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By AThomson on Monday, October 27, 2003 - 08:34 am:
Hello all
I am currently trying to formulate a rather unconventional HPLC technique.
I am dealing with organic compounds which are relatively polar, yet completely insoluble in aqueous media. I am looking for a reversed-phase technique with which to separate them, but with non-aqueous conditions (eluting with MeOH) the retention times on C18 and C8 columns are too low.
I am considering trying a more polar stationary phase such as a nitrile column, but I am not sure what would be best.
Does anyone know of a technique such as this being used?
I know that this is rather off-the-wall, but any input would be greatly appreciated.
Thanks
Andrew Thomson
-------------------------------------------------------------------------------------------------------
By juddc on Monday, October 27, 2003 - 08:48 am:
HI Andrew,
Not having even the slightest clue as to the structure of the compounds you're dealing with is going to limit the quality of the help you receive here.
In any case, might your polar compounds be soluble in MeOH or MeCN /H20 mixtures? Just because your compound isn't soluble in pure water usually does not mean that you need to go completely anhydrous with your LC mobile phase. In some cases, compounds other than solvents can be used to improve solubility as well. For instance, citric acid is good for increasing the water solubility of theobromine.
Can your compounds be ionized? pH plays a big factor in solubility.
Give more specifics and I think you'll get better information!
-------------------------------------------------------------------------------------------------------
By Uwe Neue on Monday, October 27, 2003 - 03:35 pm:
How about HILIC on a silica column? You inject the sample in an organic solvent, like methanol or acetonitrile, and your mobile phase is acetonitrile/water. To see where your compounds are leuting, run a gradient from acetonitrile to 50/50 water/acetonitrile.
-------------------------------------------------------------------------------------------------------
By Anonymous on Monday, October 27, 2003 - 11:25 pm:
You can consider also graphitized porous carbon column (e.g. Hypercarb or Zr-Carbon). This phases works well for separating polar compounds and there are practically no constraints for mobile phases.
Good Luck.
Regards
-------------------------------------------------------------------------------------------------------
By HW Mueller on Tuesday, October 28, 2003 - 12:02 am:
"Completely insoluble" often really means "soluble more than enough" for the concentrations involved on columns. Why so secretive? What are your comps?
-------------------------------------------------------------------------------------------------------
By AThomson on Tuesday, October 28, 2003 - 04:09 am:
Hello all, thanks for your suggestions.
Sorry, should have given a bit more info on the nature of the compounds etc:
I am working with amide-based rotaxanes and other interlocked architectures. These compounds are generally rather amphiphilic, and do not have any ionisable groups. In general they are not (particularly) soluble in neat DCM, but will go into DCM with a little polar cosolvent such as THF, MeOH or MeCN. It is possible that they could dissolve in MeOH (but not MeCN) with a little water present. It is possible to run conventional analytical HPLC conditions (MeCN/H2O and C18), but I need a technique that I can scale up to prep, and this is where the solubility really becomes a problem.
The compounds may seem a little weird, but their solubility/polarity is comparable to that of any other organic molecule with a preponderance of amide groups.
These ref's may give an idea of what I have to work with!
A.M. Brouwer, C. Frochot, F.G. Gatti, D.A. Leigh, L. Mottier, F. Paolucci, S. Roffia and G.W.H. Wurpel, Science 291, 2124-2128 (2001)
D.A. Leigh, J.K.Y. Wong, F. Dehez and F. Zerbetto, Nature 424, 174-179 (2003); "Unidirectional Rotation in a Mechanically Interlocked Molecular Rotor"
-------------------------------------------------------------------------------------------------------
By Uwe Neue on Tuesday, October 28, 2003 - 03:22 pm:
Ahh prep? You may want to look at our at-column dilution technique, which is designed to maximize the load in prep even when the solubility is too low (which it always is in prep). Chromatographia Supplement Vol 57, 2003, pages S121 - S127 (the issue from last years ISC).
If you can't get Chromatographia, I'll send you a copy.
-------------------------------------------------------------------------------------------------------
By AllsepTech on Tuesday, October 28, 2003 - 09:06 pm:
It looks like you need normal phase chromatography, or HILIC, or polar organic chromatography - whatever name you chose.
We have a brand new variation of this chromatography. Our company offers a stationary phase Primesep B, which is, in fact, a reverse phase brush type SP with a basic functional group in the middle of the chain. When the column is operating in the normal mode with addition of acid to the mobile phase, the stationary base and the acid are forming a very polar ion pair complex. This complex interacts with polar analytes by different polar mechanisms. By changing an acid in the mobile phase, you are changing the structure of the complex. As result, the polar properties of the complex can be tuned to separate difficult to resolve pairs of compounds. You can see our application on resolution of structural isomers of xanthines with different acids in the mobile phase on Primesep B at http://allsep.com/makeChr.php?chr=Chr_002. It is a reverse mode separation, but in the normal mode the effect is even more significant.
Please note that to replace all retained acid on this type of columns you値l have to equilibrate a column with a new acid for at least 1 hr with 20 mmol acid in the mobile phase. You can do it in 10 min if you use 100 mmol, and then switch to the working concentration 5-20 mMol or so.
-------------------------------------------------------------------------------------------------------
By HW Mueller on Friday, October 31, 2003 - 03:52 am:
Uwe, couldnｴt find your article, donｴt you have something of this sort on the Waters web page? Did you load the sample in a strong solvent? Prep columns seem to take quite a bit of such treatment if the separation is not borderline.
Hans
-------------------------------------------------------------------------------------------------------
By Uwe Neue on Saturday, November 1, 2003 - 12:06 pm:
HW,
There are restrictions from the journals on what one can put on web pages. You can put a journal article on a personal web page, but not on a commercial web page. That's why you did not find it there. However, I can provide you with a copy, if you e-mail me at the address above.
-------------------------------------------------------------------------------------------------------
By perica on Friday, May 28, 2004 - 01:18 am:
Hello all,
I am working analisys beverages with HPLC and other compounds they are into soft drinks.Does anyone work with hplc silmilar matrix?
-------------------------------------------------------------------------------------------------------
By Maja on Friday, May 28, 2004 - 03:12 am:
Yes, we are determining artificial sweeteners, preservatives and coffein in soft drinks.
What is exactly your question?
-------------------------------------------------------------------------------------------------------
By Constantine Sychov on Tuesday, June 1, 2004 - 07:11 am:
To A.Thompson
If you want to get reversed phase like order of elution you should use non-polar phases with higher hydrophobicity than C18s have. I think only C30s can meet your requirements (polymeric beads swell in organic solvents). PGC "Hypercarb" has too low efficiency (to my mind). The only polymeric phase for RP HPLC of choice in this case is Chromalite 5HGN (www.purolite.com), the one pached with 5mkm hypercrosslinked polystyrene, that can work in all conditions.
If you want normal phase like order of elution, you can use highly hidrophilic phases like (as Uwe has said) HILIC phases. CN phase wont provide necessary retention in pure organic solvents.
Hello all
I am currently trying to formulate a rather unconventional HPLC technique.
I am dealing with organic compounds which are relatively polar, yet completely insoluble in aqueous media. I am looking for a reversed-phase technique with which to separate them, but with non-aqueous conditions (eluting with MeOH) the retention times on C18 and C8 columns are too low.
I am considering trying a more polar stationary phase such as a nitrile column, but I am not sure what would be best.
Does anyone know of a technique such as this being used?
I know that this is rather off-the-wall, but any input would be greatly appreciated.
Thanks
Andrew Thomson
-------------------------------------------------------------------------------------------------------
By juddc on Monday, October 27, 2003 - 08:48 am:
HI Andrew,
Not having even the slightest clue as to the structure of the compounds you're dealing with is going to limit the quality of the help you receive here.
In any case, might your polar compounds be soluble in MeOH or MeCN /H20 mixtures? Just because your compound isn't soluble in pure water usually does not mean that you need to go completely anhydrous with your LC mobile phase. In some cases, compounds other than solvents can be used to improve solubility as well. For instance, citric acid is good for increasing the water solubility of theobromine.
Can your compounds be ionized? pH plays a big factor in solubility.
Give more specifics and I think you'll get better information!
-------------------------------------------------------------------------------------------------------
By Uwe Neue on Monday, October 27, 2003 - 03:35 pm:
How about HILIC on a silica column? You inject the sample in an organic solvent, like methanol or acetonitrile, and your mobile phase is acetonitrile/water. To see where your compounds are leuting, run a gradient from acetonitrile to 50/50 water/acetonitrile.
-------------------------------------------------------------------------------------------------------
By Anonymous on Monday, October 27, 2003 - 11:25 pm:
You can consider also graphitized porous carbon column (e.g. Hypercarb or Zr-Carbon). This phases works well for separating polar compounds and there are practically no constraints for mobile phases.
Good Luck.
Regards
-------------------------------------------------------------------------------------------------------
By HW Mueller on Tuesday, October 28, 2003 - 12:02 am:
"Completely insoluble" often really means "soluble more than enough" for the concentrations involved on columns. Why so secretive? What are your comps?
-------------------------------------------------------------------------------------------------------
By AThomson on Tuesday, October 28, 2003 - 04:09 am:
Hello all, thanks for your suggestions.
Sorry, should have given a bit more info on the nature of the compounds etc:
I am working with amide-based rotaxanes and other interlocked architectures. These compounds are generally rather amphiphilic, and do not have any ionisable groups. In general they are not (particularly) soluble in neat DCM, but will go into DCM with a little polar cosolvent such as THF, MeOH or MeCN. It is possible that they could dissolve in MeOH (but not MeCN) with a little water present. It is possible to run conventional analytical HPLC conditions (MeCN/H2O and C18), but I need a technique that I can scale up to prep, and this is where the solubility really becomes a problem.
The compounds may seem a little weird, but their solubility/polarity is comparable to that of any other organic molecule with a preponderance of amide groups.
These ref's may give an idea of what I have to work with!
A.M. Brouwer, C. Frochot, F.G. Gatti, D.A. Leigh, L. Mottier, F. Paolucci, S. Roffia and G.W.H. Wurpel, Science 291, 2124-2128 (2001)
D.A. Leigh, J.K.Y. Wong, F. Dehez and F. Zerbetto, Nature 424, 174-179 (2003); "Unidirectional Rotation in a Mechanically Interlocked Molecular Rotor"
-------------------------------------------------------------------------------------------------------
By Uwe Neue on Tuesday, October 28, 2003 - 03:22 pm:
Ahh prep? You may want to look at our at-column dilution technique, which is designed to maximize the load in prep even when the solubility is too low (which it always is in prep). Chromatographia Supplement Vol 57, 2003, pages S121 - S127 (the issue from last years ISC).
If you can't get Chromatographia, I'll send you a copy.
-------------------------------------------------------------------------------------------------------
By AllsepTech on Tuesday, October 28, 2003 - 09:06 pm:
It looks like you need normal phase chromatography, or HILIC, or polar organic chromatography - whatever name you chose.
We have a brand new variation of this chromatography. Our company offers a stationary phase Primesep B, which is, in fact, a reverse phase brush type SP with a basic functional group in the middle of the chain. When the column is operating in the normal mode with addition of acid to the mobile phase, the stationary base and the acid are forming a very polar ion pair complex. This complex interacts with polar analytes by different polar mechanisms. By changing an acid in the mobile phase, you are changing the structure of the complex. As result, the polar properties of the complex can be tuned to separate difficult to resolve pairs of compounds. You can see our application on resolution of structural isomers of xanthines with different acids in the mobile phase on Primesep B at http://allsep.com/makeChr.php?chr=Chr_002. It is a reverse mode separation, but in the normal mode the effect is even more significant.
Please note that to replace all retained acid on this type of columns you値l have to equilibrate a column with a new acid for at least 1 hr with 20 mmol acid in the mobile phase. You can do it in 10 min if you use 100 mmol, and then switch to the working concentration 5-20 mMol or so.
-------------------------------------------------------------------------------------------------------
By HW Mueller on Friday, October 31, 2003 - 03:52 am:
Uwe, couldnｴt find your article, donｴt you have something of this sort on the Waters web page? Did you load the sample in a strong solvent? Prep columns seem to take quite a bit of such treatment if the separation is not borderline.
Hans
-------------------------------------------------------------------------------------------------------
By Uwe Neue on Saturday, November 1, 2003 - 12:06 pm:
HW,
There are restrictions from the journals on what one can put on web pages. You can put a journal article on a personal web page, but not on a commercial web page. That's why you did not find it there. However, I can provide you with a copy, if you e-mail me at the address above.
-------------------------------------------------------------------------------------------------------
By perica on Friday, May 28, 2004 - 01:18 am:
Hello all,
I am working analisys beverages with HPLC and other compounds they are into soft drinks.Does anyone work with hplc silmilar matrix?
-------------------------------------------------------------------------------------------------------
By Maja on Friday, May 28, 2004 - 03:12 am:
Yes, we are determining artificial sweeteners, preservatives and coffein in soft drinks.
What is exactly your question?
-------------------------------------------------------------------------------------------------------
By Constantine Sychov on Tuesday, June 1, 2004 - 07:11 am:
To A.Thompson
If you want to get reversed phase like order of elution you should use non-polar phases with higher hydrophobicity than C18s have. I think only C30s can meet your requirements (polymeric beads swell in organic solvents). PGC "Hypercarb" has too low efficiency (to my mind). The only polymeric phase for RP HPLC of choice in this case is Chromalite 5HGN (www.purolite.com), the one pached with 5mkm hypercrosslinked polystyrene, that can work in all conditions.
If you want normal phase like order of elution, you can use highly hidrophilic phases like (as Uwe has said) HILIC phases. CN phase wont provide necessary retention in pure organic solvents.
