Headspace Concentration Calculations using SPME

[Lawborne]

Thanks for those who answered my previous question in another post.

I'm a little new to this (biology grad student learning about SPME). We want to know if we can use SPME to determine accurately how much of 2-methyl-2-butenal is in the headspace of a sample. With SPME we have been able to see that the concentration drops in an exponential pattern if the sample is left out for a couple of weeks but we would like to be able to quantify those amounts if possible.

I have been able to determine that if I make a concentration of 'x' ppm of 2-methyl-2-butenal in isopropyl alcohol it produces an area under the curve of 'y' using a MS.

I've looked at some of the equations but have gotten bogged down. I know Chemspider has a lot of useful information but I want to know what I need to know out of that. Which equations/information should I be using?

My set-up: I place 3mL of 'x' ppm 2-methyl-2-butenal/isopropyl alcohol in a 22mL sealed container. I have had a lot of luck with 65um PDMS/DVB, fused silica SPME fiber with an equilibrium time of 30 minutes and an injection time of 30 seconds.

My advisor wants to continue to use SPME but our lab manager suggests SPE.

Thanks in advance!

[rb6banjo]

What is your sample matrix? If your analyte is already dissolved in an organic solvent, why not just inject it directly into your chromatograph?

SPME is made for determination of low concentrations of materials contained in a substantially aqueous medium. For your small aldehyde, it should work very well. I generally do not use headspace-SPME to determine an analyte in an organic matrix like IPA. Here's the calibration data for a piece of work I did to help someone out with determination of benzaldehyde in a hand soap (1 g of the soap diluted to 100 mL with water):



The diluted sample ended up containing 3 ppb (ng/mL) of benzaldehyde. So at a 1:100 dilution, it was about 0.3 µg/g of soap. SPME is very sensitive. Your sensitivity will decrease if you're trying to determine your analyte in an organic solvent (your analyte will likely be too soluble in the solvent thus hindering the partitioning into the vapor phase in the vial).

Just a tip. I've learned that to get the most accurate results, it's best to calibrate in your matrix.

[Lawborne]

What we are interested in is how much of the molecule can be found in the headspace, not the organic solvent itself.

The aldehyde we are working with is a pheromone and only works if it is volatilized. They add it to products like collars, toys, etc. to attract animals. I was able to show with SPME that if the collar containing the aldehyde is left in the open air that the pheromone can be detected by the GC-MS for a certain amount of days. I left the collar out, snipped a piece of it every week and placed it in a 22mL glass container and used a SPME fiber to analyze the headspace around the fiber.

I was hoping that I could actually quantify the numbers I obtained. From what I've been reading I can do it via Henry's law but I wanted some guidance.

I hope this clears that up a little. Our chemistry expert says that we will have to use SPE and collect a sample of the air. My boss thinks that we can use SPME but he is not a chemist.

[rb6banjo]

SPME is what you want. The calibration part is what's going to be tough. It sounds like what you're interested in is the vapor phase concentration of the aldehyde. When you have the sample of the collar in the vial, what's happening is that that aldehyde partitions out of the collar and into the headspace. The distribution coefficient for that equilibrium dictates to what extent that happens. If you add some of the aldehyde to the vial with the collar and allow it to equilibrate with the collar and analyze it again, what you're really measuring is the concentration of the aldehyde in the collar. You're using Henry's Law to get there indirectly.

If you want to know the gas phase concentration of the aldehyde in the vial, you're going to have to find a way to get the collar out of the equation during the calibration step. Making gas standards is tricky business. If it were me, I'd look to a company like Restek (restek.com) to see if they can make me a standard. Then, I'd use the SPME to calibrate in an empty vial with my standard and collect the samples the same way I extracted the standards.

It's not going to be perfect as whenever the collar and the SPME fiber are present together in the vial, the collar is going to try to replenish the aldehyde that is removed by the SPME fiber. This would be true for a dynamic-headspace sampling (purge-and-trap) scenario as well. You'll get close but it won't be perfect.

If you're just trying to find out how long the collar will emit the analyte then it gets much easier. That seems to be what you were doing in your "snipping" experiment(s).

[Don_Hilton]

There is one issue you need to be careful with in snipping portions of sample to analyze: The mass of one portion may be different than another. And, given that you are dealing with partitioning between the collar and the air in a vial, the ratio of the quantity of air to the quantity of collar may have some impact on what you measure. A variation on your experiment might be to take a jar that is large enough to hold the collar with room to measure head space. Put a hole in the cap that will just admit the SPME sampling device - and perhaps seal the opening with a septum. Put the entire collar in the jar, equilibrate it and the SPME fiber for an hour or so, and run the GC/MS run and then take the collar out of the jar and let it be exposed to the environment to which you normally have it exposed and then at the end of the week use the same jar, collar, SPME fiber etc. and measure again the same way. There is no variability in how much sample you use - it is the same sample, just aged, and there is no change in ratio of solid to gas in the sampling vial. And if there is any variation in application of the pheromone along the material in the collar, it does not matter. You need to do the equilibration with the SPME fiber for the same length of time and at the same temperature each time - because how much pheromone makes it out of the collar depends on diffusion rates (time) and then how much makes it out of the air and into the fiber again depends on rates (time again). And both equilibrium and rate change with time and temperature.

This leaves the issue of instrument calibration. The sensitivity of the mass spec can change over time. You need a reference sample to check or calibrate the GC/MS. You can make up a solution of the pheromone in a suitable solvent and put a few microliters in to a headspace vial to measure the response. Note that the solvent can change the partitioning into the SPME fiber, so this will demonstrate consistency of response for a given level of pheromone, but cannot be depended on for measurement of gas phase concentration without further proof.

One other thing to consider: If you put the collar onto an animal, the collar will pick up volatile compounds from the animal, which will become part of the partitioning process. And these compounds can swamp the fiber, reducing the response of the pheromone. But as long as the material you are sampling is consistent except for the volatilization of the compound of interest, your experiment should give you a good idea of the decrease in concentration of the pheromone over time. There is debate among some about the utility of SPME for quantification. And the safest method is to have an internal standard that partitions exactly like the analyte of interest. An isotope labeled internal standard is the best way to do this. And, if the partitioning of the analyte is changed by an excess of other stuff changing the partitioning, the internal standard is affected the same way and the ratio gives you the information you need. But, we’ll assume that other volatile components are not changing – at least for now…

[Peter Apps]

You need to think carefully about the experimental design.

The collars are presumably designed to go on dogs or cats (I am assuming that although you are using a rabbit pheromone this it is the more common and commercially attractive species that you are aiming at), and so are exposed to a slightly elevated temperature during use, which you should probably simulate in your experiments. The columns remain more or less intact while on the animals, which means that as the aldehyde in the surface layers is depleted the emission rate declines because aldehyde now has to diffuse through the material of the collar to get to the surface before it can be emitted. By cutting the collar you expose fresh, aldehyde-rich surface and cause a temporary increase in emission rate.

The most straightforward way of doing these measurements would be to continuously purge an intact collar with air, and to repeatedly take samples (by any convenient repeatable method) as the emission rate decreases. If you plot peak area vs time you can fit an exponential curve, and the area under the curve is proportional to the quantity of aldehyde in the collar (This is the same as repeat injections in equilibrium headspace analysis). Presumably you know how much gets put in there (unless you are reverse engineering someone else's product) and so you can calibrate area under exponential curve vs quantity. Then for any given time period you determine the area under you fitted curve, and calculate how much was (or would be) emitted during that time.

Peter