Anion exchange chromatography problem

[Edde]

I followed a paper (Br J Pharm 1995:114:1621-1624) using IC-Pak A 50mm, 1mM phosphate buffer pH8 mobile phase, injection volume: 20uL, UV detection at 214nm, for analysis of nitrate and nitrite. Detection limits for Nitrite and nitrate are 4.6ppb and 31ppb respectively.

I have an IC-Pak anion HC 4.6 x 150mm, so I used it instead. I perform on an Agilent HPLC 1200
Mobile phase is prepared as follows:- 1mM KH2PO4 adjusted to pH8 with NaOH,
When I analyze 1ppm aqueous nitrate and 1ppm nitrite, there was no peak.

I don't know what was wrong. Anyone can kindly help please?

[Markus Laeubli, Metrohm]

The conditions are OK. I actually do not know the capacity difference between the two columns. Anyway your column will show longer retention times. Make sure that you run long enough chromatograms. you might use a stronger eluent.
Also check the purity of your water, phosphate and NaOH.

[Edde]

I actually do not know the capacity difference between the two columns

How does the capacity affect chromatography?

your column will show longer retention times. Make sure that you run long enough chromatograms. you might use a stronger eluent.

I already run isocratic for 25min and see nothing and not even in the next run for another standard. The RT stated in the paper are 6.5 and 13 min for nitrite and nitrate respectively.
What is a stronger eluate? Can a stronger eluant help?

Also check the purity of your water, phosphate and NaOH.

How purity can affect?

Sorry that I ask so many basic questions? I am new to ion exchange chromatography.
Thank you very much.

[Markus Laeubli, Metrohm]

Dear Edde

The capacity of the column directly influences the retention time similar to e.g., column length.
A column with doubled capacity but same dimensions will double the retention times.

As I do not know the columns by experience, it is difficult for me to tell any suitable run time. Actually I would start with e.g. 4-times the eluent strength, means 4 times the concentration of the eluent.

Purity: you need to make sure that there is not anabsorbance at 214 nm by any of the chemicals. You need to avoid e.g., any acetone.

[Edde]

Thank you.

Actually I would start with e.g. 4-times the eluent strength, means 4 times the concentration of the eluent.

In addition to the capacity difference, how about the column length difference? Do you count both factors when suggesting 4 times eluent conc?

4 times eluent conc. = 4mM KH2PO4 adjusted to pH8 with NaOH ?

[tom jupille]

As near as I can tell from Waters web site, both of those columns use the same packing (capacity is listed as 30 μeq/mL) the only difference being the column length (50 vs 150 mm). At equal flow rate your retention on the longer column should have been three times longer.

If you're sure you didn't see any peaks (i.e., you didn't see a big blob at t0), then a series of experiments with stronger (= higher ionic strength) buffers is indeed the next thing to try. In ion exchange, the relationship between retention and ionic strength is approximately log-log [i.e., log(k') is a linear function of log(salt concentration).

I'm a little more conservative than Marcus. If it were my problem, I would double the salt concentration each time (i.e, 1 mM, 2mM, 4mM, . . . etc.) until I saw peaks. If you go that route, be prepared to see some ugly chromatography at first once you get a strong enough eluant, because you have the analytes from a lot of injections to wash off!

Since you're doing UV detection, you might also consider a gradient run, increasing from 1mM to say 30 mM phosphate. That would let you estimate an appropriate ionic strength for elution from your column.

[Edde]

Thank you very much, Tom.
I have some more questions:
How to determine the system equilibration time? How can I wash the ion exchange column after analytical run? And in what mobile phase should I store it when it is not in use?

[tom jupille]

I've never worked with those specific columns, but generally speaking, it takes about 10 times the column volume to get complete wash-out of a column, so that's the minimum. As long as you're staying with the same ionic form (phosphate in this case), equilibration should not take any more time. Changing to a more strongly bound form (e.g., phosphate at a higher pH) is usually quick. Changing to a more weakly bound form (phosphate at lower pH, or anything monovalent) would take longer. How much longer can only be determined experimentally (change the mobile phase, equilibrate, inject a sample, equilibrate some more, . . . etc.). Waters may be able to provide some more specific recommendations.

Unless your samples contain more strongly bound ions (e.g. something divalent, like sulfate) or non-ionic junk that might be retained by adsorption or hydrophobicity there should be no need to wash the column. In the case of strongly bound ions, a higher concentration (maybe 10x ?) of phosphate would generally be appropriate. Neutrals are more problematic; methanol/water or even straight methanol come to mind as possibilities, but this is another area where I would check with Waters first.

I sound like a broken record: check with Waters for their recommendation or, failing that, use whatever was in it when they shipped the column to you. I've used 100% methanol for silica-based anion exchange columns, but these are polymer (methacrylate) based, so that may not be appropriate.

[Edde]

Thanks Tom.
Your answer is comprehensive enough.
My samples would be ultrafiltered serum, urine and some vegetable and food materials. I guess there should be plenty of ions and neutrals in there that might or might not be visible by UV. Hence, I think washing is required. The Waters column insert recommends methanol should be avoided and ACN should be <20%. I think I should make up some higher phosphate buffer (10x that of eluent) in 20% ACN to wash the column.

[tom jupille]

20% ACN / buffer makes sense to me. If you're going to be running a lot of those types of samples, a guard cartridge might be a good idea as "insurance".

[Edde]

Thanks for your comments.

[Edde]

I prepared MPA 1mM KH2PO4 and MPB 30mM KH2PO4 pH8.0 and run the following LC program at 1mL/min flow:

time (min) B%
0 0
50 100
60 0
85 0

However, there was no nitrite and nitrate peak with a 1ppm aq. standard injection.
I can't figure out what the problem is. Could somebody help please?

[prepcolumn]

Have you ever tried higher concentraions?

I searched the paper you mentioned. The concentrations you calculated are correct but to my experience, when a chromatogram is not given in a paper, i cannot trust the LOD levels (at least i couldn't get the levels yet).

Although nitrite and nitrate give reasonably high UV response at low wavelengths, given LOD levels are quite low. Besides, you use a longer column and generally cation and anion peaks height sharply decrease with increasing retention. This may also be the reason for not getting your peaks.

[Edde]

Thank you very much for your comment.

Have you ever tried higher concentraions?.

Do you mean a higher conc. of nitrate and nitrite aq. standard?

you use a longer column and generally cation and anion peaks height sharply decrease with increasing retention.

What is the rationale behind please?

[Consumer Products Guy]

Agree, typically start with higher concentrations, adjust chromatography, then determine what levels you can "really" measure.