Separation of tartaric acid and oxalic acid [May 20, 2004]
Posted: Wed Aug 25, 2004 3:43 am
By Leticia on Thursday, May 20, 2004 - 11:41 pm:
We are trying to separate oxalic acid and tartaric acid.
We tried some polar columns such as Synergy Hydro-RP and Aqua C18, with a phosphate buffer at pH 2.5 as mobile phase, and these two compounds separated but eluted very close to the void volume and oxalate co-eluted with nitrate.
Later we tried a 0.01M phosphate+0.01M tetrabutyl ammonium (pH6): acetonitrile (85:15) at 25C with a Hypersil BDS C18 column. The acids two were now well retained and separated from nitrate, but the resolution between them was not great, the best we got was USP resolution 1.1.
Has anyone got any ideas? we are trying to avoid using Ion Chromatography.
-------------------------------------------------------------------------------------------------------
By Vojtech on Friday, May 21, 2004 - 06:37 am:
Recently I turned acids into a propylesters. I used this method just for identification and I am not sure about yield of the reaction. My samples were strongly acidic with high content of nitrate. When I tried to dry the sample the matrix destroyed everything. So I pipetted about 100 ul of sample into 1 ml of n-propanol and added 10 ul of H2SO4. The sample was heated (80°C) and vortexed for about 10 minutes (in reaction vial). I primarily used GC for identication so I had to use l-l extraction or SPE for clean-up. Maybe if you will not use extraction step the reproducibilty of reaction will be good. This is a simple method and I was really surprised that water had not a detrimental effect on esterification. Good Luck, Vojtech.
-------------------------------------------------------------------------------------------------------
By AllsepTech on Friday, May 21, 2004 - 08:47 am:
Tartaric acid and oxalic acid are very different in pKa and hydrophobicity. We have two columns for the separation of acids. Using our column you can change the elution order of oxalic acid and tartaric acid, depending on the amount of modifier in the mobile phase (TFA, sulfuric or any other strong acid) and amount of organic component (ACN). Please check the following links and find similarities:
http://allsep.com/makeChr.php?chr=Chr_031
http://allsep.com/makeChr.php?chr=Chr_037
http://allsep.com/makeChr.php?chr=Chr_023
We can run a quick method for your and show you results for your particular case. We need to know your detection technique, preferred mobile phase, concentrations, etc.
You do not have to derivitize your acids and use ion pairing reagent and your two acids will be "a mile" apart.
-------------------------------------------------------------------------------------------------------
By David Blais on Friday, May 21, 2004 - 09:07 am:
I recommend trying any of the following columns:
Shodex RSPak, KC-811, 7.9mm x 300 mm
Varian Metacarb H Plus, 7.8mm x 300mm
Varian Polaris, C18-Ether, 4.6mm x 250mm
PolymerLabs, PLRP-S, 4.6mm x 250mm
PolymerLabs, PL Hi-Plex H, 7.7mm x 300mm
I have used all of these columns for the analysis of organic acids. I found the Shodex RSPak column yielded the best retention and resolution for my work (glycolic acid, lactic acid). You can check out their respective websites and see the example chromatograms or speak with their sales reps/tech support people.
Hope this helps. Good luck!
-David
-------------------------------------------------------------------------------------------------------
By Constantine Sychov on Sunday, May 23, 2004 - 06:03 am:
No, you dont need to try any neutral polymeric or ion exclusion/ion exchange columns! But you also must not believe in advertisments of different firms... polar Synergy is TOO polar column. You only have to take unendcapped C18 with high carbon load... may be, LUNA or old phases like LiChrospher... Just try ANY OTHER C18 packing you have!
By the way - it is simple mistake to apply TBA to separate week acids - it will be no good.
-------------------------------------------------------------------------------------------------------
By leticia on Sunday, May 23, 2004 - 04:41 pm:
Hi Constantine,
I read your message. Just a couple of questions:
1) When I tried the sinergy column the retention was not great, in fact, one of the acids eluted very close to the negative peak at the void volume, sometimes on top of it. Wouln't this get worse with a non-polar column?. Isn't there the possibility that the acids will not be retained at all?
2) I have been using tetrabutyl ammonium as an ion pair reagent. In your message you mention that it will not separate two weak acids. Why is that? Can you suggest a better alternative?
-------------------------------------------------------------------------------------------------------
By Chris Pohl on Sunday, May 23, 2004 - 08:41 pm:
letica,
One of the main weaknesses of ion pair is that it has poor charge distribution selectivity. Ion exchange consistently beats ion pair in separations involving divalent, trivalent and higher valency analytes due to the fact that it has much better selectivity for molecules with multiple charges at various points. This is because the ion exchange sites are fixed geometry in the stationary phase which renders it more selective for polyvalent species which "fit" this stationary phase distribution than for species that more poorly match the charge distribution of the phase. In ion pair, the ion pair reagent molecules are free to adopt "optimal" positions for all polyvalent species, eliminating this spacial selectivity present in ion exchange.
If you want to perform this separation with an ion pair reagent, lower the pH sufficiently to create a condition where oxalate is divalent but tartrate is not (sorry, I'm not near a reference with the pKa data for these analytes so I can't be more specific).
-------------------------------------------------------------------------------------------------------
By din on Monday, May 24, 2004 - 05:54 am:
Suggest to try following conditions,
Column : Inersil ODS-3V, 250 x4.5, 5 micron
M.Phase: 0.1 M Ammonium dihydrogen phosphate+phosphoric acid (pH 2.5)
Flow ; 1 ml/min
column Oven : 40
UV=210
These conditiones seperates most of the organic acids. The resolution of tartaric acid and oxalic acid is about 2.
Good luck
-------------------------------------------------------------------------------------------------------
By Constantine Sychov on Monday, May 24, 2004 - 06:53 am:
To Leticia, the answers are
1) Dont worry, everythinds gonna be alright! Do as din suggested, for example
2) Chris already has commented, but a few words more. Imagine - hydrophobicity of two compounds is very close, their pKas are almost identical. Hence, the formation of two week ion pairs (mmmm, there are no any pairs in fact, but it is simple to consider situation in this way) will yeild the likehood of two compounds increased!!! So, you have to use RP mode or ion chromatography, but not IP HPLC in this case. OR you are to regulate pH very carefully to achieve good selectivity, but you may lose the efficincy of such systems. The simple rule is - use TBA to separate strong acids, SDS to separate strong bases!
-------------------------------------------------------------------------------------------------------
By Constantine Sychov on Monday, May 24, 2004 - 07:01 am:
By the way.. If it is interesting: I achieved the selectivity oxalic/tartaric acids of about 2 (!) on experimental polymeric sorbent. The oxalic acid was on 7-8 min, and tartaric on 12-13 min.
-------------------------------------------------------------------------------------------------------
By Anonymous on Monday, May 24, 2004 - 09:39 am:
Since when pKa of oxalic and tartaric acids are almost identical? As far as literature says pKa of oxalic is 1.23 and pKa of tartaric 2.98 (first pKa), so oxalic is 57 times stronger acid.
-------------------------------------------------------------------------------------------------------
By leticia on Monday, May 24, 2004 - 04:29 pm:
Thanks to everyone for their reply.
About the pKa that is in fact the same data I have corresponding to the first pKa for oxalic acid and tartaric acid. The second pKas are the ones that are similar (about 4.3 for both acids).
Now changing the subject:
Din, What retention time did you achieve for oxalic acid and tartaric acid? I can separate them with some of my columns but the retention is very low, eluting almost at the void volume. This means irreproducible integration and that other poorly retained ions, such as nitrate, coelute with oxalate.
-------------------------------------------------------------------------------------------------------
By Anonymous on Monday, May 24, 2004 - 10:47 pm:
liticia,
Retention times,
Oxalic Acid : about 2.7 min
Tartaric acid : about 3.2 min
There is one more ineresting condition you can try ,
Column : Novapack RP-18 ,15 cm x 3.9 mm 4 micron Mobile phase: 0.1 M Sodium sulphate buffer, ph about 2.5 by dil. H2SO4
Flow rate: 0.8 ml / minute
Detector: UV -230 nm or 210
Good luck
-------------------------------------------------------------------------------------------------------
By Constantine Sychov on Thursday, May 27, 2004 - 09:01 am:
2.7 min is almost the void volume. So, the "permanent" solution is to use SAX columns, or polymeric PR columns like PLRP-S (you may find even cheaper ones of this kind)
-------------------------------------------------------------------------------------------------------
By Uwe Neue on Thursday, May 27, 2004 - 03:42 pm:
Constantine:
I don't know... The V0 of this Nova-Pak C18 column is 1 mL. A retention time of 2.7 on this column is a retention factor of about 1.17. I would not call this unretained.
On the other hand, I would use Atlantis dC18 under the same conditions to get double or triple the retention (Atlantis dC18 has been designed to give maximum retention in fully aqueous mobile phases).
-------------------------------------------------------------------------------------------------------
By leticia on Thursday, May 27, 2004 - 11:23 pm:
Thanks to everyone for their replies.
We eventually separated those oxalic and tartaric acid by IC. The resolution is very good and nitrate doesn't interfere anymore.
-------------------------------------------------------------------------------------------------------
By Constantine Sychov on Saturday, May 29, 2004 - 08:21 am:
Uwe, I was inattentive... 1 min is appr. Vo for 15x3 of course...
I heard about C30, but never used it in my practice. People say it can be applied not only for analyzing of very hydrophobic, but also very hydrophilic substances...
-------------------------------------------------------------------------------------------------------
By Uwe Neue on Saturday, May 29, 2004 - 10:17 am:
I do not know if a C30 would work. Have not worked with it. But I do know that C18 packings that either by accident (Resolve C18) or by design (Atlantis dC18) work in 100% water will give better retention of these very polar compounds.
-------------------------------------------------------------------------------------------------------
By Constantine Sychov on Monday, May 31, 2004 - 05:44 am:
Uwe,
you`re saying "by accident" and "by design" on the base of manufacturer`s information, your own experience or you know something about proprietary chemistry of these phases? I`m just interested... you may not answer.
-------------------------------------------------------------------------------------------------------
By Uwe Neue on Monday, May 31, 2004 - 10:52 am:
Sychov,
The first phase, Resolve C18, I designed myself. For the second phase, Atlantis dC18, I was peripherally involved in the creation.
Uwe
-------------------------------------------------------------------------------------------------------
By Constantine Sychov on Tuesday, June 1, 2004 - 07:19 am:
Oh, I see. I suspected something of this kind.
We are trying to separate oxalic acid and tartaric acid.
We tried some polar columns such as Synergy Hydro-RP and Aqua C18, with a phosphate buffer at pH 2.5 as mobile phase, and these two compounds separated but eluted very close to the void volume and oxalate co-eluted with nitrate.
Later we tried a 0.01M phosphate+0.01M tetrabutyl ammonium (pH6): acetonitrile (85:15) at 25C with a Hypersil BDS C18 column. The acids two were now well retained and separated from nitrate, but the resolution between them was not great, the best we got was USP resolution 1.1.
Has anyone got any ideas? we are trying to avoid using Ion Chromatography.
-------------------------------------------------------------------------------------------------------
By Vojtech on Friday, May 21, 2004 - 06:37 am:
Recently I turned acids into a propylesters. I used this method just for identification and I am not sure about yield of the reaction. My samples were strongly acidic with high content of nitrate. When I tried to dry the sample the matrix destroyed everything. So I pipetted about 100 ul of sample into 1 ml of n-propanol and added 10 ul of H2SO4. The sample was heated (80°C) and vortexed for about 10 minutes (in reaction vial). I primarily used GC for identication so I had to use l-l extraction or SPE for clean-up. Maybe if you will not use extraction step the reproducibilty of reaction will be good. This is a simple method and I was really surprised that water had not a detrimental effect on esterification. Good Luck, Vojtech.
-------------------------------------------------------------------------------------------------------
By AllsepTech on Friday, May 21, 2004 - 08:47 am:
Tartaric acid and oxalic acid are very different in pKa and hydrophobicity. We have two columns for the separation of acids. Using our column you can change the elution order of oxalic acid and tartaric acid, depending on the amount of modifier in the mobile phase (TFA, sulfuric or any other strong acid) and amount of organic component (ACN). Please check the following links and find similarities:
http://allsep.com/makeChr.php?chr=Chr_031
http://allsep.com/makeChr.php?chr=Chr_037
http://allsep.com/makeChr.php?chr=Chr_023
We can run a quick method for your and show you results for your particular case. We need to know your detection technique, preferred mobile phase, concentrations, etc.
You do not have to derivitize your acids and use ion pairing reagent and your two acids will be "a mile" apart.
-------------------------------------------------------------------------------------------------------
By David Blais on Friday, May 21, 2004 - 09:07 am:
I recommend trying any of the following columns:
Shodex RSPak, KC-811, 7.9mm x 300 mm
Varian Metacarb H Plus, 7.8mm x 300mm
Varian Polaris, C18-Ether, 4.6mm x 250mm
PolymerLabs, PLRP-S, 4.6mm x 250mm
PolymerLabs, PL Hi-Plex H, 7.7mm x 300mm
I have used all of these columns for the analysis of organic acids. I found the Shodex RSPak column yielded the best retention and resolution for my work (glycolic acid, lactic acid). You can check out their respective websites and see the example chromatograms or speak with their sales reps/tech support people.
Hope this helps. Good luck!
-David
-------------------------------------------------------------------------------------------------------
By Constantine Sychov on Sunday, May 23, 2004 - 06:03 am:
No, you dont need to try any neutral polymeric or ion exclusion/ion exchange columns! But you also must not believe in advertisments of different firms... polar Synergy is TOO polar column. You only have to take unendcapped C18 with high carbon load... may be, LUNA or old phases like LiChrospher... Just try ANY OTHER C18 packing you have!
By the way - it is simple mistake to apply TBA to separate week acids - it will be no good.
-------------------------------------------------------------------------------------------------------
By leticia on Sunday, May 23, 2004 - 04:41 pm:
Hi Constantine,
I read your message. Just a couple of questions:
1) When I tried the sinergy column the retention was not great, in fact, one of the acids eluted very close to the negative peak at the void volume, sometimes on top of it. Wouln't this get worse with a non-polar column?. Isn't there the possibility that the acids will not be retained at all?
2) I have been using tetrabutyl ammonium as an ion pair reagent. In your message you mention that it will not separate two weak acids. Why is that? Can you suggest a better alternative?
-------------------------------------------------------------------------------------------------------
By Chris Pohl on Sunday, May 23, 2004 - 08:41 pm:
letica,
One of the main weaknesses of ion pair is that it has poor charge distribution selectivity. Ion exchange consistently beats ion pair in separations involving divalent, trivalent and higher valency analytes due to the fact that it has much better selectivity for molecules with multiple charges at various points. This is because the ion exchange sites are fixed geometry in the stationary phase which renders it more selective for polyvalent species which "fit" this stationary phase distribution than for species that more poorly match the charge distribution of the phase. In ion pair, the ion pair reagent molecules are free to adopt "optimal" positions for all polyvalent species, eliminating this spacial selectivity present in ion exchange.
If you want to perform this separation with an ion pair reagent, lower the pH sufficiently to create a condition where oxalate is divalent but tartrate is not (sorry, I'm not near a reference with the pKa data for these analytes so I can't be more specific).
-------------------------------------------------------------------------------------------------------
By din on Monday, May 24, 2004 - 05:54 am:
Suggest to try following conditions,
Column : Inersil ODS-3V, 250 x4.5, 5 micron
M.Phase: 0.1 M Ammonium dihydrogen phosphate+phosphoric acid (pH 2.5)
Flow ; 1 ml/min
column Oven : 40
UV=210
These conditiones seperates most of the organic acids. The resolution of tartaric acid and oxalic acid is about 2.
Good luck
-------------------------------------------------------------------------------------------------------
By Constantine Sychov on Monday, May 24, 2004 - 06:53 am:
To Leticia, the answers are
1) Dont worry, everythinds gonna be alright! Do as din suggested, for example
2) Chris already has commented, but a few words more. Imagine - hydrophobicity of two compounds is very close, their pKas are almost identical. Hence, the formation of two week ion pairs (mmmm, there are no any pairs in fact, but it is simple to consider situation in this way) will yeild the likehood of two compounds increased!!! So, you have to use RP mode or ion chromatography, but not IP HPLC in this case. OR you are to regulate pH very carefully to achieve good selectivity, but you may lose the efficincy of such systems. The simple rule is - use TBA to separate strong acids, SDS to separate strong bases!
-------------------------------------------------------------------------------------------------------
By Constantine Sychov on Monday, May 24, 2004 - 07:01 am:
By the way.. If it is interesting: I achieved the selectivity oxalic/tartaric acids of about 2 (!) on experimental polymeric sorbent. The oxalic acid was on 7-8 min, and tartaric on 12-13 min.
-------------------------------------------------------------------------------------------------------
By Anonymous on Monday, May 24, 2004 - 09:39 am:
Since when pKa of oxalic and tartaric acids are almost identical? As far as literature says pKa of oxalic is 1.23 and pKa of tartaric 2.98 (first pKa), so oxalic is 57 times stronger acid.
-------------------------------------------------------------------------------------------------------
By leticia on Monday, May 24, 2004 - 04:29 pm:
Thanks to everyone for their reply.
About the pKa that is in fact the same data I have corresponding to the first pKa for oxalic acid and tartaric acid. The second pKas are the ones that are similar (about 4.3 for both acids).
Now changing the subject:
Din, What retention time did you achieve for oxalic acid and tartaric acid? I can separate them with some of my columns but the retention is very low, eluting almost at the void volume. This means irreproducible integration and that other poorly retained ions, such as nitrate, coelute with oxalate.
-------------------------------------------------------------------------------------------------------
By Anonymous on Monday, May 24, 2004 - 10:47 pm:
liticia,
Retention times,
Oxalic Acid : about 2.7 min
Tartaric acid : about 3.2 min
There is one more ineresting condition you can try ,
Column : Novapack RP-18 ,15 cm x 3.9 mm 4 micron Mobile phase: 0.1 M Sodium sulphate buffer, ph about 2.5 by dil. H2SO4
Flow rate: 0.8 ml / minute
Detector: UV -230 nm or 210
Good luck
-------------------------------------------------------------------------------------------------------
By Constantine Sychov on Thursday, May 27, 2004 - 09:01 am:
2.7 min is almost the void volume. So, the "permanent" solution is to use SAX columns, or polymeric PR columns like PLRP-S (you may find even cheaper ones of this kind)
-------------------------------------------------------------------------------------------------------
By Uwe Neue on Thursday, May 27, 2004 - 03:42 pm:
Constantine:
I don't know... The V0 of this Nova-Pak C18 column is 1 mL. A retention time of 2.7 on this column is a retention factor of about 1.17. I would not call this unretained.
On the other hand, I would use Atlantis dC18 under the same conditions to get double or triple the retention (Atlantis dC18 has been designed to give maximum retention in fully aqueous mobile phases).
-------------------------------------------------------------------------------------------------------
By leticia on Thursday, May 27, 2004 - 11:23 pm:
Thanks to everyone for their replies.
We eventually separated those oxalic and tartaric acid by IC. The resolution is very good and nitrate doesn't interfere anymore.
-------------------------------------------------------------------------------------------------------
By Constantine Sychov on Saturday, May 29, 2004 - 08:21 am:
Uwe, I was inattentive... 1 min is appr. Vo for 15x3 of course...
I heard about C30, but never used it in my practice. People say it can be applied not only for analyzing of very hydrophobic, but also very hydrophilic substances...
-------------------------------------------------------------------------------------------------------
By Uwe Neue on Saturday, May 29, 2004 - 10:17 am:
I do not know if a C30 would work. Have not worked with it. But I do know that C18 packings that either by accident (Resolve C18) or by design (Atlantis dC18) work in 100% water will give better retention of these very polar compounds.
-------------------------------------------------------------------------------------------------------
By Constantine Sychov on Monday, May 31, 2004 - 05:44 am:
Uwe,
you`re saying "by accident" and "by design" on the base of manufacturer`s information, your own experience or you know something about proprietary chemistry of these phases? I`m just interested... you may not answer.
-------------------------------------------------------------------------------------------------------
By Uwe Neue on Monday, May 31, 2004 - 10:52 am:
Sychov,
The first phase, Resolve C18, I designed myself. For the second phase, Atlantis dC18, I was peripherally involved in the creation.
Uwe
-------------------------------------------------------------------------------------------------------
By Constantine Sychov on Tuesday, June 1, 2004 - 07:19 am:
Oh, I see. I suspected something of this kind.