Chromatography Forum

Is it necessary to have an ion suppressor?

This may depend upon how sensitive you need to have your assay. We do plenty of IC here without suppressors. We use Agilent systems here with other companies' conductivity detectors; you can use anybody's detectors with agilent software if you have the 35900 A/D converter, either a board inside the computer (35900D) for older computers or external box (35900E) for newer computers and LAN-connected instruemnts. Contact your Agilent rep for those.

I just received a Waters' ion chromatography method note that HPLC-UV is 5x more responsive to nitrate and nitrite than is conductivity detection. Seems that HPLC-UV is my choice because I don't have to buy additional hardware.

Kostas, you're right - I had not seen this...

For complex matrices, I recommend the technique in the AOAC publication, since it is using a very specific detection technique that gets around the problem with intereferences: post-column derivatization. This technique is very specific, contrary to other more generic techniques, from UV detection to conductivity detection. Very little sample prep is required - essentially just to be free from stuff that would block the column. The detection at 525 nm is very specific.

In the AOAC vol 89, issue 2 2006(p.447-451) method, there are many additional hardware components such as post column pump, post column derivatizing agent, mixing tee, post column reactor. All fittings, unions, and specialty parts and tubings must be made of HCl-resistant PEEK material. If I use an HPLC-UV method, I only need to select a suitable column (I tend to use SAX). Do I need to use PEEK tubings for all connections by HPLC-UV method? Simple sample preparation by boiling for 15 min and filtering is done. A reference protocol states that SPE by RPC18 is necessary only if interfering peak is present. I don't know how to detect interfering peak if a single wavelength is monitored.
I don't know if my rationale is valid. Please kindly comment. Thanks.

I would try the UV-method with the matrices you have.
You can find out if you have interference by a standard addition to your samples.
If there is interference you can try a simple cartridge SPE clean-up (eg one of these http://www.dionex.com/en-us/columns-acc ... s2757.html).

In my lunch break I read an application from Metrohm. They use in-line dialysis before IC of food samples. Sounds good to me. Our Autoanalyzer for N-determination also uses a dialysis mebrane, so never has matrix problems.
Here is the link: http://lifeletter.metrohm.com/ll_metroh ... 50244&CFTO

I would be concerned about using a silica-based ion-exchanger that has been designed before 1980...

I would try the UV-method with the matrices you have.
You can find out if you have interference by a standard addition to your samples.
If there is interference you can try a simple cartridge SPE clean-up (eg one of these http://www.dionex.com/en-us/columns-acc ... s2757.html).

That means I have to run 2 samples: one neat sample plus another one with standard added? The SPE you suggested is an anion exchange resin. How is it compared to RPC18 SPE? Thanks!

I would be concerned about using a silica-based ion-exchanger that has been designed before 1980...

Is the Whatman SAX designed before 1980? Then I would have to look for some other column. But is an anion exchange column a better choice than RPC18 or something else?

Well, you have two options, sample preparation or post-column derivatization. Post-column derivatization is very selective, and you can get away with minimal concerns about sample prep. You just need to do enough not to clog up your column. On the other hand, a very good sample prep method will allow you to use UV detection, hopefully with very little interferences.

Here is my sample prep method that will work best for you: you want to retain everything in the world on your sample prep device, and you want to pass the nitrate and nitrite ions through the SPE device (we have done similar things for other very polar compounds). Here is the method:

Use an Oasis HLB cartridge (from Waters). Activate it with some methanol, followed by water. Then load your aqueous sample onto the cartridge. All inorganic ions will break through, together with sugars, which you do not see in the UV. All the rest of the world is left behind on the Oasis cartridge. Inject this onto your HPLC column and monitor at a suitable UV wavelength that is not too low to avoid seeing the interferences. You may get some further improvements if you use an Oasis MCX cartridge (cation exchanger AND hydrophobic), but this may depend on your sample and what kind of junk is in it.

Having worked for Dionex back in the 80s my experience then was that UV based methods were greatly affected by matrix interferences. The Dionex Ion exchange methods with a supressor and conductivity detection worked great for me. I note you do not actually state the levels you wish to reach and if sensitivity is an issue ? By all means try the UV method if it is a no cost option but speak to Dionex/Metrohm, they will probably be happy to run a sample for you to show if their method is better. Filter and shoot was all we ever did for water based samples.

Dear Edward; this might come a little late.
I used to analyze Nitrate and Nitrite by UV HPLC from a Shrimp Farm; Very dirty sea water, and use to work fine. the only problem is IP reagent you might need time to stabilize the column; not interference from Sulphate and pigments good retenteion times (10 min.)

I hope this be usefull.

Regards.

Oscar

Dear Edward; this might come a little late.
I used to analyze Nitrate and Nitrite by UV HPLC from a Shrimp Farm; Very dirty sea water, and use to work fine. the only problem is IP reagent you might need time to stabilize the column; not interference from Sulphate and pigments good retenteion times (10 min.)

I hope this be usefull.

Regards.

Oscar

Thank you very much indeed. My sample type can range from medicine to vegetables, so I think the matrix interference can be huge. I don't have experience working with this and so I have a question that basically I guess nitrate and nitrite are not unique in having UV absorbance at a single wavelength. I even don't know when the peak is due to interference or when it is due to nitrate or nitrite. How sure I can tell the clinician that the patient's illness is due to toxicity by nitrate or nitrite ?

Dear Edward; this might come a little late.
I used to analyze Nitrate and Nitrite by UV HPLC from a Shrimp Farm; Very dirty sea water, and use to work fine. the only problem is IP reagent you might need time to stabilize the column; not interference from Sulphate and pigments good retenteion times (10 min.)

I hope this be usefull.

Regards.

Oscar

Thank you very much indeed. My sample type can range from medicine to vegetables, so I think the matrix interference can be huge. I don't have experience working with this and so I have a question that basically I guess nitrate and nitrite are not unique in having UV absorbance at a single wavelength. I even don't know when the peak is due to interference or when it is due to nitrate or nitrite. How sure I can tell the clinician that the patient's illness is due to toxicity by nitrate or nitrite ?

Edward,

Don't take offense from what I am about to say, but as you acknowledge that you do not know anything about how to analyze chromatographically those compounds in a matrix component but your job function require that you make such important calls as to tell the clinician whether the patient illness is due to toxicity from those compounds, you need to either:

a) Receive adequate traning on the subject or,
b) Hire people that knows the "how to" or,
c) Hire consulting services of a proffesional.

Asking questions on this forum can give you some tentative directions, but you need to make sure that the end result is correct and the forum itself is not enough...

I am not providing training or consulting services or looking for a job so I feel free to make those suggestions...

Let me emphasize again: in complex matrices, the best approach is to use specific detection. This is why mass spectrometry has become such a popular detection technique in LC. However, you won't be able to use MS for your analysis of nitrate and nitrite.

However, the post-column derivatization technique has the specificity that you need. This is the reason why this silly technique with knitted tubes etc. is still around...

I wonder who came up with such a silly idea

Let me emphasize again: in complex matrices, the best approach is to use specific detection. This is why mass spectrometry has become such a popular detection technique in LC. However, you won't be able to use MS for your analysis of nitrate and nitrite.

However, the post-column derivatization technique has the specificity that you need. This is the reason why this silly technique with knitted tubes etc. is still around...

Thank you very much. I shall read and digest my available references before I go ahead. Grateful for all your comments and advices.