sampler. Hope this could help. thanks.