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By Todd Wheeler on Thursday, December 7, 2000 - 06:56 am:
I am running a simple method for the dertermination of a high mw aliphatic solvent (~30%) in a polymer matrix. The LC is set up as follows: Econosphere NH2 150x4.6mm 5um, 100% hexane(isocratic), 0.8mL/min, 10uL inj.vol.. When the mobile phase is injected, there is a negative peak on the RI which elutes just after the void volume. The peak of interest elutes with the void volume and proceeds to go below the baseline, then comes back up. No peaks (+ or -) are observed on UV (210, 230, or 254). How can a simple injection of the mobile phase cause a neagtive peak?
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By hinsbarlab on Thursday, December 7, 2000 - 08:08 am:
A few thoughts:
1. Pressure or flow fluctuation on injection.
2. Residual needle wash solvent if needle wash is used and hexane is not the needle wash solvent.
3. Mobile phase in sample vial is not the same as in mobile phase reservoir (degassed vs. not degassed?).
Another suggestion:
The peak of interest should not elute with the void volume. Adjust chromatography to get a k prime of 1 to 20 for the peak of interest.
Best Regards,
Michael Hinsberg
http://www.hinsbarlabs.com
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By chromgod on Thursday, December 7, 2000 - 11:59 am:
The peak you speak of is the injection peak, which you will always see with a DRI detector. Notice the "D" in DRI, which stands for differential which means the sample cell is compared to a reference cell filled in your case with degassed hexane. There is an inherent amount of air from your injector or sample and the detector will "see" a difference when this injection peak passes through the sample cell. If the sample is eluting in the neg peak region, it is not being retained at all by the column. If you will define your polymeric matrix and high mw aliphatic solvent, I can suggest the proper chromatography to use. Most likely you will have to use an acetone/hexane mixture or change the column type.
If your polymer is high enough MW, then you can also use a GPC column to separate out the solvent. Some may think otherwise, but we've been doing it for 12 years to control a cgmp process.
regards
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By bill tindall on Friday, December 8, 2000 - 05:26 pm:
I would agree with doing this analysis on a GPC column as suggested above. GPC is a wonderfully simple, fast way of doing low molecular weight stuff-monomer, plasticizer, solvent, stabilizer, etc- in polymers. You might even get away with doing it on a cheap GPC guard colum. For mixtures of low MW things, for example stabilizers, the high efficiency GPC columns offer surprising selectivity of low MW compounds (so long as they differ in MW!).
You are at great peril doing the analysis as you discribed. Lots of things can blow off unretained and interfer, as you have discovered.
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By Todd Wheeler on Monday, December 11, 2000 - 12:40 pm:
Thanks all for you support! I was more or else using the method as stated in order to duplicate another companies method. I was indeed considering trying GPC. However, before I could, the column was switched to another Hypersil APS column, 4mm x 250mm, 5um. The flow rate was increased from 0.8 to 1.0mL/min., all other conditions were the same. Now the chromatogram usually does not have a negative peak. A neg. peak is still occurring, but now it is random and happens only once every ~6 injections. I feel this is likely a component that is very slow to elute and therefore ghosts into future injections. The degassing theory was interesting, but I've never seen serious gas problems with non-polar solvents. I think the pressure theory might be correct in this case, since the new column has a bit higher backpressure.
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By biotech on Thursday, March 25, 2004 - 07:04 am:
dear sir,
i am a research scholar persuing research in delhi univeristy.
i would be very greatful if could solve me the problem of using a perfect column to analyze the compound GLUCOSYLGLYCEROL which is a polyol and having a moluclar weight of 254.
thank you
veer raj naidu
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By Uwe Neue on Thursday, March 25, 2004 - 04:11 pm:
Hydrophilic interaction chromatography (HILIC)will do. You need a very polar stionary phase, and a mobile phase of about 75% acetonitrile, 25% waters. When you increase the water concentration, the retention of your analyte decreases. Columns for HILIC are Atlantis HILIC from Waters, ZIC-HILIC, or amino columns from various manufacturers. If you want to go with an amino column, purchase one that has been qualified for carbohydrate separations.
I am running a simple method for the dertermination of a high mw aliphatic solvent (~30%) in a polymer matrix. The LC is set up as follows: Econosphere NH2 150x4.6mm 5um, 100% hexane(isocratic), 0.8mL/min, 10uL inj.vol.. When the mobile phase is injected, there is a negative peak on the RI which elutes just after the void volume. The peak of interest elutes with the void volume and proceeds to go below the baseline, then comes back up. No peaks (+ or -) are observed on UV (210, 230, or 254). How can a simple injection of the mobile phase cause a neagtive peak?
------------------------------------------------------------------------------------------------------------
By hinsbarlab on Thursday, December 7, 2000 - 08:08 am:
A few thoughts:
1. Pressure or flow fluctuation on injection.
2. Residual needle wash solvent if needle wash is used and hexane is not the needle wash solvent.
3. Mobile phase in sample vial is not the same as in mobile phase reservoir (degassed vs. not degassed?).
Another suggestion:
The peak of interest should not elute with the void volume. Adjust chromatography to get a k prime of 1 to 20 for the peak of interest.
Best Regards,
Michael Hinsberg
http://www.hinsbarlabs.com
------------------------------------------------------------------------------------------------------------
By chromgod on Thursday, December 7, 2000 - 11:59 am:
The peak you speak of is the injection peak, which you will always see with a DRI detector. Notice the "D" in DRI, which stands for differential which means the sample cell is compared to a reference cell filled in your case with degassed hexane. There is an inherent amount of air from your injector or sample and the detector will "see" a difference when this injection peak passes through the sample cell. If the sample is eluting in the neg peak region, it is not being retained at all by the column. If you will define your polymeric matrix and high mw aliphatic solvent, I can suggest the proper chromatography to use. Most likely you will have to use an acetone/hexane mixture or change the column type.
If your polymer is high enough MW, then you can also use a GPC column to separate out the solvent. Some may think otherwise, but we've been doing it for 12 years to control a cgmp process.
regards
------------------------------------------------------------------------------------------------------------
By bill tindall on Friday, December 8, 2000 - 05:26 pm:
I would agree with doing this analysis on a GPC column as suggested above. GPC is a wonderfully simple, fast way of doing low molecular weight stuff-monomer, plasticizer, solvent, stabilizer, etc- in polymers. You might even get away with doing it on a cheap GPC guard colum. For mixtures of low MW things, for example stabilizers, the high efficiency GPC columns offer surprising selectivity of low MW compounds (so long as they differ in MW!).
You are at great peril doing the analysis as you discribed. Lots of things can blow off unretained and interfer, as you have discovered.
------------------------------------------------------------------------------------------------------------
By Todd Wheeler on Monday, December 11, 2000 - 12:40 pm:
Thanks all for you support! I was more or else using the method as stated in order to duplicate another companies method. I was indeed considering trying GPC. However, before I could, the column was switched to another Hypersil APS column, 4mm x 250mm, 5um. The flow rate was increased from 0.8 to 1.0mL/min., all other conditions were the same. Now the chromatogram usually does not have a negative peak. A neg. peak is still occurring, but now it is random and happens only once every ~6 injections. I feel this is likely a component that is very slow to elute and therefore ghosts into future injections. The degassing theory was interesting, but I've never seen serious gas problems with non-polar solvents. I think the pressure theory might be correct in this case, since the new column has a bit higher backpressure.
------------------------------------------------------------------------------------------------------------
By biotech on Thursday, March 25, 2004 - 07:04 am:
dear sir,
i am a research scholar persuing research in delhi univeristy.
i would be very greatful if could solve me the problem of using a perfect column to analyze the compound GLUCOSYLGLYCEROL which is a polyol and having a moluclar weight of 254.
thank you
veer raj naidu
------------------------------------------------------------------------------------------------------------
By Uwe Neue on Thursday, March 25, 2004 - 04:11 pm:
Hydrophilic interaction chromatography (HILIC)will do. You need a very polar stionary phase, and a mobile phase of about 75% acetonitrile, 25% waters. When you increase the water concentration, the retention of your analyte decreases. Columns for HILIC are Atlantis HILIC from Waters, ZIC-HILIC, or amino columns from various manufacturers. If you want to go with an amino column, purchase one that has been qualified for carbohydrate separations.
