Quantification of volatile and non-volatile compounds with internal standards

The goal of quantification is to obtain an accurate estimate of volatile emission rates from an odour source. This is readily applicable to volatiles sampled using Super Q and Haysep Q adsorbents, and extracts (see sections 4 and 6 of this paper). One approach to quantification is to add a known amount of a synthetic chemical to a sample before processing the sample through extraction and separation (Heath and Manukian, 1994). Researchers usually select a chemical that is not present in their sample but has similar separation properties as the chemicals of interest. One internal standard used with honey bee volatiles is nonyl acetate (Carroll and Duehl, 2012). The internal standard automatically scales the peak areas to known amounts of material. One useful aspect of this internal standard is that quantitative errors in sample processing (i.e. fraction of sample injected on the GC, compound concentration or losses, pipetting errors) are automatically factored out, as both the internal standard and the sample compounds experience similar changes. 

To make nonyl acetate internal standard:

  1. Add 92.6 μl (80.0 mg) of nonyl acetate to 9.907 ml of the same solvent used for filter extraction (dichloromethane or hexane).
  2. Vortex for a few seconds. 
  3. Add 100 μl of this stock solution to 9.900 ml solvent.
  4. Vortex for a few seconds. 
    The concentration of this internal standard solution is 80 ng/μl.
  5. Aliquot the internal standard solution into working amounts of 500 to 1,000 μl.

For long term storage, aliquot the internal standard solution into glass ampules and seal.

It is very important that solvent evaporation of internal standard solutions be kept to an absolute minimum to maintain an accurate concentration. Store the internal standard solutions in a -80°C freezer between uses. 

The sample chromatogram will have a nonyl acetate peak that represents the 400 ng of nonyl acetate (5 µl of 80 ng/µl internal standard solution) that was added to the original sample during rinsing with solvent containing internal standard. Run a sample containing only the internal standard to determine the retention time and peak characteristics of the internal standard. Because the amount of internal standard added to the sample is known, conversion rates can be made between peak areas and compound amounts.  Sample compound amounts are calculated from peak areas against the standard as: