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By Ricardo on Monday, July 26, 2004 - 04:06 pm:
Please can anyone tell me what pH mobile phase I should use for organic acids? I have made a phosphoric acid buffer with NaH2PO4.H2O. It is 10 Mm and has a pH of 2.57. Will this work?
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By syx_gf on Tuesday, July 27, 2004 - 01:20 am:
Depend on the pKa value of your substance. You need pKa - 2 unit pH for good retention.
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By Ricardo on Tuesday, July 27, 2004 - 10:33 am:
The analytes are acetic and lactic acid, pKas of 4.74 and 3.08 respectively. Sounds like the acetic acid should be fine but the lactic may not be well retained. Is this correct?
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By X.L. on Tuesday, July 27, 2004 - 01:04 pm:
Ricardo,
I happened to work on separation of organic acids for some time. Here is what I learned from my work:
1. The column I used was Acclaim Organic Acid column (Dionex). This is a silica RP column packed into PEEK column body.
2. The chromaotgraphic conditions is 50mM phosphate buffer at pH2.6 to pH3.0(depending on acids of interest). Temp: 30 degree C. Flow rate: 0.6 mL/min, and UV detection at 210 nm.
3. The elution order of selective resolved acids in a single run: oxalic, tartaric, malic, formic, isocitric, lactic, acetic, citric, succinic, fumaric, and aconitic acids.
4. If you need to enhance the resolution between lactic and aceitc acids, try to run around pH2.9.
I can share more detailed information with you in case you need it.
Good luck,
X.L.
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By Ricardo on Tuesday, July 27, 2004 - 02:06 pm:
Thanks syx_gf & X.L. for that info. One more thing. I am using an Agilent 1100 with degasser. Does that mean I don't need to degass prior to filling the reservoirs? There are a lot of bubbles in freshly poured eluant, especially aqeous. I am assuming that the degasser takes care of this. I am also not filtering any eluant. Is the millipore water/organic solvents sufficiently filtered? I have installed an Agilent same phase guard column before the column. I apologize for these basic questions but am fairly new to LC.
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By me/myself&I on Tuesday, July 27, 2004 - 11:00 pm:
Hi,
With an online degasser (and inlet filter) I would never filter/degas the mobile phases. It just dirties them.
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By syx_gf on Wednesday, July 28, 2004 - 12:21 am:
Even though you use inlet filter you should filter your mobile phase, at least with 0.45 um pores size membrane filter. Pores of inlet filter are not small enough to retain "small" particle!
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By David Blais on Wednesday, July 28, 2004 - 10:21 am:
Ricardo,
The Merck Index states that Lactic Acid has a pKa of ~ 3.80, not 3.08 (perhaps you simply reversed the digits while typing). That said, I would think if you could get to pH 2.3 or lower (I've been taught to be 1.5 units from the pKa). I also analyze organic acids (lactic, glycolic) and use 0.1% phosphoric acid (pH ~ 2.1) with success.
Secondly, I would recommend you still degass your mobile phase (esp. aqueous) prior to use on the 1100. The Agilent 1100 degasser is not able to remove that much air from the solvents.
Regards,
David
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By bill tindall on Thursday, July 29, 2004 - 07:30 pm:
0.15% phoshphoric acid and either methanol or acetonitrile and nearly any column is good for most aliphatic and aromatic acids. For best retention of very hydrophilic acids I prefer the ES Industries Aquasep as it is the most retentive of the "aqueous " columns I have tried. I have even done sulfonic acids on this column.
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By Chris Pohl on Friday, July 30, 2004 - 01:01 am:
As mentioned by X.L. above, the optimum pH for the separation of acetate and lactate is dictated by more than simply what pH is necessary to fully protonate both acids. Generally, on most reversed phase columns the two analytes aren't resolved when both acids are nearly completely protonated. The trick is to raise the pH a bit to achieve separation via the differences in the pKa of the two acids. Since lactic acid is a stronger acid than acetic, moving closer to the pKa of lactate will move it forward faster than acetate, improving resolution.
Please can anyone tell me what pH mobile phase I should use for organic acids? I have made a phosphoric acid buffer with NaH2PO4.H2O. It is 10 Mm and has a pH of 2.57. Will this work?
-------------------------------------------------------------------------------------------------------
By syx_gf on Tuesday, July 27, 2004 - 01:20 am:
Depend on the pKa value of your substance. You need pKa - 2 unit pH for good retention.
-------------------------------------------------------------------------------------------------------
By Ricardo on Tuesday, July 27, 2004 - 10:33 am:
The analytes are acetic and lactic acid, pKas of 4.74 and 3.08 respectively. Sounds like the acetic acid should be fine but the lactic may not be well retained. Is this correct?
-------------------------------------------------------------------------------------------------------
By X.L. on Tuesday, July 27, 2004 - 01:04 pm:
Ricardo,
I happened to work on separation of organic acids for some time. Here is what I learned from my work:
1. The column I used was Acclaim Organic Acid column (Dionex). This is a silica RP column packed into PEEK column body.
2. The chromaotgraphic conditions is 50mM phosphate buffer at pH2.6 to pH3.0(depending on acids of interest). Temp: 30 degree C. Flow rate: 0.6 mL/min, and UV detection at 210 nm.
3. The elution order of selective resolved acids in a single run: oxalic, tartaric, malic, formic, isocitric, lactic, acetic, citric, succinic, fumaric, and aconitic acids.
4. If you need to enhance the resolution between lactic and aceitc acids, try to run around pH2.9.
I can share more detailed information with you in case you need it.
Good luck,
X.L.
-------------------------------------------------------------------------------------------------------
By Ricardo on Tuesday, July 27, 2004 - 02:06 pm:
Thanks syx_gf & X.L. for that info. One more thing. I am using an Agilent 1100 with degasser. Does that mean I don't need to degass prior to filling the reservoirs? There are a lot of bubbles in freshly poured eluant, especially aqeous. I am assuming that the degasser takes care of this. I am also not filtering any eluant. Is the millipore water/organic solvents sufficiently filtered? I have installed an Agilent same phase guard column before the column. I apologize for these basic questions but am fairly new to LC.
-------------------------------------------------------------------------------------------------------
By me/myself&I on Tuesday, July 27, 2004 - 11:00 pm:
Hi,
With an online degasser (and inlet filter) I would never filter/degas the mobile phases. It just dirties them.
-------------------------------------------------------------------------------------------------------
By syx_gf on Wednesday, July 28, 2004 - 12:21 am:
Even though you use inlet filter you should filter your mobile phase, at least with 0.45 um pores size membrane filter. Pores of inlet filter are not small enough to retain "small" particle!
-------------------------------------------------------------------------------------------------------
By David Blais on Wednesday, July 28, 2004 - 10:21 am:
Ricardo,
The Merck Index states that Lactic Acid has a pKa of ~ 3.80, not 3.08 (perhaps you simply reversed the digits while typing). That said, I would think if you could get to pH 2.3 or lower (I've been taught to be 1.5 units from the pKa). I also analyze organic acids (lactic, glycolic) and use 0.1% phosphoric acid (pH ~ 2.1) with success.
Secondly, I would recommend you still degass your mobile phase (esp. aqueous) prior to use on the 1100. The Agilent 1100 degasser is not able to remove that much air from the solvents.
Regards,
David
-------------------------------------------------------------------------------------------------------
By bill tindall on Thursday, July 29, 2004 - 07:30 pm:
0.15% phoshphoric acid and either methanol or acetonitrile and nearly any column is good for most aliphatic and aromatic acids. For best retention of very hydrophilic acids I prefer the ES Industries Aquasep as it is the most retentive of the "aqueous " columns I have tried. I have even done sulfonic acids on this column.
-------------------------------------------------------------------------------------------------------
By Chris Pohl on Friday, July 30, 2004 - 01:01 am:
As mentioned by X.L. above, the optimum pH for the separation of acetate and lactate is dictated by more than simply what pH is necessary to fully protonate both acids. Generally, on most reversed phase columns the two analytes aren't resolved when both acids are nearly completely protonated. The trick is to raise the pH a bit to achieve separation via the differences in the pKa of the two acids. Since lactic acid is a stronger acid than acetic, moving closer to the pKa of lactate will move it forward faster than acetate, improving resolution.
