Chromatography Forum

Hello everyone,

In an analysis of naphthalene in honey I observe a peak with the retention time of naphthalene. This peak has a considerable area. The area is about a half of the LOQ.

Initially I thought that it is a classic case of carryover, but subsequently found that it is not so.

I tried to change: acetonitrile with methanol, a type of C18 column, the ratio of acetonitrile in mobile phase. Peak is still there. Using the gradient also failed to alter the presence of this peak.

I noticed that the purity of the mobile phase is crucial. Moreover, attempts to use a plastic ware for sample pretreatment greatly worsen the situation. I used plastic ware from various suppliers. Peak becomes even higher.

Finally, another instrument was used in another laboratory. Of course, I changed everything: supplier of solvents, glassware ... everything.
And guess .... YES ... the peak was there.

Currently, I have to work with glassware and wash it in advance with the mobile phase, which complicates the analysis.

I am wondering if anyone has observed a similar phenomenon.

The HPLC conditions are:
Mobil Phase: AcCN:H2O (50:50, v/v);
Column: C18 4.6 mm x 150 mm, 5 um;
Flow rate: 1.5 mL/min;
Detection: Fluorescence Excitation 275 nm, Emission 330 nm;
Injection volume: 50 uL;
Sample and standards are dissolved in mobile phase.

Thanks in advance for your opinions and findings.

Wow, 50 ul seems a lot to inject, especially when you're using fluorescence detection which should be so sensitive anyway.

I guess my question is: how close to the LOQ are real-life samples? If the real samples are significantly higher, just report a reasonable LOQ.

Hello Consumer Products Guy,

Indeed, 50 ul are rather large injection volume. But, sensitivity to naphthalene is not that high. Naphthalene peak corresponding to the LOQ has an area of 1.2 units, which is quite close to the quantity allowed to be present in honey.

However, the area of contamination is from 0.12 to 0.6 depending on the purity of the mobile phase used glass and plastic. This makes the analysis much .... how to say .... questionable.

I might try:

substituting methanol for ACN, maybe start with 60% methanol

adding acid such as acetic acid or phosphoric acid to the aqueous portion of the mobile phase; the naphthalene peak shouldn't move, but maybe the interference will

Is this some standard test procedure you're using?

Hello again,

One of the first things I did was to put 60% methanol instead of 50% acetonitrile. The situation has not changed.

The method is not standard. It does not appear in any set of standards (ISO, EN ...etc.)

If I understand this correctly then there is a peak at the naphthalene rt when there should not be one. Does the detector allow to run excitation and emission spectra? If the peak can be identified as naphthalene then it is certain that there is contamination.

Hi

Well as a PAH, there are a bunch of articles if you google about naphtalene in in/outdoor air which potentially may impact trace/low ppm analysis.

Also naphthalene sulfonic salts are used in the manufacture of naphthalene sulfonate polymer plasticizers which are used to produce concrete and plasterboard, they are also used as dispersants in synthetic and natural rubbers, but I am unsure if there are any "leach" studies done on those.

Naphtalene has even been detected in breath (low ppm per m3) on people handling certain jet fuels.

Edit: Joggled memory a bit and at the university I attended a lot of PAH analysis was made with GC/MS and my GC column cataluge (Agilent/J&W)list naphtalene in conjunction with references to a few EPA methods:
8270
8021
502.2
might be useful for conformation.

Hello HW Mueller,

You understand fully a problem. The detector allows and I use diferent lengths of emitted light. The emmited wavelength does not affect the result. Surely this is a pollution, but pollution moves with naphthalene irrespective of the mobile phase and gradient used. It is so doubtful. The probability of such an event is, ... quite small, I would say tends to zero. Why does this happen to me ...... (( Just kidding, of course.

Hello krickos,

Thank you for your comments. I am not familliar with the production of plastics, but I am sure that I use either PP or PE plastics ware. I think so ...

Consumer Products Guy,

Perhaps you're right about the pH. I will try to add some acid to the mobile phase. I have not got high expectations, but ... I have to try. If this is a contamination, witch differ from naphtahalene, than I can get a right separation.

If the obtained emission spectrum of the supposed contaminant is the same as the one of the analyte (i.e. naphthalene) then it just might be naphthalene as well. And that was the main point both HW Mueller and Krickos tried to make, as I understood it.
The fact that changing the conditions (i.e. gradient etc.) did not introduce any relative changes in these “twoâ€

Hello Mr Dikov,

This discussion is an opportunity to find rational explanation of the problem.

However, I believe that acidification is the only action that I have not taken. I'd like to do everything possible to clarify whether this component is naphthalene or not.

By the way, I tried to use a GC/MS to determine the type of contamination. Unfortunately concentrations are very low and the method of sample preparation is not suitable.

Благодаря за съдействието

You have not told us whether the excitation spectrum is the same as that for naphthalene. nor did you really obtain an emission spectrum. If you do this systematically (scientifically) you need to know whether that blank peak is naphrhalene or not.

Dear HW Mueller,

I can use one Excitation wavelength (275 nm) and up to four Emission wavelengths (namely 310, 325, 330 and 350 nm). Only at 310 nm peak was not observed. Neither in blank, nor in the naphthalene containning sample.

Dear Consumer Products Guy,

As expected, acidification of the mobile phase does not lead to a change in the situation.

Like the others suggested: you just may have trace level contamination. That's a big issue these days, as nothing is really "zero".

What surprised me is:
"I noticed that the purity of the mobile phase is crucial. Moreover, attempts to use a plastic ware for sample pretreatment greatly worsen the situation. I used plastic ware from various suppliers. Peak becomes even higher."

Is one to suspect that naphthalene is everywhere? Or that DID contaminated everything? If this can be ruled out than it is still possible that the peak is something other than naphthalene, and since DID has no means to characterize the peak independently it should be worthwhile to vary the chromatographic conditions much more strongly than he did. If the peak is not naphthalene DID will still be trying to clean up his act in 100 years.
Also, I don´t understand why only 275nm can be used for excitation. Or why not enough can be collected to do GC.