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By Anonymous on Thursday, June 10, 2004 - 11:12 am:
Can somebody provide suggestions for an HPLC method to quantitate glycerin in the 2-5% range in the presense of peptides at 100mg/mL?
Thanks in advance.
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By Consumer Products Guy on Thursday, June 10, 2004 - 11:27 am:
Sorry, but GC is much easier at those levels, even if your samples are aqueous. See K. Molever, "Quantitative Determination of Glycerin in Soap by Capillary Gas Chromatography", JAOCS, Vol. 64, No. 9 (September 1987), pp. 1356-1357. He details trimethylsilylation followed by capillary GC on any non-polar column and FID. In my lab, we do this for propylene glycol and sorbitol as well, routinely. In his publication, he does list a reference for someone who had tried to do glycerin by HPLC-RI, but used standard additions, if memory serves.
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By Einar Pontén - SeQuant AB on Thursday, June 10, 2004 - 01:03 pm:
You may retain glycerin (1,2,3-propanetriol) on a HILIC phase. Since it seems to be relatively high concentrations RI detection can be an alternative, or MS, or ECD.
Depending on the matrix silylation and GC may be nice (or the opposite).
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By Anonymous on Thursday, June 10, 2004 - 01:50 pm:
From BioRad using a Fermentation Monitoring Column using 1 mM sulfuric acid at 0.8 mL/min, column temp=65C, and an RI detector, glycerol has an r.t. of about 4.5 minutes. You should be able to dilute the sample quite a bit to help keep the peptide load down and easily see the glycerol.
Regards,
Mark
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By Brad on Friday, June 11, 2004 - 12:26 pm:
In my experience of fermentation borth, glycerol co-elute with lactate peak, even with lesser rate 0.5ml/min, 55C, 10mM H2SO4, and 30cm column. Any idea how I can get better resln?
Thanks
Brad
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By A.Mouse on Friday, June 11, 2004 - 02:39 pm:
I think this technique is for the most part a SEC technique. You won't get anywhere with changing conditions on this column.
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By Bob on Friday, June 11, 2004 - 03:27 pm:
what is SEC please?
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By Anonymous on Friday, June 11, 2004 - 06:43 pm:
Size Exclusion Chromatography
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By Anonymous on Monday, June 14, 2004 - 08:05 am:
The techinque is not size exclusion but ion exclusion.
Regards,
Mark
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By tom jupille on Monday, June 14, 2004 - 05:01 pm:
Actually, those "ion exclusion" columns operate via a mixed mechanism that involves actual ion exclusion and (apparently) liquid-liquid partition. Glycerin is non ionic, and is retained past the total permeation volume of the column, so the mechanism for this compound is arguably partition.
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By Anonymous on Wednesday, June 16, 2004 - 05:18 pm:
An amino column can work reasonably well with this. 90% MeCN / 0.1% H3PO4 / QS Water / 2 mL/min with RI detection is what I've used for cosmetics samples and it works well. RT is about 5 min and the peak is well shaped. It's not the most sensitive method, but I don't need ultimate sensitivity. ~10ug injected gives me a peak height of about 100 mV with a mid-range sensitivity setting on the RI. Hope this helps.
Can somebody provide suggestions for an HPLC method to quantitate glycerin in the 2-5% range in the presense of peptides at 100mg/mL?
Thanks in advance.
-------------------------------------------------------------------------------------------------------
By Consumer Products Guy on Thursday, June 10, 2004 - 11:27 am:
Sorry, but GC is much easier at those levels, even if your samples are aqueous. See K. Molever, "Quantitative Determination of Glycerin in Soap by Capillary Gas Chromatography", JAOCS, Vol. 64, No. 9 (September 1987), pp. 1356-1357. He details trimethylsilylation followed by capillary GC on any non-polar column and FID. In my lab, we do this for propylene glycol and sorbitol as well, routinely. In his publication, he does list a reference for someone who had tried to do glycerin by HPLC-RI, but used standard additions, if memory serves.
-------------------------------------------------------------------------------------------------------
By Einar Pontén - SeQuant AB on Thursday, June 10, 2004 - 01:03 pm:
You may retain glycerin (1,2,3-propanetriol) on a HILIC phase. Since it seems to be relatively high concentrations RI detection can be an alternative, or MS, or ECD.
Depending on the matrix silylation and GC may be nice (or the opposite).
-------------------------------------------------------------------------------------------------------
By Anonymous on Thursday, June 10, 2004 - 01:50 pm:
From BioRad using a Fermentation Monitoring Column using 1 mM sulfuric acid at 0.8 mL/min, column temp=65C, and an RI detector, glycerol has an r.t. of about 4.5 minutes. You should be able to dilute the sample quite a bit to help keep the peptide load down and easily see the glycerol.
Regards,
Mark
-------------------------------------------------------------------------------------------------------
By Brad on Friday, June 11, 2004 - 12:26 pm:
In my experience of fermentation borth, glycerol co-elute with lactate peak, even with lesser rate 0.5ml/min, 55C, 10mM H2SO4, and 30cm column. Any idea how I can get better resln?
Thanks
Brad
-------------------------------------------------------------------------------------------------------
By A.Mouse on Friday, June 11, 2004 - 02:39 pm:
I think this technique is for the most part a SEC technique. You won't get anywhere with changing conditions on this column.
-------------------------------------------------------------------------------------------------------
By Bob on Friday, June 11, 2004 - 03:27 pm:
what is SEC please?
-------------------------------------------------------------------------------------------------------
By Anonymous on Friday, June 11, 2004 - 06:43 pm:
Size Exclusion Chromatography
-------------------------------------------------------------------------------------------------------
By Anonymous on Monday, June 14, 2004 - 08:05 am:
The techinque is not size exclusion but ion exclusion.
Regards,
Mark
-------------------------------------------------------------------------------------------------------
By tom jupille on Monday, June 14, 2004 - 05:01 pm:
Actually, those "ion exclusion" columns operate via a mixed mechanism that involves actual ion exclusion and (apparently) liquid-liquid partition. Glycerin is non ionic, and is retained past the total permeation volume of the column, so the mechanism for this compound is arguably partition.
-------------------------------------------------------------------------------------------------------
By Anonymous on Wednesday, June 16, 2004 - 05:18 pm:
An amino column can work reasonably well with this. 90% MeCN / 0.1% H3PO4 / QS Water / 2 mL/min with RI detection is what I've used for cosmetics samples and it works well. RT is about 5 min and the peak is well shaped. It's not the most sensitive method, but I don't need ultimate sensitivity. ~10ug injected gives me a peak height of about 100 mV with a mid-range sensitivity setting on the RI. Hope this helps.
