If you want to use an internal Standard, it is usually recommended, that the molecul is not too far away from  the target molecule...on the other hand: you would not use  more than one, maximum too internal standards per method, you mostly calculate your results according too one reliable value. It is more important that the standard itself is showing a good and stable reproducibility, a second internal marker might not increase  the precision of your method. Perhaps you place it to the average middle value of all searched peaks. In case of GC you mostly use alkane standads, Octane, Nonane, Decane etc...

Hello Joe

You do not necessarily need to use only one or a few ions to quantify your target analytes. If your peaks are well separated (and since you can identify the compounds I assume that this is the case) you can quantify on the basis of peak areas on the Total Ion Chromatogram (TIC). You only need selected ions if your peaks are overlapped, or if you have high levels of background noise.

If you want to measure the concentrations of your mite compounds in the headspace, then the most straightforward way will be to calibrate peak area vs analyte mass per peak by a series of injections of standards made up at different concentrations in solvent - a conventional GC liquid injection. Splitless injections will provide better accuracy than split injections, since the split ratio for the analytes is not the same as the split ratio for the carrier gas.

From that calibration and the area of the peaks from your headspace samples, you will know the mass of each analyte in the headspace sample, and from that and the volume of the sample you can work out mass per volume, and with molecular weight you can get mole fraction.

Hello Peter,

Thanks for taking the time to respond to my question, it is much appreciated. I have well separated peaks and should therefore be able to use the total ion chromatogram as you have suggested.

I think I will have to construct a number of calibration plots for the variety of VOCs that I am interested in quantifying. If I undertake a linear regression of the plots, I can reaarange y=mx + c to solve for x and therefore my concentration...is that correct?

Joe

Beside Frank and Peter, I want to address another issue to quantify VOC without raw standard. If you have GC-MS, GC-FID and some VOC standard in your laboratory, you can quantify any VOC without standard. First you need to run your available raw standard in GC-FID to draw calibration curve. Once you have response factor values, you can derive effective carbon number (ECN) of compound lacking standard. You can use those ECN number to quantify unknown VOC present in your sample. To identify unknown VOC's, simply use GC-MS library after running your sample into GC-MS...

Thanks for your response. The issue I have is that I only have access to GC-MS and not GC-FID. How does quantification work with ECN?

Hi Joe

Your calibration algebra is correct.

How many compounds are you interested in ?, and is it a problem to get them as standards ? If you can get them, then calibrating with the actual compounds you are interested in is by far the most accurate approach. Although you can use substitute standards it involves potential pitfalls.

As long as the peaks separate you can put all your standard compounds into one solution; you do not have to make separate standards for each compound.

I don´t know how accurate your results need to be, how much plant you put in your headspace vial, how high the headspace temperature is compared to the boiling point of your analytes and if you analyse very different plants.

Multiple headspace extraction is a way to quantify your analytes using a liquid calibration standard without having to worry about different sample matrices. Attached you find an application note, explaining the concept. Surely you can find something suitable for your device on your vendors webpage.

@Peter: from the preliminary research I have already undertaken I have around ~15 compounds of interest in my samples and all of them separate reatively well I believe. I am able to obtain all of them as standards with two exceptions. How do you decide which concentrations to make your solutions at? Thanks!

Hello Joe

To set the concentrations for your calibration you need to do some trial and error. Ideally you want your sample peaks to be close to the middle of your calibration range. You must not have sample peaks that are outside the calibration range.

Since area vs quantity is reasonably linear, if your trial standard has a peak that is about ten times too big, then you know you have to dilute your standard by ten, etc.

In headspace analysis internal standards are difficult to apply because every compound partitions differently between matrix and headspace.

Please consider in the first place if you really need to quantify these compounds in the headspace!! Most likely, you'll need to follow variations of compounds ratios in infested vs. non-infested plants and maybe relative increase/decrease in emission rates. I suggest therefore to use a reference compound as an aid to a rough quantitation and focus on ratios and ralative variations.

Quantitation makes sense in the whole sample, so you'll need the multiple extraction method, as already pointed out.

Quantifying in the headspace is actually straightforward - it requires only a calibration of peak area vs quantity, which is easy to do with liquid injections, and a known sample volume. 

Complexities arise when headspace analysis is used to quantify the composition of the condensed phase (solid or liquid) because then the partition between phases has to be taken into account, and it varies with condensed phase composition and temperature (and under some circumstances with phase volume). Internal standards are of limited value since it is extremely difficult to find internal standards whose partition behavior matches that of the analyte(s).

Dear Joseph,

We used SPME to extract VOCs from aphid-infested soybean leaves.  We used GC-MS for qualitative analyses.  This might be helpful:

Cai, L.; Koziel, J.A.; O'Neal, M.E. Studying Plant–Insect Interactions with Solid Phase Microextraction: Screening for Airborne Volatile Emissions Response of Soybeans to the Soybean Aphid, Aphis glycines Matsumura (Hemiptera: Aphididae). Chromatography 2015, 2, 265-276.

Jacek

http://www.mdpi.com/2227-9075/2/2/265

Dear Joseph,

I have been screening the previous answers and I think I may add a few points; I apologize if I happen to repeat anything that was previously mentioned.

1) You may want to use an internal standard, especially if you are working in headspace or SPME extraction. Those methods are extraction methods, therefore you need to control on "how efficient was the extraction". Your assumption here is: "if the internal standard extract to 56%, the target analytes that have a somewhat similar structure should extract to a similar rate". This allows you to decrease the matrix effect. I understand that you may be working with solid rather than liquid matter, so you should try to figure out how you could introduce an internal standard in your matrix, in a manner that it will be efficient enough to represent the behavior of your target analytes.

2) When it comes to compare your treatments you have (at least) two choices:

• you can compare your samples within your own experiment using an "approximate quantification"; this will give you a way to compare your treatments, but it won't allow you to compare that much with the literature. For example, you may use ratios of areas: you will do the ratio between the area of your analyte and the area of the internal standard. This will strictly allow you to say : "ok, treatment A has a higher level of compound Y compared to treatment C". You can't really go further without a true calibration. On the other side, quantifying compounds from a similar group is commonly achieve by doing %area (think about essential oils for exemple), but this is usually done using a FID.
• You do a complete calibration, using both internal and external standards, and then you are in better position to compare with the literature. However, if you use SPME, you may want to do some validation steps to make sure of the accuracy of your method (I won't explain that here ; you can email me if you want more details).

3) If you calibrate, the best is to use external standards (in addition to internal standards if you go for SPME or headspace extraction), but you can also used the methods mentioned in other posts if you are missing some standards, which is not uncommon when working with natural products. 

4) Choosing your quantification masses (quantifier ions): If you are working on a GC-MS, you need to use the Single Ion Monitoring (SIM) mode to have a better accuracy when you quantitate. There are a couple of steps that needs to be done before you decide on quantifier ions:

• First, use the GC-MS in Scan mode, in order to determine how good is the separation between your analytes; if your analytes are really similar to each other, or if there are other compounds with similar masses around your peaks, you need to reach a higher level of chromatographic separation because otherwise the ions from these picks will be interfering with the quantification of your analytes; 
• Once your separation if fine, choose your quantifier ions. This operation is simple yet complex to a certain point: you need your quantifiers to be unique compared to other peaks around, so you want to avoid low molecular weight masses as much as possible; on the other side, you want your masses to be as intense as possible in the mass spectra of your analytes (the base peak may be a good choice but this is not systematic). The extent to which the intensity is important to your quantitation depends on how far you are looking for traces compounds. If you are expecting your analytes to be at high concentration, just make sure your quant ions are unique and you'll be fine. If you are digging traces, make sure you get a good compromise between "unique" and "intense". What I usually suggest my students to do when they work on this topic, is to print out the mass spectra of their target analytes so they have them at hand all the time; then use software functions like "select (or "show selected") masses" and see how well define is each masse in the chromatogram. If you see masses providing double peaks or shoulder peaks, don't choose them; if you have to pick them, try to improve your chromatography (back to step 1!).

Hope that helps.

Hi Karine

We have already established that Joseph can separate all his compounds, so he does not need to use SIM for selectivity. Also, he has big enough peaks to be able to identify the compounds from their spectra, so he does not need SIM to enhance signal:noise. So why would he use SIM as you recommend ?

Peter

Hi Peter,

As I mentioned, I screened trough the previous answers so I guess I missed the fact that you stated that Joseph did not need to work in SIM. Sorry about that. However, I would add that, as far as I known, SIM is the proper way to quantify compounds in GC-MS; it is more precise in part because it increases the sensitivity of the instrument, as you mentioned. Also, I think it is safer so a certain point, when you are working with complex samples, as unpredicted compounds may arise at anytime and potentially coelute with the target analytes. On the other side, this can happen in SIM too, obviously, but you have to be unlucky enough to have another compounds showing the same mass as the one you chose. I haven't test both methods side by side though, as we use a TOF in my team; I would probably give it a try before choosing the Scan mode. In order to improve the identification in SIM, it is also usual to choose some qualifiers ions.

Also, I don't know how big Joseph's compounds are (maybe are we talking about C6 leaf volatiles here?), but if they are highly concentrated (e.g. TIC peaks look like "potatoes"), and he can't really reduce the sample size (in headspace or SPME), I would suggest to use lower intensity masses as quantifiers, and work in SIM in order to avoid over-saturating the MS detector with undesirable ions. Higher molecular weight quant masses have more chance to be unique to the analyte. 

I hope it answers your question.

Karine

Again, and following Jacek answer too, I don't think you really need to strictly quantify your compounds: put your efforts in setting up a reproducible and representative experiment