Split/Splitless Injectors

DTD devices are typically interfaced to the gas chromatograph via split/splitless injection ports. Split/splitless injectors have been developed to allow injected volatiles to be concentrated at the head of the capillary column, yet still provide sufficient gas flow to sweep out the injection port. This is accomplished by alter- ing the flow through the injector. During the splitless mode (Fig. 3a), the carrier gas enters at the top of the injector and applies pressure on the volatiles to drive them into the top of the capillary column. The majority of the carrier gas exits through the septum purge line at the top of the injector. After sufficient time is allowed to void the injection volume, the flow is changed so that the majority of the carrier gas sweeps through the injection liner but exits the sweep vent. This method works well for normal injections using syringes.

FIGURE3 Diagram of gas flow of an HP split/splitless injector (a) under normal operation

Direct thermal desorption devices alter the flow through the injector. The carrier gas mixed with the analytes now enters the injection port through the sample needle (Fig. 3b). The majority of the carrier gas and volatiles exit through the septum purge, with only a fraction of the purged volatiles going onto the head of the column. For this reason, the split ratio must be decreased as much as possible, and in some cases the split vent may be capped off. However, this results in a reduced flow of carrier gas through the injection port and can lead to carryover between runs and irreproducible results. This is especially true for volatiles being desorbed from complex food samples.

IV. APPLICATIONS

A comparison of DTD was made with purge and trap (P&T) for analyzing vola- tiles from samples of beet sugar, roasted peanuts, and grilled ground beef. Ali- quots from the same sample were used for the comparison. Method parameters

were kept the same with two exceptions: the P&T method used N2as the purge

gas, while the DTD used helium, and following P&T, the Tenax trap was ther- mally desorbed at 150°C. Samples were purged at temperatures determined ex- perimentally to be optimal.

A. Experimental

A short-path thermal desorption device (SIS, Ringoes, NJ) was installed on an HP 5890 Series II gas chromatograph. The capillary column used was a DB-5, 30 m, 0.53 I.D column with a 5µm film (J & W Sci, NJ). Following the injection port, the capillary was passed through a cryofocusing unit (SGE, Australia). A thermocouple was attached to the cryofocusing zone and the temperature held at ⫺150°C. The gas chromatograph was held at 100°C for 10 minutes, then ramped at 3°C/min to 200°C. A second ramp of 25°C/min was used from 200 to 250°C. The temperature was then held for 5 minutes for a total run time of 50 minutes. The GC program was initiated at the beginning of the desorption so compounds not retained would be observed at the detector. The capillary flow rate was 3 ml/ min with the split at 11 ml/min and the septum purge at 3 ml/min. The split/ splitless injector was operated in splitless mode for 4 minutes during the desorp- tion.

1. Purge and Trap

Sugar crystals were ground to produce free-running crystals. Frozen precooked beef patties were chopped to a fine meal. Roasted peanuts were chopped to about

tube equipped with a sparge needle. The sample was purged with nitrogen for 4 minutes at a rate of 17 ml/min. An adsorbent trap consisting of 200 mg of Tenax, held in place with glass wool, was used to collect the volatiles. The sugar was held at 150°C, the beef at 70°C, and the peanuts at 120°C. These temperatures have been determined to be the highest possible without significantly altering the sample. After purging and trapping, the Tenax trap was desorbed at 150°C for 4 minutes using the SPTD under the same conditions used for DTD.

2. Direct Thermal Desorption

Aliquots of 500 mg each of the same samples used for purge and trap were taken and placed in a glass-lined stainless steel tube. Glass wool was used to hold the sample in place. The sample was purged of atmospheric oxygen for the minimal setting of 1 second prior to injection. The thermal desorption temperature was held at ambient for 1 minute following injection. This equilibration period is needed when switching the carrier gas from the normal operation to pass through the sample. Upon equilibration, the heated blocks close around the sample tube.

The sample was heated to 150°C for sugar, 120°C for peanuts, and 70°C for

beef. Volatiles were swept directly onto the column and cryofocused at⫺150°C

utilizing liquid nitrogen. After a 4-minute desorption, the liquid nitrogen was cut off and the cryofocusing zone allowed to rise to the GC oven temperature of 100°C.

B. Volatiles from Beet Sugar

Beet sugars are prone to adsorb off-odors as a result of contact of sugar beets with soil microorganisms that produce potent off-flavors. Odor is a major factor in quality control of the acceptability of the sugar. The volatile compounds previ- ously reported in beet sugar are primarily mixtures of short-chain fatty acids, furanones, aldehydes, and alcohols (20,21). The sample chosen possessed an ex- ceptionally offensive odor and does not represent a typical chromatographic pro- file of beet sugars. The volatile composition of the sample is dominated by short- chain fatty acids and straight-chain aldehydes. Figure 4 shows a comparison of the beet sugar analyzed by the purge-and-trap method and by direct thermal de- sorption. The total amount of volatiles loaded onto the column is greater when using the purge-and-trap method. A likely explanation is that the dense packing of the sugar in the tube for DTD does not allow the purge/carrier gas to efficiently desorb the volatiles from the sugar.

Acetic acid is one of the first components to elute (Fig. 4, compound#1)

with a retention time of 6.37 minutes. Propionic, butyric, isovaleric, and hexanoic acids (compounds 2,3,4, and 5) are observed in both methods. These short-chain fatty acids are the primary causes of the offensive odor of this sugar. The concen-

FIGURE4 GC-FID trace of beet sugar. (Top) Volatiles desorbed by purge and trap: 1,

acetic acid; 2, propionic acid; 3, butyric acid; 4, hexanal; 5, heptanal; 6, methylbutyric acid; 7, 2-octenal; 8, octanal; 9, nonanal. (Bottom) Volatiles directly desorbed onto the column.

trations of these compounds are much greater using the P&T method relative to DTD. In the P&T trace (Fig. 4, top), the straight-chain aldehydes heptanal, octa- nal, and nonanal are also observed, while only trace levels are observed in the DTD chromatogram.

C. Volatiles from Peanuts

Figure 5 shows the chromatographic traces of a crushed peanut sample analyzed by the two different techniques. The upper chromatogram was obtained using the P&T method, while the bottom chromatogram was run using the DTD method. Again, the conditions were optimized for DTD and not for P&T.

The volatile composition of roasted peanuts consists of aldehydes, alkylpyr- azines, furanones, and alcohols (22). Compounds identified by standards and re- tention times are shown in Figure 5. For the peanuts, the total volatile concentra- tion is greater for the DTD method than for the P&T method. The relatively larger chunks of the peanut sample prevent close packing and result in enhanced desorption efficiency from the sample in the DTD method.

FIGURE5 GC-FID trace of roasted peanuts. (Top) Volatiles desorbed by purge and trap: 1, pentanal; 2, N-methylpyrrole; 3, hexanal; 4, heptanal; 5, 2,5, and 2,6-dimentylpyrazine; 6, 1-octen-3-ol; 7, methylethylpyrazine; 8; 2-pentylfuran; 9, phenylacetaldehyde; 10, vi- nylphenol. (Bottom) Volatiles directly desorbed onto the column.

Both methods produced large amounts of acetone, pentane, and acetalde- hyde, which are unresolved at the front end. The straight-chain hydrocarbons and aldehydes—pentane, hexane, heptane, heptenal, octenal, and nonanal—are observed in both chromatograms, with concentrations slightly greater in the DTD method. The pyrazines are observed in greater concentration in the DTD relative to the P&T chromatogram.

D. Volatiles from Grilled Beef

Figure 6 shows the chromatographic traces of the volatiles from grilled ground beef. The volatile profile has been shown to vary with purge temperature (23). A purge temperature of 70°C was selected because protein denaturation has been shown to occur at higher temperatures (3). The sample was taken from a 4-day- old refrigerated sample and is typical of samples having undergone meat flavor deterioration. The large peak at 8.5 minutes is hexanal, which overloads the capil- lary column in both the P&T trace and the DTD trace. As observed with the sugar sample, the total volatile concentration is greater in the P&T method.

FIGURE6 GC-FID trace of grilled beef. (Top) Volatiles desorbed by purge and trap: 1,

acetaldehyde; 2, pentanal; 3, hexanal; 4, heptanal; 5, 2-octenal; 6, nonanal. (Bottom) Vola- tiles directly desorbed onto the column.

Pentanal and hexanal (Fig. 6, compounds 2 and 3, respectively) are ob- served in higher concentrations using the DTD method, while heptanal, 2-octenal, and nonanal are present in relatively greater concentrations in the P&T method. These three examples show that for low-boiling compounds, DTD can be more efficient for desorbing samples, but that the concentrating power of P&T is needed for higher-boiling compounds. The relative purging efficiency of P&T versus the desorbing efficiency of DTD is sample dependent.

E. Quantitative Analysis

One of the challenges encountered with direct thermal desorption is in quantita- tion. With automated instruments, it is fairly easy to reproduce temperatures, flow rates, and purge times. However, variability can occur as a result of sample preparation, i.e., granulation, shredding, and chopping, and as a result of sam- ple packing in the desorption tube. The variation in gas flow through the sample affects the total amount of material desorbed. The efficiency of the purge/carrier gas to strip volatiles is directly related to the amount of the sample’s surface area

with which it comes into contact. The addition of a standard is often employed to calibrate the purge efficiency and the instrumental response.

There are two types of standards: a surrogate standard and an internal stan- dard. The surrogate standard is added to the sample prior to any sample manipula- tion and is used to gauge purge efficiency. These types of standards work well with liquid matrices where the standard is readily incorporated into the sample matrix. An internal standard is added to the sample tube prior to DTD and is used to gauge instrument performance. With little or no sample preparation steps, the distinction between the two types becomes blurred. The standard itself should be thermally inert and nonindigenous to the sample. If gas chromatography— mass spectroscopy (GC-MS) is being used, a stable isotope-enriched derivative of the compound being analyzed is the best standard.

Mechanical problems associated with tube seals and needle blockage may also cause difficulty with reproducibility. Since the needle remains in the injection port for several minutes (the injection period plus the desorption period), septa need to be replaced more frequently than for normal injections. A leak around the needle will result in a decrease in the sample amount loaded onto the column. With the SPTD, fast heating of the sample can result in breakthrough at the cryotrap. A large burst of volatile compounds is blown through the cryotrap as soon as the heating units are closed. A signal prior to the end of the desorption period is an indication of the volatiles breaking through the cryotrap. Break- through is more severe in the case of P&T relative to DTD. A possible explana- tion is that as the matrix is subjected to heat, the volatiles are more readily de- sorbed off the Tenax, resulting in a plug of carrier gas containing an increased concentration of volatiles. A temperature ramp on the heating blocks and/or a more efficient cold trap could eliminate this problem.

Table 2 shows the averages, standard deviation, and the relative percent error for a mixture of pentanal, hexanal, heptanal, octanal, nonanal, and decanal run three times each by DTD. The mixture was desorbed at 150°C for 4 minutes

TABLE2 Repeatability of Three Runs of a 100

ppm Mixture of Hydrocarbons by DTD

Direct Thermal Desorption Std. Dev. % Average Pentanal 32923 1831 5.6 Hexanal 41436 2145 5.2 Heptanal 33072 1395 4.2 Octanal 62003 1507 2.4 Nonanal 59425 1513 2.5

FIGURE7 GC/MS traces of aromatic rice from three consecutive runs of (top) 875, (mid-

dle) 790, and (bottom) 750-mg samples. 1, Pentane/acetone; 2, acetic acid; 3, pentanal; 4, hexanal; 5, 2-pentalfuran; 6, nonanal.

and cryofocused at⫺150°C. Peak areas were measured using an HP 3390 integ- rator. The relative percent error for pentanal is 5.6% but decreases to 2.5% for nonanal.

The chromatographic traces from the DTD-GC-MS of three aliquiots of a commercial aromatic brown rice are shown in Fig. 7. Three samples consisting of 0.50 g each of cracked rice were thermally desorbed at 70°C. The entire col-

umn was held at 0°C during the desorption (4 min) and then ramped at 5°C/

min to 200°C. No breakthrough was observed, and the first 5 minutes of the

chromatograms have been cut off. The broad peaks at the front end result from the poor cryofocusing of the low-boiling compounds. The straight-chain aldehydes resulting from lipid oxidation dominate the chromatogram. Some variability is observed between runs in the relative concentrations of the aldehydes. The amounts of acetic acid (2), 2-pentylfuran (5), and nonanal (6) remain constant between runs. However, the amounts of pentanal (3) and hexanal (4) show a slight increase between runs. These runs demonstrate the variability of the desorbing efficiency from a sample as a result of packing efficiency.

V. SUMMARY

Direct thermal desorption provides a rapid technique for the qualitative analysis of solid samples with little or no sample preparation. Volatiles are thermally desorbed from the sample and concentrated directly onto the head of a GC col- umn. Similar chromatographic profiles may be obtained using DTD relative to P&T. Since the purge and desorption times are concurrent in the DTD method, analysis times are shorter. The relative purge efficiencies are compound and ma- trix dependent. DTD in some cases may provide a greater amount of material for detection. This is especially true for low-boiling compounds with higher vapor pressures.

Food samples that have moderate moisture content can be analyzed, but these ultimately require additional steps, which may affect the analysis greatly. Quantitative analysis is possible but is dependent on the specific analyte and the matrix. The composition of the sample particles must be uniform, enabling equivalent packing between runs. Septa need to be examined and replaced more frequently, and methodology for incorporating an internal standard must be devel- oped.

ACKNOWLEDGMENTS

The authors would like to thank Mary An Godshall and J. V. Verecellotti for their editorial assistance in the preparation of this manuscript.

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