Acids on Eclipse C18XDB

[WK]

I tried to separate organic acids and aldehydes with 0.15%v/v phosphoric acid and MeCN with success except for irreproducible RTs (both run to run and sequence to sequence).
So I tried buffering KH2PO4 at 10 and 20mM and the peaks tail really bad.
The lowest analyte pKa is 4.42 and I adjusted to pH 2.5 and also 2.1 with no luck.
Why do the peaks tail when all I add is buffer which should help?
Thanks in advance

[Uwe Neue]

Before you added the phosphate your pH must have been below 2. What do you know about the pH stability of your column?

[Uwe Neue]

Oops, I had not seen that you used the XDB column. It is possible that you degrade the coating at a pH less than 2.

[WK]

Thanks Uwe for the reply.
I started using 0.15%v/v phosphoric acid again today and good peak shapes returned so the column is OK (pH of this is 2.20).
I am going to try my separation at pH3 today since XDB is recommended at pH3-8, although pH2 is tolerable. I am worried that I will not be at least 2units under the lowest pKa of my sample though (only 1.4 under)
I could use another Zorbax C18 type column for lower pH but I will need to order one in!
I have a short Hypersil BDS column but I don't get the desired selectivity for my sample with this.

[WK]

Uwe,
I have tried pH3.0 with tailing and also pH2.3 with tailing.
So I'm left with pH2.2 and below to pH2.0
It is clear I will have problems continuing with the pH2.2 with XDB so I will look to SB-Aq or even a SB-Phenyl. Something a little more stable at pH2.2
The MeCN content is 7% at the moment so an aqueous type might do well.
What do you think?

[WK]

A Genesis C18aq gives good peak shape but retention is similar to C18 (understandable).
I have not used Phenyl before so I am interested in its characteristics.

[MG]

In general, phenyl will give you less retention than C18, but with different selectivity. If you try it, try both methanol and acetonitrile as your organic modifier.

[Uwe Neue]

You are successfull with your sample, and you are getting a good peak shape at pH 2.2. I would stick with this method, until I would see results that speak against using this column.

Do not go to a phenyl column, it is generally less stable than a C18! I would also not go to an AQ column, unless I would know much more about it.

If it turns out that you need a more stable column, here are your options:
Within the Zorbax product line, I would use a Stablebond column. This column has a large side chain that gives the column a good stability at acidic pH. (Please check, if all Stablebonds have this bonding technology - I am not sure about it at this moment.) Alternatively, you can use a bonded phase with a difunctional or trifunctional bonding. Options are Atlantis dC18 and Sunfire dC18 from Waters, or Luna C18 (2).

[XL]

WK,

From my experience working with organic acid analysis, the column needs to be compatible with pure or highly aqueous mobile phase and hydrolytically stable at operating pH, say pH2. The columns that are potentially useful for you include Atlantis dC18 (Waters), Stablebond C18 (Agilent), and Acclaim OA (Dionex). I am not sure if Lune(2) C18 is compatible with 100% aqueous.

Once the column (RP) is chosen, I would consider two factors in method development stage - mobile phase pH and ionic strength. I have much experience with Acclaim OA column for organic acid analysis. Here is the chromatographic condition I used to separate oxalic, tartaric, malic, formic, acetic, lactic, citric, succinic, and fumatic acids in a single run.

Column: Acclaim OA, 5um 4x150 mm
Mobile phase: 50 mM phosphate buffer, pH2.7
Flow rate: 0.6 mL/min
Temperature: 30 C
Detection: UV at 210 nm

If you need to increase the resolution between acetic and lactic, raise pH up to 2.9. Other more hydrophobic analytes can be eluted with ACN gradient.

By above method, I haven't observed any retention time reproducibility problem so far except one time I did something wrong in preparing my mobile phase.

I hope my reply is helpful, and am willing to share more information with you.

Good luck,

XL

[Bill Tindall]

1. You are adequately buffered with the 0.15% phosphoric acid. You can separate very acidic species with this eluant- trifunctional aromatic acids and many sulfonic acids for example. I have done zillions of these analyses for acids with this eluant, usually starting with 100% aqueous.

2. Any of the "AQ" columns work well with this eluant. My preference, because of its excellent retention, is the ES Industries AQ column, either C8 or C18. I have used a Spherisorb column for years with this level of phosphoric acid as well.

3. I suspect the wandering retention times is nothong to do with the column chemistry or eluant. Probably a pump problem.

[Einar Ponten]

May I have a considerate proposal. While being on the edge of reversed phase separations and using conditions requiring almost 100% water.

Why not try HILIC mode of separation. That may give you more options and flexibility in order to get selectivity.