Anal. Chem. 1995, 67, 3300-3304
Determination of Partition Coefficients and Quantitation of Headspace Volatile Compounds Alain Chaintreau,* Andrea Grade, and Rafael Muiioz-Box Nestle Research Centre, NESTEC Ltd., P.0. Box 44, Vers-Chez-les-Blancs, 7000 Lausanne 26, Switzerland
A survey of partition coefficient values reported in the literature showed numerous discrepancies. These could be attributed to technical problems encountered during headspace measurements. Consequently, a novel method based on a combination of static headspace sampling and dynamic headspace traps was developed. The new method also includes determination of partition coefficients independently of the concentration of the reference. Once the partition coefficient is known,a simple calibration of the gas phase constituents can be performed. This headspace quantification method does not require addition of a standard. Estimation of variance showed the method to be accurate (0.5-15%, depending on component and matrix). A model mixture of flavors was investigated by the new method. Varying the concentration of one compound had no effect on the rest of the aroma profile. Small differences in the aroma profile due to changes in the matrix were investigated as exempued by alterations in flavor retention observedwhen water, lipids, or emulsions were used as the solvent. The aroma of the gas phase in equilibrium with a food or a beverage is a key parameter that influences the choice of a consumer who opens a package. Similarly, the flavor released by the food when preparing a meal or when chewing will also determine consumer preference. Both require quantitation of the aroma components in the gas phase, as well as measurement of the partition coefficients between air and the matrix. However, if we consider the literature, quantitation in the gas phase appears to be a hard task. During the last decade many improvements have been made in headspace (HS) sampling, and automated HS injectors are now commercially available. HS is generally classified into two categories: static headspace (SHS), in which the vapor phase is directly injected in a gas chromatograph, and enriched (or dynamic) headspace (E-HS), which requires trapping of the volatiles onto an adsorbant prior to GC injection. The HS analysis of plants and flowers has been reviewed by Bicchi and Joulain' while Seto recently described the biological applications.' Quantification. While E-HS offers a better sensitivity than SHS, it is generally not suitable for quantitation of headspace components as release of the volatiles in the gas phase is strongly dependent on their interaction with the matrix. In other terms, the headspace composition is related to the air-to-matrix partition coefficients of each compound. Among the different quantitation methods that have been proposed, the most appropriate seems to be multiple headspace extraction OMHE) using S H S ampl ling.^-^ Kolb4 proposed two (1) Bicchi, C.; Joulain, D. Flavour Fragrance J. 1990,5, 131-145. (2) Seto, Y. J. Chromatogr. 1994,674, 25-62.
3300 Analytical Chemistry, Vol. 67, No. 18, September 15, 1995
MHE calibration methods, using an internal or an external standard: External Standard. The calibration mixture must be contained in a vial as a gas phase only without any solvent. This limits the calibration to very volatile compounds, and it requires the measurement of very small volumes which cannot be accurate. Internal Standard. The GC response coefficients must be determined separately in the same matrix. If matrices differ, partition of the flavorings and of the standard in both phases will change. Stable isotope-labeled standards appear to be the only alternative since their behavior will be similar to that of the nonlabeled molecules. In conclusion, calibration for headspace quantitation remains either tedious (labeled standards) or inaccurate (external standards or nonlabeled internal standards). Errors in Partition Coefficient Measurements. Great discrepancies appear between the values measured by different authors (Table 1). Drawbacks were found in all methods proposed if they had to be applied to the low-concentration range found in the headspace above foods. Dynamic Methods. Burnettg used a stripping gas to extract the volatiles from the solution. The partition coefficient is mathematically deduced from a dynamic process rather than from a system at equilibrium. Efficiency of the stripping step should be previously ascertained. Pressurized Vials. Kolb et pressurized the headspace of the sample vial prior to injection. This can modify the partition of the solute between phases, and values are higher than in systems operating at atmospheric pressure (Table 1). However, other important parameters are probably responsible for the great difference of hexane values (1400%), since all authors used different operating conditions. Glass Sym'nges. Wall adsorptions and leaks occur when operating with syringes.11,12They may seriously alter the results (up to 80%nonanal absorbedg) when operating in the usual ppm range of flavors in a food matrix, which corresponds to the ppb range in the gas phase. (3) Milana, M. R; Maggio. A,; Denaro, M.; Feliciani, R.; Gramiccioni, L. J . Chromatogr. 1991,552, 205-211. (4) Kolb, B. Chromatographia 1982.15, 587-594. (5) Hagman. A;Jacobsson, S. HRC CC,J. High Resolut. Chromatogr. Chromatogr. Commun. 1988,11, 830-836. (6) Bosset, J. 0.;Gauch, R. J. Chromatogr. 1988,456, 417-420. (7) Uhler, A. D.; Miller, L. J. J. Agric. Food Chem. 1994,36,772-775. (8)Maggio. A,; Milana, M. R; Denaro, M.; Feliciani, R; Gramiccioni, L. HRC CC, J. High Resolut. Chromatogr. Chromatogr. Commun. 1991,14, 618620. (9) Burnett, M. G. Anal. Chem. 1963,35, 1567-1570. (10) Kolb, B.: Welter, C.; Bichler, C. Chromatographia 1992,34,235-240. (11) Vitenberg, A. G.; Ioffe, B. V.; Dimitrova, Z. St.; Butaeva, I. L. J . chromatogr. 1975,112. 319-327. (12) Guitart, R.; Puigdemont, A,; Airboix. M. J. Chromatogr. 1989,491, 271280. 0003-270019510367-3300$9.00/0 0 1995 American Chemical Society
Table I. Air-to-Water Partition Coefficients from Literature Data
k
2-butanone 1-butanol ethyl acetate
n-hexane
.
1.9 x 10-3
3.9 10-3 2.3 10-3 8.5 x 2.95 x 10.' 3.6 10-4 1.6 x 0.9 x 10-2 7.14 58
temp PC) ref 25 30 25 30
25 25 40 40 40 40
13 10 11 10 9 13 10 12 10 12
method gasloop/atmpres pressurizedvial synnge/atmpres pressurizedvial shipping/atm pres gasloop/atmpres pressurizedvial syringe/atmpres pressurizedvial syringe/atmpres
Calibration. AU methods are faced with this diffifultyalready mentioned above. Most calibratethe gas phase by fully vaporizing a small amount of the pure compound in an empty Accurate measurement of small volumes is difficult, and full vaporization is limited to highly volatile components. Direct injection of the standards in a solvent is sometimes used?J5 It assumes that the GC response factors must be identical when a vapor phase or a solution is injected. Theoretical Calculations. Rohrschneider starts from a calculated partition value.16 Unfortunately, Voilley et al. have shown discrepancies of 23-M)%between experimental data and calculated coefficients." In addition, thermodynamic models do not apply when the solutes are highly water soluble, which often happens with flavors. Dilution. In spite of the good linearity of the calibration curves, EmUeiler's apparatus, used by Gratis did not seem suitable because sampling large volumes would lead to a dilution of the gas phase by air entering the system. EXPERIMENTAL SECTION Sampling Cell. The sampling cell F i r e 1) is formed of a 5 MI x 15 cm glass tube (d) (Omni6t Cambridge, U.K.). The bottom end piece (e) and the end of the piston (0 were made in-house with Teflon and Won seals. A removable glass receptor (g) was inserted into (e), to receive up to 2 mL of the solution to be equilibrated with the headspace. Before use, all glass pieces of the modified syringe were silanized with Sylon CT (5% dimethylchlorosilane in toluene) (Supelco, Bellefonte, PA)to avoid any adsorption effects on the glass walls. The volume has been calibrated by filling the cell with water, pressing the piston down, and weighbg the expelled water. There was almost no dead volume when the piston was at the bottom. To ve* that no significant gas diffusion occurred through the tnbe 0 ,a Tenax trap was connected, and the cell was equilibrated for 30 min with a 22 ppm solution of Zbutanone in the receptor. The trap was then disconnected without pressing the piston and desorbed into the GC. No peak was detected. (13) Buttery, R.: Ling. L C.; Guadagni. D. G . / . agrie. Fwd Chm. 1969. 17, 385-389. (14) Nelson. P. E.: Hoff,J. E./. Food Sd.1968.33.47C-482. (15) LeTanh. M.:Pham Thi. S.T.: Voillqr, A Sd.Alinmfr 1992.12.581-592. (16) Rohrschneider. L And. Chen. 1973.45.1241-1247. (17) LeTanh. M.:Lamer.T.: Voillqr. A: Jose.1.1.Chin. p)s;Chim. Bid. 1993. 90,545-560. (18) Graf. E.: de Rms K B. In 7hcrnnlly Genemlcd Flauors: Parliment. T. H.. Morello. M.J.. McComn. R I.. Eds.; American Chemical Society: Washington,DC. 1991: pp 4 3 - 4 4 8
Figurr I. Headspace cell.
AU solutions were prepared at concentrations between 2 and 150 ppm. For the measurement of the inrluence of increasing one compound, the concentration of this compound was chosen to be 5000 ppm. The solvents used were deionized water, medium chain higlycerides (Ma soja ,oil, or milk as an oil in water emulsion. The milk contained 2.8 g of milk fat in 1dL and was partially skimmed. Headspace Sampling. At the beginning of each experiment, a blank of the sampling cell was made. After that 1 or 2 mL of the prepared solutions was added. m e dead volume in the .receptor was negligible, 0.4%error, when 1 mL of solution was used.) The sampling cell was then closed with the piston at the top and equilibrated for exactly 30 min in a water bath at 30 "C. After this time, the gas phase was trapped by pressing down the piston at a constant flow of 50 mWmin into a stainless steel tnbe containii 60-80 mesh Tenax as adsorbent The amount of Tenax in the different traps was between 160 and 170 mg, and the traps were cleaned for 24 h at 350 "C before use. The loaded traps were desorbed by an ATD 400 (F'erldn Elmer, Beaconsfield. U.K.) desorption unit and analyzed by gas chromatography. The desorbed traps were cleaned by a special program of the ATD 400. The sampling cell was washed with EtOH and dried in a vacuum oven for at least 1h at 60 "C. To detennine reproducibility of cell sampling, measurements were repeated three times at each concentration level under the same conditions. Analytical Chemisty, Vol. 67, No. 18, September 15, 1995
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Instrument Conditions. A Perkin-Elmer adsorption thermal desorption system (ATD 400) coupled on line with a HewlettPackard 5890 gas chromatograph (Palo Alto, CA) was used. The GC was equipped with a flame ionization detector (FID) and an apolar widebore CPSIL 5 CB fused silica capillary column (50 m x 0.53 mm, 1.0 pm film thickness; Chrompack, Middleburg, The Netherlands). Helium was used as the carrier gas at 10 mL/ min. Operating parameters for the two instruments were as follows: ATD 400. The traps were desorbed at 300 "C for 15 min. The volatiles were focused in a cold Tenax trap at -30 "C and then desorbed at 280 "C for 3 min. There was no split between the traps and the GC column, and the transfer line was maintained at 150 "C. After each injection the traps were cleaned at 350 "C for 30 min. GC. The column temperature was held at 50 "C for 2 min and programmed at 4 "C/min to 200 "C. The detector was maintained at 300 "C. Curve Fitting. The parametric equations were adjusted to the experimental data using a regression software, Tablecurve 2D for Windows (Jandel Scientific, Erkrath, Germany). DISCUSSION
Sampling Cell. From the analysis of the literature, a suitable headspace cell should fit the following requirements: (1) sampling of the vapor phase at equilibrium, (2) no pressure change during sampling or separation of the liquid and the gas phase, (3) no dilution while sampling. In addition, measurement of low partition values and low headspace concentrations implies sampling of large volumes which cannot be directly injected onto a gas chromatograph. Therefore a method combining static headspace and purge and trap technique was chosen (Figure 1). The volatiles equilibrate between the sample cell (a) and the headspace chamber 6). Pressing the piston pushes the gas through the Tenax cartridge (c) in which the flavors are trapped. To detect a possible breakthrough, the headspace sampling was performed using two traps in series. Flows exceeding 100 mL/min through the cartridge gave insdcient adsorptions in the first trap. Therefore a 50-80 mL/min flow was chosen for the other experiments. In addition, a higher sampling flow rate would give cause a pressure variation in the cell, which could alter the phase equilibrium. A typical 30 min equilibration time was used as longer times did not increase the GC peak area. Peak Area Linearity and Accuracy. (All symbols used are listed in Table 2.) Defining the partition coefficient as k, = C,/Cl with C, and C, the concentrations of the solute in the headspace and in the liquid, respectively, and V, and VI the headspace and the liquid volumes. Since the initial mass, m,, is distributed between both phases: m, = mg + ml,the concentration in the vapor phase is C, = k,
mo - cgvg
v,
Or, using the initial concentration in the solution C I ~= m , / K 3302 Analytical Chemistry, Vo/. 67, No. 18, September 15, 7995
Table 2. Symbols Used
definition
symbol
volume of the gas phase volume of the liquid phase mass of the flavor in the gas phase at equilibrium mass of the flavor in solution at equilibrium initial mass of flavor in the solution flavor concentration in the gas phase at equilibrium flavor concentration in the liquid phase at equilibrium initial flavor concentration in the liquid phase air-to-solutionpartition coefficient area of the GC peak corresponding to a flavor in the headspace response factor of the GC detector slope of the GC area curve as a function of the initial Concentration in the solution slope ratio for two different amounts of the same solution in the headspace cell
c, = 1 +kVCP kvVg/V)
(3)
For a given volume, V,, of injected vapor phase, the area A of the GC peak is proportional to the concentration C, in the headspace:
A = ICg
I being the GC response factor
(4)
from (3) and (4):
A=
AkvCp 1 + kvV,/V)
(5)
Equation 5 shows a linear relationship between the GC area and the initial concentration GOin the solution. The experimental data using a solution of 2-butanone in water fit well this relationship (Figure 2). From 0 to 83 mg/L a least-squares fitting gave the following results: (1) coefficient of determination 0.996; (2) standard error of the slope 1.1%. No residual (in absolute value) exceeded 10%of the corresponding estimated value. As replicate experiments were included in the regression procedure, a lackof-fit test was performed giving a p-value of 0.55, which confirms the adequacy of the h e a r model. As the regression line was based on 15 experiments, more than 1week was required to achieve the injections, and possible variations of the GC detector could not be excluded. However, the repeatability of the vapor phase sampling appeared satisfactory (Table 3). Determination of Partition Coefficients. Ideally, the determination of partition coefficients should avoid previous headspace calibration, since the use of standards has been shown to be tedious. We present hereafter a regression method. Equation 3 is a straight line calibration function: A = pCp with p = Akv/(l kVVg/m. Starting from two different sample volumes Kl and I+ having the same initial concentration Cp,two slopes $1 and $2 can be obtained. If the volume of the headspace V, and the response coefficient 1 are considered to be constant, the ratio of the slopes is
+
Table 4. Slopes and Partition Coefficient of ?.Butanone in Water
3et06e!
a
2.5et06-
4
2et06-
PI
value slope (1 mL) slope (2 mL)
8 1.5et06-
30840 37115 2.1 10-3
kv
std error 122 176
9.5
coeff of
determination residuals
-= 1%
0.9998
0.9998
