Analysis of 15 carbamate compound automatically by GPC

[WESAM]

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[carls]

The problematic, interfering peak (#2) is an impurity in methanol? Have you tried other lots or brands of methanol? Perhaps using acetonitrile instead of methanol will do the trick. Your analyte of interest (propham) is a neutral and from what you have described so is the interference. If that's the case pH is not going to help.

[tom jupille]

L There are six ways to control selectivity (peak spacing) in reversed-phase:
- solvent strength
- temperature
- solvent type
- column type
- pH
- additives (e.g., ion pair reagents).

So far, you have looked at two of them. You have four to go.

[SIELC_Tech]

Use mixed-mode column. It will provide you with completely different selectivity. You have a mixture of neutral and basic compounds:
http://www.sielc.com/Technology_2D_Properties.html


or send me a sample. We can resolve these within couple of hours.

[WESAM]

ok thanks

[bisnettrj2]

Which column types have you tried? Have you tried running a gradient instead of isocratic?

[WESAM]

ok thanks

[bisnettrj2]

I should have been more clear - what column phases (silica, amide, diol, C8, C18, phenyl, phenyl-hexyl, pentafluorophenyl, etc.) have you tried? If you're looking for different selectivity, you're probably going to get the biggest changes with a different column phase or with a pH change or with both.

What kind of system do you have? The type of pump you're running will determine if you can run a gradient. You'll need a binary or quaternary system to run a gradient.

[carls]

Since carbamates (IPC and CIPC) are not ionizable and you cannot get rid of the interference by switching solvents you are left with changing the column chermistry. Best bet would be to change to a phenyl or PFP phase. You can easily move chloroaniline around (if needed) using pH.

[HW Mueller]

How did you measure the flow rate? It looks to me that it is lower than 1mL/min and that your aniline is at ~tm, because otherwise the aniline should come earlier with pH 3 as compared to pH 7. Another possibility is that your pH are not what you think they are.

[WESAM]

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[carls]

Were the chromatograms shown run on a 150x4.6mm column?

Is the interfering peak present when pure water, pure methanol or mobile phase are injected ?

What detector are you using, UV? If UV, what wavelength?

The interfering impurity could be coming from the water instead of the solvent. Try making a new mobile phase using a new unopened bottle of HPLC grade water from a reputable manufacturer (glass bottle). Be sure to clean and thoroughly rinse all glassware with HPLC grade methanol then HPLC grade water prior to use.

3-chloroaniline is a very weak base with a pKa in water of ~3.5. In 75% methanol it will be an even weaker base, perhaps pKa ~2.5.

The pKa of phosphoric acid will increase (i.e. become a weaker) in methanol and thus the pH of a 75% methanol mobile phase made with an aqueous pH 3 phosphate buffer will be greater than 3, perhaps pH 4-5. The observed pH change from pH 3 to pH 6 is larger than what I would expect but is likely the due to the combined effect of the pKa change and dilution. In either case the mobile phase pH is likely ~2 pH units greater than the pKa of the analyte and therefore the analyte is neutral whether 25% water or 25% pH 3 aqueous phosphate buffer is used in the mobile phase

The 2 earliest peaks that were resolved with water merge when buffer is used. Any idea what these are or what is going on there?

[HW Mueller]

What do you mean by saying that you are sure of the flow rate? Did you assume this by checking pump setting/reading , or did you measure it? i am still not convinced that you are far enough away from tm.
Also, it might help if you go to lower pH (maybe 1.5) and higher phosphoric concentration (there are better ways to prepare buffers than what you did), also, reducing the MeOH might be useful (you don´t need to go to 55% MeOH).

[WESAM]

ok thanks

[HW Mueller]

You saw above that dilution + solvent effect raised the pH. I don´t know how the solvent effect works out on column stability, but the dilution should certainly be enough here. The next experiment would then be a buffered pH near 7 experiment with less than 75% MeOH.