Volatile sampling in the headspace environment

The complete flow path for a closed push-pull flow system (described without connecting lines) is 1) air source-pressure regulator, 2) air filter, 3) air heater, 4) air humidifier (bubbler), 5) air flowmeter(s), 6) enclosed odour source, 7) adsorbent filter volatile trap (in protective jacket), 8) vacuum flowmeter(s), 9) vacuum pressure regulator, and 10) vacuum source (see Fig. 1). Each setup may not necessarily have all these features. A step-by-step guideline to the selection, assembly and sampling of headspace volatile is provided below.

  • Collect volatiles in a headspace environment that mimics core colony conditions (Winston, 1987; Kraus and Velthuis, 1997).
  • Use incubators and glass in-line bubblers to control temperature and humidity. 
  • To minimize contaminants, construct all enclosures, lines, and tubing before the volatile trap out of odourless glass, Teflon/PFTE (polytetrafluoroethylene), copper, or stainless steel. All line materials under significant pressure or vacuum should be constructed of metal tubing. Materials after the volatile trap can be made of “dirty” (e.g. odiferous) materials such as Tygon plastics.
  • Control the air and vacuum flow rates in closed and partially-closed collection systems with flowmeters. Flowmeters are necessary for quantitative comparisons of volatile emissions.
  • Filter the main air source with gas filters (molecular sieve and activated charcoal) to eliminate water and oil contaminants from air pumps.  Clean air can also be purchased in pressurized tanks.
  • Provide airflow to ventilate subjects without creating disturbance.  A flow rate that exchanges the air in the container about once every 20 minutes is sufficient.
  • Protect SPME fibres, adsorbent traps, and other sensitive objects from direct contact with bees by shielding the devices with a screen or Teflon shield. 
  • Collect appropriate odour “blanks” to account for background odours originating from the equipment, the air supply, and the surrounding environment. Use hive equipment of similar age (preferably less than two years old) whenever possible.
  • Limit background odours by eliminating unnecessary odour sources. Avoid hive materials containing dead or diseased bees, rotten food stores, active small hive beetle infestations, or odiferous treatments such as essential oil patties.  Limit the use of smoke. If use of smoke is unavoidable, pass clean air through the collection system for 30 minutes before volatile collection, or collect appropriate background samples.
  • If needed, scale up the amount of volatiles sampled either by extending the sampling period or increasing the sample size by forming homogenous patches (e.g. single age brood cohorts, pollen patches, and simultaneous infection of brood).
  • Avoid accidental contamination of collection equipment by household cleaners and detergents. Use GC grade solvents (ethanol, methanol, hexanes, and acetone), water, or unscented detergent to clean equipment. Equipment can be baked at 100°C for 1hour to remove most volatile contaminants. 

Fig. 1. In situ collection from a whole frame face with an observation frame containing pupae and adult workers. Air flows sequentially from the 1) air source through the 2) bubbler humidifier, 3) air flowmeter array, 4) air line, 5) air port connector, 6) port connector extension tubes into the 7) observation frame.  Sample headspace is pulled through the 6) extension tubes through the 8) vacuum port connector, 9) adsorbent filter volatile trap, by the 10) vacuum line which is regulated by the vacuum flowmeters (hidden by the observation frame). The collection equipment is kept in a temperature controlled environmental chamber to keep the frame near colony temperature (32°C).  Some flow system features such as the air source pressure reducer, air filter, air heater, vacuum pressure regulator and the vacuum source are not visible in this picture.