302 Analyst, May, 1974, Vol. 99, pp. 302-305 A Method of Collecting and Concentrating Headspace Volatiles for Gas- chromatographic Analysis By R. E. HURST (Fisheries Research Board of Canada, Vartcouver Laboratory, 6640 N . W. Marine Drive, Vancouve, 8, B.C., Canada) Headspace analysis by gas chroinatography has been widely used in the field of food science in order to characterise flavours and odours, and as an index of quality. One of the major difficulties encountered in this work lies in obtaining a sufficient concentration of volatiles to permit detection and identification. A method of collecting and enriching headspace volatiles and of introducing the total concentrate on to a gas chromatograph is described. GAS-CHROMATOGRAPHIC analysis of the headspace over various foods has been used for such purposes as the characterisation of flavours and odours, and as an index of quality.Direct sampling of the volatiles with a gas-tight syringe is the most elementary procedure used in headspace analysis and for certain applications is very sati~factory.l-~ Generally, however, this method does not provide a sufficient concentration of volatiles for adequate detection by gas chromatography, and various devices and techniques have been developed to overcome this Hughes4 used a sweeping gas to carry volatiles from cooked herring into a U-trap cooled with liquid oxygen. When the collection was complete the condensed volatiles in the U-trap were expanded into a glass syringe of large capacity (50 or 100 ml) as the gas warmed to ambient temperature. Samples were then removed by syringe through a septum in the system and injected in a convenient way on to a gas chromatograph.The method described here is a modification and extension of this procedure, which permits volatiles at ambient temperature to be collected and the total volume to be injected on to the column, METHOD GAS CHROMATOGRAPHY- Analysis was performed on a Microtek MT220 gas chromatograph with flame-ionisation detection. The injection port septum nut of the instrument was modified by adding a Teflon- lined tube on the top to act as an additional seal for the injection needle (Fig. 1). Further, a Whitey toggle valve was fitted to the carrier-gas line so that the flow could be shut off during injection. A 1.8-m long column was used, constructed of heavy-walled glass tubing, 8 mm 0.d.x 2 mm i.d., with 6 mm 0.d. heavy-walled tubing on both ends to accommodate the &-inch Swagelok fittings at the inlet and detector port of the instrument. The column was packed with Porapak Q, 80 to 100 mesh. Temperature programming was used in the range of 50 to 180 "C at a rate of 2 "C min-l and the carrier gas was nitrogen with a flow-rate of 25 ml min-1. The instrument was attenuated at 1 x 16 so as to give maximum sensitivity with an accept- able signal to noise ratio. PROCEDURE- A detailed description of the apparatus is given in Fig. 1. The sample flask with two side-arm tubes containing septa is connected by Vacutainer 22G needles ( l i inches) (Becton, Dickinson and Company, Rutherford, New Jersey) and silicone rubber tubing to a 50-ml glass syringe and to a collection tube consisting of a heavy-walled test-tube (9 mm i.d., 13 mm 0.d.and 70 mm long with a volume of approximately 3 ml) stoppered with a 4-ml Vacutainer stopper. The sample flask is charged with sample which, in the two examples given, was glycerol containing a mixture of alcohols of low relative molecular mass at concentrations of 40 p.p.m. and liquor from canned salmon. The syringe plunger is withdrawn so as to give a 50-rnl capacity and a glass stopper placed securely in the neck of the sample flask so that the system is sealed and the syringe is held at the 50-ml mark. Liquid nitrogen @ SAC and the author.HURST 303 - contained in a small vacuum flask is brought into contact with the collecting tube so that approximately the bottom two thirds of the tube is immersed, which ensures that the upper area of the tube does not become too cold and thus cause freezing-out at the inlet needle.Stirrer Vacutainer stopper 'Collection tube -Vacuum flask T -Syringe 50 40 - - 30 2c - 1c - B n Aluminium cap Septum (1 3 mm) Hole Vacutainer needle PTFE Modified septum nut Septum (1 Column 6mm 0.d. 8mm 0.d. 2 mm i.d. 3 mm) Fig. 1. Schematic diagram of t h e collection device As the headspace gas from the sample flask condenses in the collection tube, the syringe plunger moves down to compensate for the reduced volume of gas in the system. When the syringe plunger has moved sufficiently to replace 50 ml of condensed headspace, the liquid nitrogen is removed from the collection tube, and as the temperature rises the syringe plunger moves upwards in order to accommodate the expanding gases.When the expanded volume has reached 45 ml, the liquid nitrogen is again brought into contact with the collection tube and the procedure is repeated so that a further 45 ml of headspace is condensed. It should be noted here that the principal condensation was of 50 ml; however, expansion to a volume of only 45 ml was permitted, thus ensuring that a sufficiently low temperature is maintained within the collection flask so that the condensed volatiles essentially remain during expansion. The temperature at this stage is approximately -50 "C. After an appropriate number of condensations have been made, which in this work was four, the needle is withdrawn from the septum of the collection tube while the latter is still immersed in liquid nitrogen.The tube is then removed from the liquid nitrogen, immediately inserted into the aluminium jacket and the cap quickly screwed down tightly (Fig. 2A). The rubber stopper in the collection tube is now held securely in place so that no volatiles escape as the pressure within the tube increases with warming. The jacketed tube is placed in a water-bath at 50 "C ready for injection on to the gas chromatograph. Carrier-gas flow to the gas-chromatographic column is shut off by means of the toggle valve, which will cause a negative response on the recorder. The long end of a Vacutainer 22G needle (14 inches) is inserted into the modified injection nut and through the septum.Next, the cap on the aluminium jacket containing the collection tube is brought down on to304 [Analyst, Vol. 99 the protruding short end of the Vacutainer needle in such a way as to enter the hole in the cap and pierce the cap septum and tube stopper (Fig. 2B). This procedure discharges the volatiles under pressure on to the column and an immediate pressure spike will ensue on the recorder. When the pen drops back to the negative response position, the carrier gas is turned on and the needle is withdrawn from the septum of the instrument, leaving the upper end of the needle to remain in the collection tube. The instrument is then set to temperature programme, HURST: A METHOD OF COLLECTING AND CONCENTRATING HEADSPACE DISCUSSION Gas chromatograms of alcohols of low relative molecular mass are shown in Fig.3. Headspace was collected above glycerol containing 40 p.p.m. each of methanol, ethanol, propan-1-01 and butan-1-01. Sampling by the standard method using a 10-ml Hamilton 1010 gas-tight syringe is compared with a single condensation and with four condensations in order clearly to demonstrate the enrichment by this method. Identification of volatiles associated with canned salmon is currently being undertaken and the method described above was developed for this purpose. Gas chromatograms of headspace volatiles collected from the liquor in canned salmon are shown in Fig. 4. The enrichment of volatiles from successive condensations can be seen by comparing trace 1 from a single 50-ml condensation with trace 2, which represents four condensations totalling 185 ml. Single 50-ml condensations have been successfully used in a routine manner to demonstrate, by gas-chromatographic profiles, differences related to the quality of product.Care should be taken in the procedure to ensure that the needle entering the collection tube just passes through the Vacutainer stopper so that plugging will not occur from freezing during condensation. Once the collection tube has been secured in the aluminium jacket with the cap, no problem has been encountered with the escape of gas prior to injection, although no attempt has been made to store the collected volatiles in this manner. It was necessary to modify the septum nut of the instrument in order to prevent the escape of volatiles during the pressure injection, which is approximately 16 atm in the collection tube at ambient temperature.This amount of pressure has presented no hazardous problems. The apparatus has been thoroughly tested by using a thin-walled glass collection tube instead of the heavy-walled tube so as to encourage breakage under pressure. In every instance in which the aluminium cap was not in place the rubber stopper released the pressure in much the same manner as the pressure valve in some domestic pressure cookers. Once the tube is secured in the jacket any breakage would be contained; however, the use of a thick-walled collection tube makes this possibility remote. Choice of a column packing material centred on the porous polyaromatic polymer type of beads because of their compatibility with water.The Porapak series, P, Q, S and T (Waters Associates), were tested and the best results were obtained on 80 to 100-mesh Porapak Q. A I B Fig. 3. Gas chromatograms of alcohols: 1, methanol; 2, ethanol; 3, propan-1-01; and 4, butan-1-01, collected from the headspace above glycerol containing 40 p.p.m. of each alcohol, A, 10-ml sampling with gas-tight syringe; B, one 50-ml conden- sation ; and C, four condensationsFig. 2. A, Exploded view showing the assembly of the collection tube, aluminium jacket and cap; and B, the jacketed collection tube in position during injection [To face p. 304May, 19741 VOLATILES FOR GAS-CHROMATOGRAPHIC ANALYSIS 305 Fig. 4. Gas chromatograms of headspace collected from canned salmon liquor.Trace 1, one collection of 50 ml; and trace 2, enrichment from four collections (total 185 ml) of headspace Recently, Krumperman* has drawn attention to possible erroneous peaks associated with Porapak Q when the latter is used to trap volatiles. This material was therefore checked in order to determine if an analogous problem would be encountered when it was used as column packing. The column was conditioned for 4 days at a temperature of 250 "C and a carrier-gas flow-rate of 30 ml min-l. The collection tube, sample flask and syringe were carefully cleaned, and the condensation procedure was then carried out without a sample in the sample flask. No peaks were observed after the pressure spike. This system has been used for the past 5 months in our laboratory and it has not presented any maintenance or operational problems. 1. 2. 3. 4. 5. 6. 7. 8, REFERENCES Buttery, R. G., and Teranishi, R., Analyt. Chem., 1961, 33, 1439. Guadagni, D. G., Bomben, J. L., and Hudson, J. S., J . Sci. Fd Agric., 1971,22, 110. Lorenz, K., and Maga, J., J . Fd Sci., 1971, 36, 936. Hughes, R. B., J . Sci. Fd Agric., 1964, 15, 290. Mendelsohn, J. M., Steinberg, M. A., and Merritt, C., J . Fd Sci., 1966, 31, 389. Jennings, W. G., and Nursten, H. E., Analyt. Chem., 1967, 39, 521. Jennings, W. G., Wohleb, R., and Lewis, J. M., J . Fd Sci., 1972, 37, 69. Krumperman, P. H., J . Agric. Fd Chem., 1972, 20, 909. Received July 23rd, 1973 Amended December loth, 1973 Accepted January 14th, 1974