Chromatography Forum

Hi,
I am trying to determine benzene in gasoline 0%-1% with HPLC UV. My mobile phase is ACN60%-H2040% and my column is a Vydac (Avantor) 250mmx2.1mm, 120A, 5μm. The detector is set at 205nm since benzene absorbs intensively at 205nm and the other compounds such as olefins etc not. Also, the solvent (1:100 sample dilution) is ACN.

I have a very good separation between benzene and toluene, but I notice great RSD in my injections. When I inject the same vial, i have area drift and the area of benzene reduces with every injection. This happens in both samples and standard solutions.

I tried to have the same sample in different vials. Then I notice RSD (4-9%) between 5 vials (not drift with reducing area). Then i tried to have a vial being injected and quickly replace the cap for the next injections. Still the area would reduce with every injection.

Maybe i should check my injector and seal packs, which I haven't yet but,

Do you think there is a problem with my solvent. Has anyone have any solvent issues when the matrix is gasoline?

Thanks in advance

I think evaporation of benzene is your problem, this is why you observe the drift when injecting multimple times from the same vial. Also during replacement of the caps, evaporation will happen. So if your autosampler can be cooled, try if it helps to set it at 8 °C instead of room temperature.
What type of septa do you use? Avoid pre-slit septa, try out PTFE-lined rubber septa.
The high RSD between different vials is most likely also caused by evaporation during pipetting.

I think evaporation of benzene is your problem, this is why you observe the drift when injecting multimple times from the same vial. Also during replacement of the caps, evaporation will happen. So if your autosampler can be cooled, try if it helps to set it at 8 °C instead of room temperature.
What type of septa do you use? Avoid pre-slit septa, try out PTFE-lined rubber septa.
The high RSD between different vials is most likely also caused by evaporation during pipetting.

I am using pre slit vials. My autosampler is set at 7degrees C. So if i there is evaporation during pipetting, there is nothing i can do about it? Because i have also tried acetone, dichloromethane, methanol and DMSO as solvents but i still have drift and rsd

Hi Petro,

I think bunna has the correct idea here. It's a good idea to lower your AS temp, but it would probably be a better idea to make your sample immediately before injection. I know you say you inject out of the same vial, but I would simply make your solution and aliquot them. Simply remove them from the fridge/freezer when they need to be run. Personally, I have never done any analysis of benzene myself so take my response with a grain of salt, but I do have something to potentially add.

205 nm is a somewhat "poor" wavelength to observe anything really. It is just prone to having a lot of background noise which can potentially impact your readings and chromatography. I'm not saying that this is your issue, however you may be noticing RSD issues because of that as well. I'm sure all your solvents are HPLC grade so you should not worry too much about those types of interferences.

A quick google search gave me a great result from someone doing something similar to your analysis and they suggest dissolving samples in methanol (50% to be exact) as it provides an RSD of about 1.7% visualizing at 210 nm.
Here's a link to that article: https://pubs.rsc.org/en/content/article ... d1ay00083g
Hopefully it will help you in preparing a good sample solvent or as a guide for this analysis!

Normally we do benzene analysis by purge and trap GC/MS and for that we use vials with zero headspace. After they are punctured the benzene will begin to lower in the solutions.

You could try using low volume inserts and fill them to the top, and seal with a rubber septa, that should reduce the loss of benzene from standards and samples. One injection per vial would be best. If you make 5 vials and they all have different amounts of headspace you will see them come to equilibrium with different levels in each liquid sample, that could be where you are getting the variation.

As James says, reduce the headspace in the vials so that less benzene evaporates out of the liquid phase. There are some precautions you can take against evaporation during sample prep, it is usually a problem with evaporation of voaltile solvents, but the same principles apply to voaltile analytes; https://www.researchgate.net/publicatio ... ileContent

GC is a much more suitable technique for analysis of benzene in gasoline than HPLC. The issue is not benzene itself but the matrix. If the sample is diluted 100x as mentioned, then the matrix (generally containing C4-C12 hydrocarbons) concentration is 1%(10000 ppm). Some of these hydrocarbons elute before benzene and some elutes much later than benzene. If the run time is not long enough for the isocratic method, hydrocarbons will accumulate in the column.

Since the diluent is ACN and the mobile phase composition is ACN-water, there is a good chance that the more hydrophobic hydrocarbons in the matrix do not stay in solution once injected.

As pointed out above, the wavelength selected is also questionable.

GC is a much more suitable technique for analysis of benzene in gasoline than HPLC. The issue is not benzene itself but the matrix. If the sample is diluted 100x as mentioned, then the matrix (generally containing C4-C12 hydrocarbons) concentration is 1%(10000 ppm). Some of these hydrocarbons elute before benzene and some elutes much later than benzene. If the run time is not long enough for the isocratic method, hydrocarbons will accumulate in the column.

Since the diluent is ACN and the mobile phase composition is ACN-water, there is a good chance that the more hydrophobic hydrocarbons in the matrix do not stay in solution once injected.

As pointed out above, the wavelength selected is also questionable.

I use a PDA. I have the same RSD in every wavelength from 200-260nm. So it is not the wavelength i suppose. My column is non polar C18 so it is possible that many hydrophobic hydrocarbons stay on column. But i guess i would notice pressure increasing over time.

I guess i should examine having greater run times?

May be I should try a gradient with 100% ACN to elute the hydrophobic hydrocarbons, but i do not see how that affects the peak of benzene.

Most of the more hydrophobic compounds in gasoline are liquid, for example, m.p. of dodecane is ~-10C and m.p. of unsaturated C12s are expected to be even lower, therefore, the column pressure is not expected to increase even these compounds are not eluted from the column. Particulates (solids) are more likely to cause column pressure increases.

The wavelength does not cause the peak area to decrease, a higher wavelength should be considered since many unsaturated hydrocarbons show uv absorption @205 nm.

If evaporation caused the benzene peak area to decrease, the effect should be subtle (assuming the run time is not too long). If significant changes in peak area were observed from injection to injection especially with a chilled autosampler, other factors could be involved.

Most of the more hydrophobic compounds in gasoline are liquid, for example, m.p. of dodecane is ~-10C and m.p. of unsaturated C12s are expected to be even lower, therefore, the column pressure is not expected to increase even these compounds are not eluted from the column. Particulates (solids) are more likely to cause column pressure increases.

The wavelength does not cause the peak area to decrease, a higher wavelength should be considered since many unsaturated hydrocarbons show uv absorption @205 nm.

If evaporation caused the benzene peak area to decrease, the effect should be subtle (assuming the run time is not too long). If significant changes in peak area were observed from injection to injection especially with a chilled autosampler, other factors could be involved.

Well my injector works fine. I did a calibration test with my technician on his column on another analysis. And the RSD was lower than 1%. So the injection system works fine.

I get the same RSD on higher wavelengths so wavelength parameter is not the problem. There are significant changes in peak area but i do not know what other factor i should consider.

My chemistry knowledge is getting very rusty as I get older, but could the benzene be slowly reacting away in the presence of 100% ACN? We have some samples that have to be prepared in a ACN/H2O blend in order to preserve some impurities. You might could try sample preparation in a blend of mobile phases.