GC Derivitisation for uronic acids
Posted: Wed May 30, 2007 2:40 am
G'day all
I’ve got quite an involved procedural query for you all here. Mainly wanting to look into the reaction mechanisms for the derivitisation of uronic acid residues in the presence of other monosaccharide, in brief;
(from Ref A)
(1) hydrolysis of soil sample with 4N TFA at 120C for 2 hrs.
(2) filtration then evaporation to dryness
(3) pH adjustment to split open lactones giving sodium uronates (0.5ml of 0.5M Na2CO3)
(4) Reduction with NaBH4 in DMSO (20g/l)
(5) quench NaBH4 with acetic acid
(6) remove Na and other cations with Dowex 50W-X8, eluent is water
(7) evap to dryness
(8) remove borate with MeOH as azeotrope on RotorVap
(9) dehydrate residue to convert aldonic acids to aldonolactones
(10) derivitise uronates with pyridine and N-hexylamine
(11) dry down
(12) derivitise alditols with acetic anhydride & 1-methylimidazole catalyst
(13) run GC analysis
What i would like to do is reduce the amount of sample workup (who doesn't?)
I have a method (Ref B) that allows me (have tried this successfully) to reduce the aldoses in NaBH4/DMSO in the presence of water. In addition, this method does not have the problem of borate interference with the acetylation step (nice one!). So my queries revolve around the use off the Dionex resin and MeOH azeotrope to remove cations and borate.
Does anyone know if cations and/or borate interfere with the amide formation when reacting the aldonolactones with N-hexylamine?
Do the uronic residues need to be protonated before the dehydration step or even the hexylamine derivitisation step?
Will the acetic anhydride be consumed by the hexylamine? That is, can i go with the PYR + hexylamine derivitisation, followed (without any dry down step) by addition of acetic anhydride (X’s) and catalyst to effect acetylation? The X’s acetic anhydride will be quenched with water, and the derivatives back extracted into DCM for GC and GCMS analysis.
Obviously i will check this effect experimentally, but was hoping there may be someone who has done this type of dervitisation before.
Cheers
Peggsy
Ref A: Walters & Hedges, 1988, Anal. Chem. 60:988-994
Ref B: Rumpel & Dignac, 2006, Soil Biol & Biochem. 38: 1478-1481
I’ve got quite an involved procedural query for you all here. Mainly wanting to look into the reaction mechanisms for the derivitisation of uronic acid residues in the presence of other monosaccharide, in brief;
(from Ref A)
(1) hydrolysis of soil sample with 4N TFA at 120C for 2 hrs.
(2) filtration then evaporation to dryness
(3) pH adjustment to split open lactones giving sodium uronates (0.5ml of 0.5M Na2CO3)
(4) Reduction with NaBH4 in DMSO (20g/l)
(5) quench NaBH4 with acetic acid
(6) remove Na and other cations with Dowex 50W-X8, eluent is water
(7) evap to dryness
(8) remove borate with MeOH as azeotrope on RotorVap
(9) dehydrate residue to convert aldonic acids to aldonolactones
(10) derivitise uronates with pyridine and N-hexylamine
(11) dry down
(12) derivitise alditols with acetic anhydride & 1-methylimidazole catalyst
(13) run GC analysis
What i would like to do is reduce the amount of sample workup (who doesn't?)
I have a method (Ref B) that allows me (have tried this successfully) to reduce the aldoses in NaBH4/DMSO in the presence of water. In addition, this method does not have the problem of borate interference with the acetylation step (nice one!). So my queries revolve around the use off the Dionex resin and MeOH azeotrope to remove cations and borate.
Does anyone know if cations and/or borate interfere with the amide formation when reacting the aldonolactones with N-hexylamine?
Do the uronic residues need to be protonated before the dehydration step or even the hexylamine derivitisation step?
Will the acetic anhydride be consumed by the hexylamine? That is, can i go with the PYR + hexylamine derivitisation, followed (without any dry down step) by addition of acetic anhydride (X’s) and catalyst to effect acetylation? The X’s acetic anhydride will be quenched with water, and the derivatives back extracted into DCM for GC and GCMS analysis.
Obviously i will check this effect experimentally, but was hoping there may be someone who has done this type of dervitisation before.
Cheers
Peggsy
Ref A: Walters & Hedges, 1988, Anal. Chem. 60:988-994
Ref B: Rumpel & Dignac, 2006, Soil Biol & Biochem. 38: 1478-1481