Chromatography Forum

I used Waters Oasis MAX SPE (3cc, 60mg) for nitrite extraction. pKa of nitrite is 3.9. I tried on aqueous nitrite standard. Protocol as follows:-
Sample: 1mL 200ppm nitrite in water + 2mL 5% NH4OH in water (pH~11)

Condition:
2mL MeOH
2mL H2O

Sample application

Wash
2mL 5% NH4OH in water
2mL MeOH

Elution
2mL 0.46% HCl in water (pH ~1)

However, the recovery is only ~60% by manual SPE and even worse by autoSPE (~10%).
Could anyone tell me what I can do to improve recovery please?

Many thanks in advance.

I suggest to add 2mL 5% NH4OH in water in the Condition step (after water, before Sample application).

I've replaced the water step with 2mL 5% NH4OH in water in the Condition step.

Another point, I am using auto SPE, 6mL/min for conditioning steps. And washing steps are 9mL/min. Are they too fast?

Thank you.

My "intuition" said it can be too fast, but... you should verify it experimentally.
I suggest to start with 1-2 ml/min...
Retention on Oasis MAX cartridges is based on ion-exchange, so loading the sample should be also very slow.

Thanks. I'll try slower rate.

Hi Edde; 60% of recovery is very low.
Yes; you should load your sample slower 1-2 ml per minute; but also and try to analyze the eluent of each step; that is the best way to know what is failing.
Personally I think that washing with NH4OH 5% is too high; remember also that Hydroxide will displace the equilibrium NO2- + H+ <----> HNO2 to nitrite so this anion will attach better to the Substrate; that is good but 5% in higher for me.
MeOH will help you to elute organic neutral and acidic compound (remember MAX is mixed anion exchange so neutral organic are also retained through whatever pi-σ interactions). Teorically you could weave MeOH washing or wash with MeOH:H2O:NH4OH mixture; at the end when you pass 0.46% HCl in water; inorganic Anions will elute but organic bases should be neutral retained.

Please, tell us if your recovery improve.

Best regards.
Oscar

Hi oscarBAL,
Sorry for replying late. I have been trying your suggested method and many other types of SPE such as HLB, WAX, MCX, HILIC, C18, Polyimine (a weak anion exchange SPE). Both the "target analyte binding-wash-elute" and "sample flow-through" approaches had been tried in many different pH and ionic strength.
Also, the analyte behaviour differs significantly in aqueous std and in nitrite spiked urine samples. In the latter, the nitrite ion behaviour is very unpredictable from the pKa.
At last, I find that nitrite spiked urine in neutral pH applied to MAX and the sample filtrate collected for analysis is best in terms of response and least interference.
The recovery is around 80%. Since urine pH may differ from person to person, I may need to dilute with phosphate buffer and the response then will be less.
Do you think a 1:1 (urine: 0.15M phosphate buffer pH 7.5) dilution is adequate to bring urine pH to 7.5 consistently in different patients?

I apologize again for the late reply and appreciate your kind comments.

Hi Edde; indeed I believe MAX is a better option. 80% is not the best but is not bad either.

I have many Ideas but I do not know if I could be helpfull.
Could you plese describe your MAX procedure?
I believe that the differences between aqueous STD and urine spike could be due to the ionic strengh among other reasons; uirine have many anions that could be saturating (partially) your cartridge so recovery in urine is lower??

What Nitrite level are you expecting see? if you do not have sencitivity limitations you could use another approach; for example use MCX or C18 in such a way that when you load your sample you retain neutrals and cations and allow anions to pass; doing this way you eliminate many interference but you can't concentrate your sample.

Could you describe your HPLC method? what column and detector are you using?

I do not know if 1:1 dilution for the buffer is fine; this depends of the buffer capacity; I would say that 1:1 is more to enough but I do not have any fundament for that statement; you will have to try with many urins samples as you can. I wonder why pH of 7.5? why not 8 or 9? I am just concern about possible saturation of the cartridge due to high concentration of phosphate.

did I sent you this article before?

http://chromatographyonline.findanalyti ... rticle.pdf

If you can't use your column for IPC then you could concider buy a new column for this.

I think I haven't been too much usefull maybe if you send the rest of the information or any one else can help.

I hope soon I will start a project of analyzing sugar in urine samples and as I can see youw work with this can of matrix I would like to have your contact; my email is:
oscar.cortesledezma@gmail.com

good look and best Regards.

Oscar.