What can I do to improve the quality of this peak?

Chromatography Forum: GC Archives: What can I do to improve the quality of this peak?
Top of pagePrevious messageNext messageBottom of pageLink to this message  By Susan on Wednesday, November 10, 1999 - 08:30 am:

I have just received an HP 6890 with EPC and I am currently trying to copy a 1995 CLIN CHEM application titled "Simultaneous Determination of Alcohols and Ethylene Glycol in Serum by Capillary-column Gas Chromatography". I've set each parameter as listed, with the exception of using a split injection at 10:1, instead of a splitless with an adaptor to allow 0.1ul sample volume injection. This procedure does everything I need it to do, but the first peak off the column, MEOH, has a very odd shape--somewhere between chair and tailing-- when in the presence of 1,2,butanediol, the I.S. for ethylene glycol. I have improved this peak by increasing the inlet temperature from 250 to 270, but my PHD wants it to have perfect symmetry, as the other alcohols display. My column has a max temp of 280. Can someone tell me if this is really being caused by something I have done in installing the column? Or should I go higher with the inlet temp. or just tell the PHD, this is how it's gonna look. Now keep in mind this unacceptable shape is not noticeable except when decreasing the range to allow viewing of each peak and also that the area remains the same for the MEOH peak reguardless of its shape. Parameters listed below:
inlet 250
detector 250
avg velocity 100cm/s
flow 17.1ml/min
pressure 10.44
oven 40deg for1 min, ramp 70deg to 250deg for 2min.
column 3um Rtx-200,0.53mm, 30m with 5m guard
FID detector

Thanks for listening and helping a beginner!

Top of pagePrevious messageNext messageBottom of pageLink to this message  By Bruce Freeman on Wednesday, November 10, 1999 - 10:01 am:

Assuming your chromatography is good, the simple answer is to accept the odd peak shape. Before you do so, make at least three injections of sample and calulate the RSD (="CV"). If the RSD>>1%, then you have injection problems and you cannot trust your data. (The high RSD implies major problems in injection and your accuracy could be much worse than your precision). Preferably check this at both the lowest and higest levels of methanol you expect. )

I have not seen the article you refer to, so don't know how the chromatogram looked, nor any of the operating conditions. For example, what solvent did they use? Are you using the same solvent or a different one? Is water your solvent? What is your injectin volume? Do I understand correctly that the referenced article called for the use of a 250C inlet?

All these questions pertain to the change from a splitless to a split method. Although split generally is simpler to implement, splitless has a number of advantages. Why did you choose to change injection modes? I am tempted to suggest that you have lost the benefit of solvent focussing, but without knowing the sample solvent I really can't assert that.

I am surprised by the high inlet temperature. The BP's of both ethylene glycol and 1,2-butanediol are under 200C, and they both have vapor pressures of >10 torr at 100C. This suggest that not only could you reduce the injection port temperature, you may be better off doing so. However, this can also strongly depend on your solvent, etc. I say this because in my experience too hot an inlet can cause sample blow-back into the carrier inlet line or out the septum-purge vent, though this is less true for split injections than for splitless. (I don't know what effect the EPC might have on this.)

Top of pagePrevious messageNext messageBottom of pageLink to this message  By Susan on Thursday, November 11, 1999 - 06:01 am:

Dear Bruce,
In answer to your questions:

I am using the same solvent: water
0.1ul sample volume form 1ul 10:1 split ratio injection...which I altered I do not have an adapter that will allow that small of an injection

inlet 250c per method

operating conditions listed in first note

as for what the method's chromatogram looked like:
all 9 peaks are eluted within 3 mins with MEOH being the first peak and 1,2 Butanediol the last. There is good separation between peaks, good baseline resolution, but as for peak symmetry, it is hard to tell because their representative gram is pictured in spikes...with the total picture being about1.5" by 2".

My first peaks are not eluting as fast as theirs, but all are off by 3.4 minutes with good separation and good baseline resolution. They were using a 5890,non EPC with a hp 7673b autoinjector with nanoliter adaptor. I'm using a 890 with autosampler and EPC. My head pressure is lower than theirs and I figured this was due to pressure differences.
I have the GC set on const. pressure, but maybe I need to set it on const velocity???

Anyway I hope these responces help. Your input is greatly appreciated.

Top of pagePrevious messageNext messageBottom of pageLink to this message  By Bruce Freeman on Thursday, November 11, 1999 - 06:28 am:

This is a first-impression response, because I don't have time now to consider this carefully. (I'll come back to it later).

If I understand you correctly, the original method used a 0.1 uL injection, splitless. You are using a 1 uL injection, split 10:1.

Here's a possible problem: At 250C, 1 uL of water occupies more than 2 mL. Even the largest available inlet liner has less than 1 mL internal volume. This means that you might be blowing sample vapor backwards up the carrier inlet line or out the septum purge vent, or both. (I've found that the use of split injection tends to minimize this problem, but I'm used to splits much greater than 10:1.)

Try checking RSD of at least three replicate injections. If it's much greater than 1%, this is probably causing your peak shape problem, and if so, then you must correct the problem or your quantitation will be faulty.

By the way, what's the k' of the methanol peak?

Top of pagePrevious messageNext messageBottom of pageLink to this message  By Bruce Freeman on Thursday, November 11, 1999 - 01:33 pm:

Have you considered purchasing 0.5 uL autosampler syringes (made by either Hamilton or SGE for HP autosamplers -- I don't know whether HP offers their own brand) and using that to inject 0.1 uL? This would allow you to implement the method as it was published.

I've lost track of the HP6890, but on the 5890 head pressure was read on a gauge. If you used a gauge suitable for fine (eg. 0.25 mm or less) capillaries, then you really couldn't read the head pressure on a 0.53 mm column, because it was just too low for the gauge. Dual gauges would have made sense, but instead they offered an alternative. Perhaps they now have digital pressure reading -- I don't know whether that's more accurate. However my point is that the authors of the paper might have had an error in their pressure reading due to trusting a gauge that's reading too low on its scale.

Another question: What kind of liner are you using? I'd suggest a straight tube with no constrictions, about 4mm ID, with a small plug of unsilanized glass wool in the middle. (Unsilanized so water will wet it.) This may result in a more controlled evaporation of the sample, which might help peak shape. Without such a plug of glass wool, the stream of water could shoot right out the bottom of the inlet, without even evaporating (like water on a hot griddle). Konrad Grob published a book that covers this sort of thing, maybe ten years ago.

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