Chromatography Forum

Hi all,

Low plate counts of analyte peaks was observed when verifying the determination of monomers (methyl methacrylate and methacrylic acid) in methacrylic acid - methyl methacrylate copolymer, a EP8.0 method (No.1127).

HPLC system: Waters 2695
Column: phenomenex nucleosil C18, 100mm * 4mm, 5um (EP recommended), brand new use.
Flow rate: 2.0 mL/min (EP uses 2.5 mL/min)
Wavelength: 202 nm
Mobile phase: methanol: pH2.0 phosphate buffer (30:70 v/v)
Injection: 50uL
Concentration: 0.26 ug/mL
Blank solution: 50 mL of methanol + 25 ml of mobile phase

The example chromatogram is showed as below:
http://imgur.com/zZmIO9r


The first two peaks are from blank; the last two peaks are analytes (methyl methacrylate and methacrylic acid). It shows low plate count for two analytes.
Additionally, the first analytes (1.557) was interfered with the peak from blank.

We try to:
1. Reduce the interference from the blank:
Action: We have changed different brand methanol but the peaks from blank were not reduced.
2. Adjust the retention time of analytes:
Action: flow rate was changed to 2.0 ml/min as flow rate at 2.5 ml/min per EP requirement showed low k’.

Please provide any advice for further investigation, thank you in advance.

You run at 202nm? Why? Due to the UV cut off from Methanol? What is the UV max for your analytes?
Have you run the test chromatogram stated on your COA to see if the plate number is still on the same range?
EP = USP = challenging!
Good luck

You run at 202nm? Why? Due to the UV cut off from Methanol? What is the UV max for your analytes?
Have you run the test chromatogram stated on your COA to see if the plate number is still on the same range?
EP = USP = challenging!
Good luck

I know, any interference will have a great enhanced signal at such low wavelength.
We have successfullyverified USP method with plate count around 4000. though it is a different method.

2. Adjust the retention time of analytes:
Action: flow rate was changed to 2.0 ml/min as flow rate at 2.5 ml/min per EP requirement showed low k’.

With a change in flow-rate, you cannot change k´ in isocratic methods. Low retention stays low retention.

I think the main problem here is a combination of too low retention, a rather strong solvent (mobile phase: 30% MeOH, diluent: 67%MeOH) and a rather high injection volume.
Unfortunately, you don't have much influence here, since you have to stick to EP. You might try to change the mobile phase (reduce MeOH content) in the range allowed by EP (AFAIK 2%?) or reduce injection volume (but I'd suppose you'll run into sensivity issues here).

On the other hand, as far as I could see, the only SST criterion demanded by the EP monograph here is a minimum resolution between your two analytes. Is it fulfilled? Then don't bother about plate count!

The second peak is most likely dissolved oxygen (I experienced this with MeOH/H2O eluent and sample solvent: ). Try to purge your sample with helium or argon to remove oxygen from it. This will not change plate numbers, but most likely remove or greatly decrease the interference.
Edit: see this thread: viewtopic.php?f=1&t=21857

Good luck
Jörg

2. Adjust the retention time of analytes:
Action: flow rate was changed to 2.0 ml/min as flow rate at 2.5 ml/min per EP requirement showed low k’.

With a change in flow-rate, you cannot change k´ in isocratic methods. Low retention stays low retention.

I think the main problem here is a combination of too low retention, a rather strong solvent (mobile phase: 30% MeOH, diluent: 67%MeOH) and a rather high injection volume.
Unfortunately, you don't have much influence here, since you have to stick to EP. You might try to change the mobile phase (reduce MeOH content) in the range allowed by EP (AFAIK 2%?) or reduce injection volume (but I'd suppose you'll run into sensivity issues here).

On the other hand, as far as I could see, the only SST criterion demanded by the EP monograph here is a minimum resolution between your two analytes. Is it fulfilled? Then don't bother about plate count!

First, thank you all above for advising me.

It was found that the strong solvent in sample solution indeed caused one of the issue. Strong solvent made the peak fronting and split. After I modified the solvent used for sample preparation ( same ratio as mobile phase), the interference reduced, the peak shape of analytes became sharp, the plate count of analytes raised to 1500 and 3000 respectively, but the retention time reminded around. The interference was from MeOH (Merck, LC gradient grade) after i injected buffer and MeOH respectively.
However, the sample did not dissolve in the solution of MeOH:buffer = 30:70.

About the aerated issue, i tried to deaerated mobile phase and sample solution with Helium before use, but same result showed.

Here comes the question, how can I reduce the interference and sharpen the peaks without modifying sample preparation per EP monograph.

Is there any higher grade than "Merck, gradient LC grade"? How about spectroscopy grade? Can it be used for mobile phase preparation?
Thanks again.