Chromatography Forum

I am trying to separate oxalic acid and NaCl so I can quantify the oxalic acid. Other organic acids being calibrated at the same time are pyruvic, lactic, acetic and propionic. Retention times for oxalic and NaCl are 1.79 and 1.85 minutes, right on top of the void volume.
I am using a Stablebond AQ from Agilent. Mobile phase is 20 mM Phosphate buffer at pH 2.92 with 1% ACN. Temp is 30 C and flow of 1.0 ml/min. I have tried different temps and tried increasing the organic content using a gradient but saw no change in resolution. Also used pH of 2.5 but still no good. Am I trying to do too wide a range of acids? if I increase the pH significantly, I think my resolution for some of the other peaks may be in question.

Hi, Roberto!

From my experience, separation of oxalic acid and NaCl ( Na+ or HCl ?? what are you looking for?) at mentoined conditions is hardly possible - no retention! I can recommend to you the system I have applied successfully for oxalic acid (not for NaCl), but, first, which detector are you using?

Ricardo, sorry, I had mess you name.

Maris

If this is at all possible with this column, you should use a fully aqueous mobile phase, and not 1 % MeCN. What detection are you using?

An alternative to RP could be HILIC.

I am using diode array detection at 210 nm. I also have an IR detector available.
In addition, I am in the process of reviewing makers of ELSD to purchase soon. Any suggestions?

If your retention for the oxalic acid peak is not increasing a lot after taking out the 1% organic, my suggestion would be to use a silica HILIC column. Oxalic acid would be retained the most, and you would get the least retention for propionic. Start with a gradient from 90% acetonitrile to 50% acetonitrile, and see if the elution pattern is satisfactory. If not, it can be manipulated depending on the results.

Ricardo,

It is quite difficult to achieve resolution of this pair using a reversed phase system. Your only hope is to lower the pH but this would require dropping the pH to around 1 which isn't great for the stationary phase stability and furthermore this pH compromises the lactate-acetate resolution.

Two other options:

Use a silver loaded resin cartridge to remove chloride prior to analysis. Such cartridges are available from a number of sources. The product available from Dionex is called OnGuard Ag.

Switch to anion exchange since chloride and oxalate are easy to separate by anion exchange. The anions in question can be separated on an IonPac AS17 from Dionex. This column has been optimized for use with a hydroxide eluent so you wouldn't be able to use ELSD with this eluent unless you pass the eluent through a suppressor device prior to the ELSD (a variety of such suppressors are available from Dionex).

Evaluation of Glycolic acid by HPLC



Reagents: Acetonitlile HPLC
Water HPLC
Phosphoric acid 85%
Hexylamine (Acros 204725000 is suitable)

Column & Packing: Inertsil C8-3 5micron 250 x 4.6 mm GL Sciences P/N 5020-01901.
Mobile phase : Solvent A: 80% Buffer : 20% ACN

Detection: UV operated at 210 nm.

Injection volume : 20 mcL

Oven temperature: 30oC

Preparation of buffer solution: Add 8 mL of Hexylamine to 1.0 L of water. Adjust the pH to 6.0 with Phosphoric acid.

When the chromatogram is recorded in the prescribed conditions, the retention time of Glycolic acid is about 8-10 minutes.

Good luck!

Try 100% aqueous 0.15% phosphoric acid on one of the ES Industries "aqueous" columns, C8 or C18. The packing is high surface area and they are the most retentive of any column I have evaluated. You might call people at ES Industires and ask about your separation. they may have tried it, I have not.

UV at 205 nm is very sensitive for orgainic acids and especially so for ones with two carbonyls. If you can get a separation for oxalic acid I will bet the detection limit is 1 ppm or there abouts.

The method proposed by me isn't a speculation, it works in at least three laboratories.

Hi Ricardo

Unless you are able to clean up the samples prior to injection as suggested by Chris Pohl, you will have NO CHANCE of separating oxalate from chloride. We routinely analyse for organic acids in plant root exudates (either soil or hydroponic based samples), and with no SPE, we don't even try to look for oxalate. We use Alltima C18, 1mL/min PDA@210nm and 100% 25mM KH2PO4 at pH2.5 to try to screen for oxalate, but no success unless the samples are very very clean. I have also tried a number of the aqueous columns on the market, again no success with respect to oxalate retention. I do not read too much into any published results for oxalate using RP-HPLC due to my personal experience.

If you are in dire need of oxalate quantification, i would siggest GC post TBDMS derivitization. This is an easy process akin to using SPE for LC analyses.

Good luck mate!

Regards
Greg

What is TBDMS? I have done GC of oxalic after derivatization with diazomethane to the dimethyl esters. It´s very clean, simple.
If you are not limited to any method, column...., oxalic (as acid) is well retained on Hypercarb (mentioned before).

Well, for the separation of such polar things, I recommend HILIC. You will have no problem separating oxalate from chloride.

TBDMS is tertiary butly dimethyl sily derivitizatives for alcohols, sugars and the like. Simple deriv process in solvent such as pyridine, MeCN, but must be water free.

CH2N2 methylation can be done in aqu. solution.