Anal. Chem. 1980, 52, 2249-2257 78-1428: National Bureau of Standards: Washington, DC, Sept 1977. (8) Walpole, R. E.; Myers, R. "Probability and Statistics for Engineers and Scientists"; Macmiilan: New York, 1972; p 190. (9) Natrelh, M. G."Experimental Statistics"; NBS Handb. ( U . S . ) . 1963, No. 91. (10) Ku, H., Ed. "Precision Measurement and Calibration"; NBS Spec. Pub/., No. 300, Vol. 1. (11) Harris, T. H.; Cummings, J. G. Residue Rev. 1964, 6 , 104. (12) . , "Handbook for Analvtical Quality Control in Water and Wastewater Laboratories": U S . Environmenfal Protection Agency, Environmental Monitoring and Support Laboratory: Cincinnati, OH, March 1979: p 10-6. (13) DeVoe. J. R.. Ed. ACS S v r n ~ Ser. . 1977. No. 6 3 . (14j Wernimont, G. ACSSymp. S e r . 1977, NO. 63, Chapter 1. (15) Youden, W. J. Assoc. Off. Anal. Chem. 1963, 4 6 , 56. (16) Youden, W. J.; Steiner, E. H. "Statistical Manual of the Association of Official Analytical Chemists"; Association of Official Analytical Chemists: Washington, DC, 1975. (1 7) "Sixth Materials Research Symposium in 1973": NBS Spec. Pub/. ( U . S . ) 1975, No. 408, 806. (18) "FDA Proposed Criteria and Procedures for Evaluating Assays for Carcinogenic Residues", FR 44, No. 55; Food and Drug Administration:
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Washington, DC, March 20, 1979: pp 17070-17114. (19) "Appendices on Tentative Nomenchture, Symbols, Units and Standards, 11. Terms and Symbols Related to Analytical Functions and their Figures of Merit-Number 28"; International Union of Pure and Applied Chemistry, Oxford, U.K., 1972. (20) Kaiser, H. H. Anal. Chern. 1970, 4 2 , 26A. (21) Currie, L. A. Anal. Cbem. 1968, 4 0 , 586. (22) Youden. W. J.; Steiner, E. H. "statistical Manual of the Association of Official Anawical Chemists"; Association of Official Analytical Chemists: Washington, DC, 1975, Chapter 4. (23) Currie, L. A.; DeVoe, J. R. ACS Symp. Ser. 1977, No. 63, Chapter 3. (24) Youden, W. J. Anal. Chem. 1960, 32, 23A. (25) Hertz, H. S.; Cheder, S. N. Ed. NBS Spec. Pub/. (U.S.) 1979, No. 519. See articles on sampling by H. Ku, p 1; 6. R . Appel et al., p 121; H. G. Eaton et al., p 213; W. Horowitz and J. W. Howard, p 231; and H. G. Lento. p 243.
David Freeman acknowledges support from the National Science Foundation for his work in connection with this project.
Distribution Phenomena of Mobile-Phase Components and Determination of Dead Volume in Reversed-Phase Liquid Chromatography R. M. McCormick and B. L. Karger" Institute of Chemical Analysis and Department of Chemistry, Northeastern University, Boston, Massachusetts 02 115
The elution behaviors of three common reversedphase organic modtflers were examined from 0 to 100% modifier in aqueous mobile phases. Elution of modifier and deuterated modifier enriched samples is complicated by retention and isotherm curvature. The former species yields a concentration pulse In the chromatogram while the latter produces two bands-a concentration pulse and a band containing the deuterated species. Injection of water-enriched samples results in vacancy peaks with the same elution behavior as corresponding modtflerenrlched samples. Injection of D,Oenrlched samples yields two bands-a vacancy peak and the D,O-rich band. The elution volume of D 2 0 provides a good estlmate of the column dead volume. Modifier dkstribution isotherms measured by several procedures show that significant quantities of organic solvent are extracted into the stationary phase, the amount being dependent on the solvent strength of the modifier and its concentration in the mobile phase.
At present, the majority of high-performance liquid chromatographic (HPLC) separations are accomplished with use of chemically bonded reversed-phase packings. The predominant mechanism of retention in reversed-phase liquid chromatography (RPLC) is hydrophobic expulsion of a solute from a mixed aqueous organic mobile phase ( 1 , 2 ) . While hydrophobic phenomena are generally associated with nonpolar solute selectivity, it is now also recognized that selectivity based on polar group differences of solute molecules can also be very significant in RPLC (3-5). The organic solvent or modifier (e.g., methanol (MeOH), acetonitrile (ACN), or tetrahydrofuran (THF)) has been shown to play a particularly important role in this behavior ( 3 , 6), and thus control of mobile-phase composition (e.g., ternary phases ( 3 , 4 ) )provides a powerful means of manipulating separation. T o work successfully with mobile-phase phenomena in the achievement of separation, one needs to understand in more 0003-2700/80/0352-2249$01 .OO/O
detail the retention process in RPLC. At the heart of this understanding, a better description of the stationary phase is required, as well as a more complete consideration of the distribution phenomena of all components in the reversedphase column. RPLC retention is more complex than simple solute interaction with the bonded n-alkyl chains. Besides the influence of unreacted and accessible silanol groups, the organic modifier, as first proposed by Knox and Pryde (7), is enriched in the stationary phase, and this extracted solvent will also interact with solute species. Scott and Kucera (8,9),Westerlund and Theodorsen ( I O ) , and Tilly-Melin et al. (11) provided experimental proof of the selective concentration of the modifier into the bonded phase. The organic solvent is expected to distribute between the mobile and stationary phase in RPLC since the modifier, as well as the solute, is subject to hydrophobic expulsion from the aqueous mobile phase. The quantity of extracted modifier will be dependent upon its solvent strength, and thus THF, for example, should extract to a much larger extent than MeOH. The composition and character of the stationary phase will vary, depending upon the composition of the mobile phase. Hence, in order to understand retention in RPLC, the distribution process of the organic modifier must be considered. The purpose of this paper is to examine in detail the distribution of organic modifiers in reversed-phase columns. Consequences of this distribution process with respect to retention of various species identical or related to the mobile-phase components (e.g., deuterated solvents) will also be examined. Since this distribution is an equilibrium process and since the organic modifier is a constituent of the mobile phase, the laws of finite concentration chromatography are applicable t o the organic solvent. On the basis of the information developed in this work, a second paper ( 1 2 ) will deal with retention phenomena of solute species (Le., nonconstituents of the mobile phase in RPLC). 1980 American Chemical Society
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T h e stationary phase not only changes character with alteration of the composition of the mobile phase but, in additon, the volume of t h a t phase varies as well. This can complicate the determination of the dead volume of a reversed-phase column, a problem of current interest (6,13,14). Prerequisite to understanding retention in RPLC is an accurate estimate of t h e dead volume of the column. Various solutes identical or related to the mobile phase have been used as nonsorbed markers, and it will be shown in this paper that most of these species lead t o an incorrect estimation of the dead volume. However, except in mobile phases of low water concentration, D20gives a reasonable estimate of the dead volume in RPLC for solutes which are not excluded from the porous matrix (15, 16), and this result is directly understandable from the distribution phenomena occurring within the column. EXPERIMENTAL SECTION Equipment and Chemicals. All chromatographic experiments were performed on an SP 8000 liquid chromatograph (Spectra Physics, Santa Clara, CA) equipped with a built-in data system. Detectors were either an SP 8310 UV detector or a Model R 401 refractometer (Waters Associates Inc., Milford, MA), thermostated via an external circulating water bath. All chromatographic measurements and equilibrations were made a t 25.0 f 0.1 "C in air or water thermostated baths. Except in those cases where noted, chromatographic measurements were performed on either a 10-cm or 20-cm column packed with octyl bonded Hypersil (Shandon Southern Instruments, Inc., Sewickley,PA), the bonding and packing being performed in our laboratory. The parent silica had a mean particle diameter of 5 wm and a specific surface area of 183 m2/g, as reported by the manufacturer. The bonding and end capping procedures were modifications of those in the literature (17). Carbon analysis of the bonded phase was performed by Galbraith Laboratories (Knoxville, TN) and 3.65 pmol of dimethyloctylsilyl groups/m* were calculated on the basis of the percentage of carbon found. Columns were packed via the balanced density slurry method using 4.6 mm i.d. polished bore stainless steel column blanks (Analabs, North Haven, CT). Liquid chromatography grade solvents were obtained from Burdick annd Jackson Laboratories, Inc. (Muskegon, MI) and J. T. Baker Chemical Co. (Phillipsburg, NJ). Solvents were degassed via a helium purge. Samples were obtained from Aldrich Chemical Co. (Milwaukee,WI) and Sigma Chemical Co. (St.Louis, MO). Gas chromatographic measurements were performed on a dual column Model 350 gas chromatograph (Tracor Instruments, Austin, TX) equipped with a thermal conductivity detector. Analyses were performed either isothermally or temperature programmed between 90 and 170 "C with helium as carrier gas. A 1.8-m glass column packed with silanized Porapak Q 80/100 mesh (Supelco, Inc., Bellefonte, PA) was used. Chromatograms were quantitated via a Vidar Autolab digital integrator (Mountain View, CA) in parallel with a recorder (Linear Instruments Corp., Irvine, CA). Maximum Column Dead Volume. The maximum dead volume of a column was determined by a weight difference procedure similar to that of Slaats et al. (18)and van de Venne (19). Approximately 100 mL of methanol and carbon tetrachloride were sequentially pumped through the column to achieve separate equilibration for each solvent. Between equilibrations, the column and its contents were weighed to the nearest 0.1 mg on an analytical balance. The maximum column dead volume was then ~ M M d H are respectively calculated by using eq 1where M C Cand v,m=
=
Mcci,
-
MMeon
PCClr - PMeOH
(1)
the masses of the column equilibrated with CC14and methanol. The density p of the methanol and carbon tetrachloride used for the equilibrations was measured a t 25.0 "C in a thermostated pycnometer and found to be in agreement with literature values. Measurement of Solute Elution Volumes. Retention of the modifier and the various solutes was determined by valve injection of a 10-pL sample into the column equilibrated with the pre-
blended solvent. The refractometer was used to monitor the column effluent. The various modifiers and solutes were dissolved in the mobile phase with which the column was equilibrated, and the concentration of the solutes was adjusted such that a discernible peak was produced at the higher detector sensitivities; solute concentrations were typically less than 1%. The low solute concentration in the sample produced symmetrical peaks (asymmetry factors measured at 10% peak height were less than 1.4), and thus retention times were measured at the point of maximum concentration in the solute band. At least five replicate measurements of the retention time of each solute were made, and the average of the five was used to obtain the retention volume. The flow rate was measured in replicate with a 5.00-mL buret and a mechanical timer. The nominal flow rate was 1 mL min-' for all experiments. (Relative standard deviations of the averaged retention volumes were less than 1.5%.) Organic Modifier Distribution Isotherms by Gas Chromatographic Measurement. The modifier distribution isotherms were produced in a manner similar to the procedure of Tilly-Melin et al. (11). Equlibration of the column was achieved by pumping approximately 100 mL of the appropriate preblended mobile phase through a 10-cm column. The mobile phase was prepared by dilution of a known mass of the modifier to a known volume with water. Thus, the mobile phase composition could be expressed in grams of modifier per milliliter of solution, which was necessary for the isotherm determinations. Conversion to volume percent was accomplished by using the known density of the mixed aqueous organic phase. The bulk mobile phase and extracted modifier were then stripped from the column by pumping 24.5 mL of dioxane through the column and collecting the effluent in a 25-mL volumetric flask to which 1-propanolhad previously been added as an internal standard. The contents of the flask were then adjusted to the calibration mark with dioxane. The collected samples were analyzed via gas chromatography and the concentrations of various modifiers in the samples were calculated on the basis of a concentration working curve for each modifier. The working curves were prepared from standards containing known concentrations of the modifiers plus the 1propanol internal standard. Gas chromatographic analyses of subsequent fractions of column effluent confirmed that all of the modifier had been stripped from the column. Water Distribution Isotherms. The distribution isotherms of water using both acetonitrile and tetrahydrofuran as the mobile phase were measured by the static procedure of Scott and Kucera (8). The octyl Hypersil was dried at 130 "C under a vacuum of 1 torr for at least 4 h prior to static equilibration with the mobile phase at 25.0 "C. The samples as well as standards were analyzed via gas chromatography on a column of Porapak Q. A correction was made for the water in the original solvent with a sample blank. RESULTS A N D D I S C U S S I O N The Maximum Column Dead Volume. T h e maximum column dead volumes Vmmmof the 10- and 20-cm columns packed with n-octyl bonded Hypersil were determined from the weight difference of the columns equilibrated with methanol and carbon tetrachloride. For the 10-cm column, the maximum accessible volume was found to be 1.19 f 0.02 mL. Likewise, for the 20-cm column, the maximum accessible column dead volume was 2.42 f 0.01 mL. These values reflect the dead volume of the column proper. When comparing these values to those obtained by dynamic chromatographic measurements, the extracolumn dead volume contributions from the connection tubing, detector, etc. were taken into account. The magnitude of the dead volume obtained by this measurement reflects the maximum volume within the column accessible t o a molecule of a size comparable to the above solvent molecules. Molecules of dimensions significantly larger than the aforementioned may experience dead volumes smaller than the maximum value due to steric exclusion from the porous matrix. T h e value of Vmmm serves as a criterion for evaluation of solute retention, since elution volumes that are larger than the maximum dead volume are indicative of retention. Moreover, the value of Vm- can provide a reference point to aid in the understanding of changes in the column
ANALYTICAL CHEMISTRY, VOL. 52, NO. 14, DECEMBER 1980
dead volume arising from factors such as the distribution of the mobile-phase components into the bonded phase as discussed in the next section. Measurement of O r g a n i c Modifier Distribution Isot h e r m s by Gas Chromatography. As previously noted, a distribution of organic modifier will occur between the nonpolar bonded phase and the aqueous mobile phase in a reversed-phase LC system. The amount of organic modifier extracted into an octyl bonded phase was measured in this study by equilibration of a 10-cm column with mobile phases of varying modifier concentrations and subsequently measuring the total quantity of modifier contained within the column by the gas chromatographic procedure described in the Experimental Section. From the quantity of modifier contained within the column and a knowledge of the column dead volume V,, the quantity of organic solvent extracted into the stationary phase can be calculated from eq 2, where M , M , = M , - C,V, and M , are respectively the masses of modifier in the stationary phase and the total column proper and C, is the concentration of modifier in the mobile phase. Since the column dead volume V, changes with variations in the composition of the mobile phase, the following iterative procedure was used to determine the amount of modifier extracted into the bonded phase. For a first approximation, one can assume that V, equals Vmma. Tentatively, a mass of extracted modifier can then be calculated from eq 2 by using the maximum dead volume. From the resulting mass, the volume occupied by the extracted organic modifier in the stationary phase can next be calculated. This volume is then subtracted from the maximum column dead volume to yield a revised column dead volume V,' as in eq 3, since any ex-
Vmr= Vmmax- M , / p
(3)
tracted organic modifier will reduce the maximum dead volume. The revised dead volume obtained from eq 3 is then used to revise M , in eq 2. This procedure is repeated until the mass of extracted modifier and the resulting dead volume attain constant values, yielding a dead volume Vmrwhich reflects the reduction in dead volume from Vmma as a result of the extraction of the organic modifier. (For this calculation, the density of modifier extracted into the stationary phase was assumed to be the same as the density of the modifier in the bulk liquid.) The isotherms for the distribution of methanol, acetonitrile, and tetrahydrofuran between bulk mobile phase and the bonded stationary phase at 25.0 "C attained by this procedure are presented in Figure 1. Of the three modifiers studied, methanol is least extracted into the stationary phase, whereas T H F is extracted to the greatest extent and acetonitrile exhibits intermediate behavior. This order is expected on the basis of the relative reversed-phase solvent strengths of the three modifiers. The mobile-phase concentration ranges for the isotherms in Figure 1 are limited a t the high modifier concentration end because of increasing imprecision in the measurement of the small quantity of extracted modifier on the ever-increasing background of organic solvent in the mobile phase. Yet, the isotherms do illustrate the general trend of curvature at higher modifier concentrations and thus the retention volumes of the organic solvents in this region will depend upon the concentration of modifier in the mobile phase. This dependency is investigated in the next section. C h r o m a t o g r a p h i c Elution Behavior of the O r g a n i c Modifiers. The distribution isotherms presented in Figure 1 reveal considerable variation in the magnitude of the distribution coefficients for the three modifiers studied. These differences will be manifested in the elution behavior of the
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'20/TT7-7 8
1
1 i
60
1
0
B
20
0
i 60
0
10
20
30 40
% MODIFIER
50
Figure 1. Organic modifier distribution isotherms obtained by GC analysis.
three solvents. Plots of the elution volumes of modifier-enriched mobile-phase samples as a function of the volume percentage of each respective modifier in the mobile phase are reproduced in Figure 2. Also included in this figure are the analogous plots for the fully deuterated analogues of the three organic solvents. The positive and negative signs in the plots indicate the relative directions of the peaks in the chromatograms as measured by refractive index detection. The chromatographic peaks for methanol and acetonitrile change direction at a concentration of about 60% (v/v) modifier in the mobile phase (Figure 2a,b) due to maxima in the refractive indices of the mixtures at this concentration. The maximum dead volume of the columns as measured by the previously described procedure is represented in the plots by the dashed horizontal lines. As expected from the isotherms, the elution volumes of the nondeuterated modifiers in Figure 2 exhibit a strong dependence upon the concentration of modifier in the bulk mobile phase. For mobile phases with modifier concentrations less than about 30% (v/v), the elution volumes of the unlabeled modifiers are generally greater than the previously defined maximum column dead volume. Retention of the modifier by the stationary phase is obviously occurring, and this is substantiated by the isotherm data in Figure 1. As the concentration of modifier in the mobile phase is increased, the elution volumes of the modifier-enriched samples of mobile phase decrease due to several interrelated factors. First, the distribution coefficient of the modifier decreases as ia result of curvature in the isotherm. In addition, the quantity of modifier extracted into the stationary phase increases, resulting in a decrease in the column dead volume and a concomitant reduction in the elution volume of the modifier. At high coincentrations of modifier (greater than -60% v/v) in the mobille phase, the elution volumes of the modifiers in Figure 2 pass through a minimum and then increase to the maximum column dead volume in mobile phases of 100% modifier. At the other end of the concentration scale, retention rapidly increases as the modifier concentration in the mobile phase approaches zero. However, with pure water as eluent, the elution volumes of methanol and CDBODare considerably lower than the maximum dead volume of the column (Figure 2a). Karch et al. (20)have noted a similar decrease in elution volume with water as eluent using particles with 60-A pore diameters and have attributed the phenomenon to a change in the conformation of the alkyl chains bonded to the siliceous support. Presumably such a change may block access to a portion of the intraparticle pore volume. This decrease in
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ANALYTICAL CHEMISTRY, VOL. 52, NO. 14, DECEMBER 1980 I
I
I
I
40
60
I
I
I
1
1
I
t .44 t .40
i 1.24
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1 1 1 1 1 1 ) 0
20 40 60 80 % MeOH ( v / v )
100
0
20
% ACN ( v / v )
80
1000
20
40 60 80 % THF ( v / v )
100
Flgure 2. Plot of elution volume of (a) methanol- and CD,ODenriched mobile-phase samples as a function of YO (v/v) MeOH in the mobile phase (10-cm column); (b) acetonitrile- and CD,CN-enriched mobile-phase samples as a function of YO (v/v) ACN in mobile phase (20-cm column); (c) tetrahydrofuran- and THF-d,-enriched mobile-phase samples as a function of YO (v/v) THF in mobile phase (20-cm column).
elution volume in 100% water is typical of several of the solutes in this study; further details and a fuller explanation will be given under the heading of Elution of D 2 0 in 100% Water as Mobile Phase in a later section. The curves in Figure 2 characterizing the elution behavior of the deuterated modifiers differ from those for the corresponding nondeuterated solvents. For the injection of a deuterated modifier-enriched sample of mobile phase, two peaks are usually observed in the chromatogram produced by refractive index detection. One of these peaks has the same retention behavior as the modifier concentration pulses in Figure 2 , while the other marks the elution of the band containing the deuterated modifier molecules. The former peak can be eliminated from the chromatogram by adjusting the total concentration of modifier and deuterated analogue in the injection sample so that it exactly equals the concentration of modifier in the bulk mobile phase. It is also noted that the curves for the deuterated modifiers approach the maximum column dead volume, reaffirming the validity of the maximum dead volume measurements. The results in Figure 2 can be understood in more detail from a consideration of the distribution of the organic modifier between the stationary and mobile phases over the whole composition range, as illustrated for the general case in Figure 3. Two definitions of the distribution coefficient K at any value of C, on the isotherm can be given, viz. K* = C,/C, (4)
K** = SC,/SC,
(5)
where eq 4 represents the slope of the chord connecting any point on the isotherm (e.g., point M or N in Figure 3) to the origin and eq 5 is the slope of the tangent to the isotherm at that point. The two values K* and K** are equal only over the portion of the isotherm which is linear from the origin. The value of K , of course, is significant in determining the migration rate of a chromatographic band through the column. The general expression for the velocity, u , of a band at finite concentration in chromatography was derived by DeVault (21) and is given in revised form (22, 23) as
where K is either K* or K**, u, is the mobile phase velocity, and V, and V , are the respective stationary and mobile-phase volumes.
Figure 3. Generalized distribution isotherm illustrating the difference between the distribution coefficient for a concentration pulse and for individual molecules. Concentration pulses, such as those produced by injection of modifier-rich plugs of mobile phase, have a distribution coefficient given by the slope of the tangent at any given point on the isotherm (24). The velocity of the pulse is described by eq 5 in conjunction with eq 6. At high mobile phase modifier compositions, the slope of the tangent (e.g., point N in Figure 3) may become negative. Injection of a sample of modifier-enriched mobile phase in this region will produce a band with an elution volume less than the column dead volume, since the denominator of eq 6 will be less than unity and thus the band velocity will be greater than the mobilephase velocity. If the modifier used to initiate the pulse is distinguishable from that of the bulk mobile phase (e.g., isotopically labeled modifier), then migration of this band will be described by eq 6 in conjunction with eq 4 (assuming no isotope effect on retention) (22). This arises from the fact that while the mobile- and stationary-phase systems will be established in part by the distribution of the organic modifier, the isotopically labeled modifier will essentially undergo linear elution conditions in that chromatographic system. For the convex-type isotherm illustrated in Figure 3, K** will always be less than K* in the nonlinear region of the isotherm. Therefore, the concentration pulse of organic modifier will travel through the column faster than the isotopically labeled modifier, as seen in Figure 2. As Helfferich has noted (23, 24), the retention of the isotopic sample is equivalent to that of the individual modifier molecules, which is greater than or equal to the retention of a concentration pulse of the unlabeled molecules in the mobile phase for the isotherm shape in Figure 3.
::Ti
ANALYTICAL CHEMISTRY, VOL. 52, NO. 14, DECEMBER 1980
C
1.36
3.401
C
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+I
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I
+I
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2253
1.32 1.28
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2 60 1.20
v,max
~
2,40:
1.16
2 20-
-
1
1
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0
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1
I
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2.00-
2 0 4 0 6 0 8 0 1 0 0
0
20
60
40
80
1000
20
40
60
80
100
(v/v) % ACN (v/v % THF ( v / v ) Flgure 4. Plot of elution volume of (a) H,O- and D,O-enriched mobile-phase samples as a function of % (v/v) MeOH in mobile phase (10-Cm column); (b) H20- and D,O-enriched mobile-phase samples as a function of % (v/v) ACN in mobile phase (20-cm column); (c)H,G and D,O-enriched moblle-phase samples as a function of % (v/v) THF in mobile phase (20-cm column). Note: D,O produces two bands. One band is D 2 0 and the other is identical in elution volume to H,O. %MIOH
The generation of two chromatographic peaks from the injection of the deuterated modifier also arises from the difference in the migration velocity. The first peak is a concentration pulse of organic solvent produced by displacement of nonlabeled organic solvent from the stationary phase upon injection of the sample. T h e second peak is produced by elution of the band containing the deuterated modifier molecules which can be detected by the refractive index monitor. Elution Behavior of Water-Enriched Samples of Mobile Phase. The elution volume curves of water-enriched samples of mobile phase (i.e., samples with a slightly lower organic solvent concentration than the mobile phase) in the three modifier systems are shown in Figure 4. Also included in these plots is the elution behavior of D 2 0 in each of the mobile phase systems. The curves for the water-enriched samples in these plots are essentially identical to the curves for the corresponding modifier-rich samples in Figure 2 except that the refractive index of the water-rich bands is opposite t o that for the modifier-rich bands. It may be surprising to observe retention of water in the hydrophobic alkyl stationary phases, especially since, in the past, water has been considered a nonsorbed species. The apparent retention of water can be explained by the rules of vacancy chromatography (22,Z-28) and from the existence of the extracted modifier in the bonded phase. Injection of a sample of the mobile phase containing a slightly higher concentration of water than the bulk mobile phase disrupts the equilibrium distribution of modifier between the stationary and mobile phases a t the beginning of the column. The system reinstates the original equlibrium by extracting modifier from the stationary phase, leaving a modifier-deficient region in the bonded phase. The original mobile-phase composition is reestablished in the original injection band, and, subsequently, this band becomes obscured by the identical mobile phase surrounding it. The modifier-deficient band, on the other hand, is detectable. This band travels through the column with essentially the same distribution behavior as that of the modifier-rich band (i.e., eq 5) and thus both bands leave the column with the same elution volume. Indeed, injections of modifier-rich and water-rich samples yield concentration pulses, both of slightly different concentration than the bulk mobile phase, but in opposite directions, and as such, the same elution behavior should be
A.
I!
0 TIME, minutes
Figure 5. Chromatogram from injection of (a)0.3% D20dissolved in 20 % (v/v) ACN/H,O into a column equilibrated with a mobile phase of 20% (v/v) ACN in water; (b) 0.5% (v/v) H,O dlssolved in a moblle phase of 20% (v/v) ACN in water.
expected. The only difference between the two bands is that the refractive index signals of the two bands are in opposite directions. A study of the elution behavior of deuterium oxide permits a test of the presence of vacancy peaks in the reversed-phase chromatography. For the injection of the water-rich sample in the previous discussion, the original injection band eluted from the column undetected. However, for the case where deuterium oxide molecules are used to produce the water-rich sample, the original injection band is distinguishable from the bulk mobile phase, since deuterium oxide has a higher refractive index than HzO. Thus, an a priori prediction of two peaks in the chromatograms of deuterium oxide samples is found to be the case, as illustrated in Figure 5 . One peak in the chromatogram possesses behavior identical with that obtained from injection of a sample of H20-rich mobile phase; this is the vacancy peak produced by the injection of a water ( H 2 0 D20)-richsample. The presence of this vacancy peak can be eliminated if the total water concentration (i.e., [H20]
+
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ANALYTICAL CHEMISTRY, VOL. 52, NO. 14, DECEMBER 1980
,
2701 ACN R l C h SAMPLES
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14CN IDEFICIENT (SAMPLES
7
1
2401
2 35
0
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M
40
50
€0
70
80
90
100
% v l v D 2 0 IN INJECTION PLUG
Flgure 6. Elution volumes of DO , and concomitant vacancy/pulse band a s a function of % (v/v) D,O in injection sample: injection volume, 10 pL; mobile phase, 80120 (v/v) H,O/ACN.
+
[D20])in the injected sample exactly equals the concentration of H 2 0 in the bulk mobile phase. The other peak in the chromatogram arises from elution of the band containing the deuterium oxide molecules. As noted above, injection of a sample of mobile phase containing D 2 0 produced two peaks in the chromatogram. From Figure 4,it is seen that the retention volume of the D20 band is approximately equal to the value of the maximum column dead volume a t low modifier concentrations, and for all three organic solvents the retention volume of D 2 0 noticeably decreases as the mobile phase modifier concentration is increased. Moreover, the elution volume of D 2 0 in Figure 4 is seen to decrease most for mobile phases of aqueous T H F while only a slight reduction is noted for aqueous methanol mobile phases. Recalling that T H F is significantly extracted into the stationary phase while MeOH is extracted only to a small extent, it is reasonable to postulate that the reductions in the elution volume of DzO follow the decrease in the column dead volume resulting from the extraction of the modifier into the stationary phase. Thus, D 2 0 would potentially appear to be an unretained solute for measuring dead volumes in RPLC (29). This conclusion presumes that deuterium oxide molecules have full access to the pore volumes experienced by more lipophilic molecules and that D 2 0 does not strongly interact with the residual, water-deactivated silanols. The results in Figure 4 are based on a 10-pL injection of a sample of the mobile phase slightly enriched in DzO. It was of interest to examine the influence of injected samples that differed significantly in modifier concentrations from the bulk mobile phase on elution of the D 2 0 and vacancy/pulse bands. Since the vacancy/pulse band was attributed to a disruption of equilibrium at the head of the column, a composition change of the injected sample would be expected to alter the elution volume of the concentration pulse. T h e elution volumes of the D 2 0 and vacancy bands for 1O-hL samples ranging from 0 to 100% D 2 0 in ACN were measured in a column equilibrated with a bulk mobile phase of 20% (v/v) ACN in water. The results are presented in Figure 6 where elution volume is plotted vs. the volume percent D 2 0 in the injected sample. The dotted line in the figure marks the sample of 80% D 2 0 composition which is the analogue of the mobile-phase concentration. T h e segment from 0 to 80% D 2 0 corresponds to injected samples enriched in ACN relative to the bulk mobile phase. Since the modifier distribution isotherm is convex (see Figure l),the concentration pulses arising from injected samples of 0-80% D 2 0 will experience K** values lower than the value a t 20% ACN, with a resultant decrease in the elution volume, as seen in the upper curve of Figure 6. For points between
80 and 100% D20, a vacancy peak is produced which experiences a higher K** value than samples with lower water concentrations and thus elutes with a larger retention volume. Obviously, a plot identical with the upper curve in Figure 6 would result from the injection of sample containing 0-100% water in ACN. Turning next to the elution behavior of the D20 band (lower curve in Figure 6), a smaller dependence of the elution volume on mobile-phase composition is observed relative t o the vacancy/pulse band. The small decrease from 80% to 10% D20 in the injected sample corresponds to a variation of only 0.1% in the elution volume of the D 2 0 band, which thus may be considered constant. This result is expected if DzO is a reasonable dead volume marker. Organic Modifier Distribution Isotherms by Dynamic Methods of Measurement. In order to understand in more detail the stationary-phase systems in RPLC, we determined the organic modifier distribution isotherms over a wide composition range of MeOH, ACN, or T H F in the mobile phase. In addition, the hypothesis that the elution volume of D 2 0 reflects the true dead volume of the column could be tested by comparing isotherms measured by dynamic methods using D 2 0 as a dead volume marker with the isotherms measured by gas chromatography. As recalled, the latter approach estimated the dead volume via an iterative procedure. Agreement between the two methods of isotherm measurement would serve as a check that DzO is unretained in the RPLC systems. The dynamically measured distribution isotherms were calculated by using three different approaches. In the first approach, isotherms were directly calculated from the elution volume of the D 2 0 band and knowledge of the maximum column dead volume by 103[vmmax
CS(4) =
-
vR~ 2 0 ( 4 ) 1 ~ ~
M
(7)
where C,(4) is a point on the isotherm a t any modifier concentration, 4, given as milligrams of modifier per gram of bonded phase. The term VRDzo(4)is the elution volume of D,O in the mobile phase of concentration 4 from a column of maximum dead volume Vmmmcontaining M grams of bonded phase. As before, the density of the extracted modifier po was assumed to be equal to the bulk density of the pure modifier. In the second approach, which is referred to as elution on a plateau (30,31),isotherms were calculated from the elution volume curves of the organic-modifier pulses shown in Figure 2. These curves are the first derivatives of the distribution isotherms, and a point on an isotherm a t any concentration 4 can be computed from
where VR0(4)is the uncorrected elution volume of an organic modifier concentration pulse a t any mobile phase modifier concentration 4, and the other symbols have their usual meanings. In the final method, isotherms were calculated from the corrected retention data of the deuterated modifiers (30) by using the following expression and assuming a negligible isotope effect (9) where VNDso($)is the net retention volume of the deuterated-modifier molecules in a mobile phase of modifier concentration 4. The deuterated modifier obeys this simple relationship since the migration of the deuterated molecules is not complicated by finite concentration phenomena such as isotherm curvature. Although an isotopic effect is known to
ANALYTICAL CHEMISTRY, VOL. 52, NO. 14, DECEMBER 1980
Figure 7. Distribution isotherms of (a) methanol, (b) acetonitrile,and (c) tetrahydrofuran in water measured by (0) GC method, (A)elution on a plateau, (0)elution volume of deuterated modifier, and (0) elution volume of D,O.
exist for compounds containing protium and deuterium (32, 33), the effect would be expected t o be small for deuterated acetonitrile and methanol, since these isotopically labeled substances contain only three and four deuterium atoms, respectively. On the other hand, deuterated T H F contains eight deuterium atoms, and an isotope effect may be more significant. The distribution isotherms for MeOH, ACN, and T H F as calculated by eq 7-9, along with those of the gas chromatographic (GC) approach, are shown in Figure 7 . As already noted, the amount of extracted organic solvent is seen t o be in the order MeOH < ACN < T H F , i.e., the solvent strength order. The three isotherms appear to reach a maximum with respect to the amount of organic modifier extracted into the stationary phase, in a manner as illustrated in Figure 3. As the percentage of water in the mobile phase is reduced, the driving force for extraction of modifier into the stationary phase is correspondingly diminished. The elution behaviors of solutes identical with or related to components of the mobile phase have been explained on the basis of Figure 3 which is a generalized picture of the isotherms in Figure 7. Separate studies a t the high organic content region (>6O% (v/v)) are currently under way in order t o confirm the result of the isotherm maximum and to define more clearly the dead volume measurement with pure organic modifier as mobile phase (see below). The agreement between the isotherms calculated by the GC approach (which only measures up to 40% v/v organic modifier) and the dynamic approaches is fairly good, strengthening the argument that DzO is unretained. T o illustrate, the typical error between the GC method and the dynamic methods is about 10 pg of modifierlg of bonded phase, which roughly corresponds to a discrepancy of about 12.5 MLor an error of less than 1%in the column dead volume. Further examination of the isotherms for ACN (Figure 7b) reveals good agreement for the approaches using D 2 0 (eq 7) and CD&N (eq 9). The small discrepancy, particularly at the isotherm maximum, between these two approaches and the elution on a plateau method (eq 8) may arise from an accumulation of errors over the composition range which is inherent in the integration approach. With eq 7 or 9, individual points on the isotherm are measured so that an error in one point will not affect other points. In the case of T H F (Figure 7c), a difference in the isotherm calculated by the integration method (eq 8) relative to the measurement of the isotherm based on D 2 0 (eq 7) is again observed. While propagation of error in the integration probably causes the discrepancy, the difference in the amount of T H F in the stationary phase a t 60% (v/v) T H F is only 50 pL, which represents an error of roughly 2.0% in t h e dead volume and thus errors appear to be magnified in the isotherms. Deuterated T H F was not included in this study because of t h e possibility of a significant isotope effect on retention.
2255
Elution of DzO in 100% Water as Mobile Phase. It was previously noted that a marked decrease in the column dead volume occurred in a mobile phase of pure water as measured by the elution of DzO (Figure 4a). Karch et al. (20) reported a similar observation for particles with 60-A pores and attributed the decrease to the collapse of the alkyl bonded phase structure, resulting in blockage of some of the internal pore structure of the siliceous particles. This phenomenon was examined in more detail. T h e column dead volume a t 100% H 2 0 was first investigated by t h e mass difference method used previously t o evaluate the maximum column dead volume. Methanol and water were sequentially used as the solvents to equilibrate the column at 25.0 f 0.1 "C. For a column equilibrated with water, the following mass summation equation can be written
Mz = Mk
+ e V m m a x f J+~ (1 e ~-~e ) V m m a X P H z O
(10)
where M z is the mass of the column equilibrated with water, Mkis a column constant reflecting the mass of column tubing, end fittings, bonded phase, etc., Vm- is the maximum column dead volume as determined previously with two organic solvents, and 8 is the fraction of Vmmmaccessible to methanol but not t o water. For a column equilibrated with methanol, the following expression can be written (11) MI = Mk + VmmarPMeOH where MI is the mass of the column equilibrated with MeOH. Combination of eq 10 and 11 yields
e=i-
M2 -
MI
Vmmax(pH20 - PMeOH)
(12)
T h e maximum dead volume of the 10-cm column was measured with carbon tetrachloride and methanol by the previous technique and found to be 1.19 mL. T h e fraction 8 of dead volume inaccessible when HzO was the mobile phase was then determined by successive equilibrations with methanol and water and found to be 0.577, i.e., 57.7% of the maximum dead column dead volume or 0.70 mL was inaccessible to water. This value of 8 can be compared with the intraparticle (ti) and interparticle (to) porosities of the bonded phase. The value of eo is typically 0.4 for a well-packed column of porous silica particles (34). For a 10 cm X 4.6 mm i.d. column, the empty column volume is 1.6 mL, and when packed, an interparticle volume of -0.6 mL (0.4 x 1.6 mL) is present. Hence, for this column, the intraparticle pores contribute roughly 1.20 - 0.6 (Vmm - 0.6 mL) = 0.6 mL, which is approximately the same value as the pore volume inaccessible t o water. Presumably, water cannot gain access to a significant fraction of the internal pore structure due to poor wetting conditions. The dead volume of the column with 100%water as mobile phase was further investigated as a function of the mobilephase flow rate and hence the column head pressure. After column equilibration with methanol, the mobile phase was changed to water, and the column was then equilibrated a t a flow rate of 0.3 mL/min until a stable base line from the column effluent monitored with the refractometer was obtained. T h e elution volume of DzO was assumed to be the column dead volume. The flow rate was then successively increased, and the elution volume of D 2 0 was measured a t each flow rate. The value of the dead volume of the column, as measured by the weighing procedure, was used as the value of 0 mL/min (or 0 psig). From these elution volumes, Figure 8 was constructed. T h e dead volume is seen to increase with pressure. This behavior can be explained by an increasing ability of the water to penetrate the pores at higher pressures. The water is forced into the intraparticle pores in much the same manner as mercury is forced into the pores of nonwetted silica particles (34). This phenomenon will be dependent upon the extent
2256
ANALYTICAL CHEMISTRY, VOL. 52, NO. 14, DECEMBER 1980
40 -
20 O
.
'
B
J
J
I
530
1
,
/
loo0
O
I
,
I
I
1500
,
2000
V'
0
I
I 7 0
Table I. Retention Volume (mL) of Reversed-Phase Organic Modifiers in Pure THF and ACN Mobile Phases solute THF-d, CD,CN ACN MeOH DZO HZO
mobile phase THFa ACN 1.18 2.4 2
1.24 1.34 1.38 1.40
a 10-cm column. 20-cm column. rated analogue of THF; C,D,O.
2.67 2.77 2.79
The fully deute-
of coverage of the surface of the silica by nonpolar alkyl moieties as well as the pore structure of the silica itself. The deep recesses of conical mesopores and micropores of the siliceous particle may be terminally blocked in pure H 2 0 , as indicated by the fact that the plots attain a maximum value 0.18 mL lower than the maximum column dead volume. In addition, because a pressure drop exists across the column, the outlet end of the column will experience lower pressures, and water may thus not be forced into the pores in this region. The low value of the accessible dead volume in 100% HzO was noted earlier from the elution volumes of MeOH and CDBODin this mobile phase. The reduction was not apparent in Figure 2 for ACN and T H F , since it was obscured by the higher retention of these modifiers. Also, it was seen previously that the elution volume of D 2 0 increased to approximately Vmmaxupon addition of roughly 1%modifier to the bulk mobile phase. Apparently, the addition of a small quantity of organic modifier causes a significant change in the wettability of the support by the mobile phase. Elution in 100% ACN and THF. An interesting feature of the elution behavior of the various solutes in pure ACN and T H F mobile phases is that the elution volumes are greater than the maximum column dead volume (Figure 4). As noted above, an elution volume that is larger than the maximum column dead volume is indicative of retention. The retention volumes of various solutes in 100% T H F and ACN as mobile phases are summarized in Table I. It is seen that the elution order of these solutes is the same as the order of solvent strength in normal-phase chromatography (35),e.g., tetrahydrofuran is the weakest solvent of the group in adsorption chromatography and deuterated T H F has the lowest retention volume in pure T H F (Table I), while water is the strongest solvent and has the largest retention volume in both T H F and ACN. These results can be explained in terms of a partial return t o a normal-phase chromatographic system in pure organic modifier mobile phases due to the interaction of the solutes with the residual silanols of the siliceous support. Though the reversed-phase packing material used for this work had a high coverage of bonded alkyl groups (3.65 pmol m-2 capped with trimethylsilyl groups), the material still possessed residual
I
1
2
3
H20 ( " / " )
Figure 9. Water distribution isotherm between the n-alkyl bonded phase and acetonitrile or tetrahydrofuran.
silanols that yielded a small amount of polar-phase character. A supporting piece of evidence for interactions between the solutes and the siliceous support is the poor peak shape for water and deuterium oxide with the pure modifier as mobile phase. The asymmetry factor (band width ratio a t 10% peak height) for the water peak was 4.3, while for the other peaks, it was approximately 1.4. Addition of small amounts of water to the mobile phase reduced the elution volume of water and deuterium oxide to the maximum dead volume and significantly improved the symmetry of the peaks to the value cited in the Experimental Section. As further evidence of the importance of water in deactivating polar sites of the bonded-phase silica support, the distribution isotherms of water a t very high ACN and T H F concentrations were measured by using the static GC procedure described in the Experimental Section (8). This method is justified for the higher modifier concentrations for the following reasons. First, the particles were easily equilibrated with the mobile phase a t the higher organic solvent concentrations. Second, the column stripping procedure used for the previous isotherm measurements could not be employed, since commercial HPLC solvents contain varying amounts of water (3Ck200 ppm). Thus, the stripping solvents would have added a substantial water background to the measurements, in addition to not fully removing the water from the column. The isotherms for the distribution of water in both T H F and ACN mobile phases are shown in Figure 9. It is seen that a small quantity of water is extracted into the bonded phase, presumably due to interactions with the residual silanol groups a t the base of the bonded phase. Slight differences are found between the two isotherms, which may arise in part from the fact that the water peaks were slightly tailed on the GC column. Nevertheless, the quantity of extracted water is small, and the distribution appears to plateau at concentrations greater than -3% HzO in the mobile phase. The fact that the quantity of extracted water is roughly constant above 3% provides further justification that water is only very weakly retained. Finally, it is worth noting that the sensitivity of retention to unreacted silanols in pure organic modifier indicates that this effect may be significant in nonaqueous reversed-phase liquid chromatography (36),particularly for solutes which possess functional groups that can interact with silanol groups.
LITERATURE CITED (1) Hofiath, C.;Melander, W.; Molnar, I. J . Chromatogr. 1976, 725, 129-156. (2) Karger, B. L.; Gant, J. R.; Hartkopf, A.; Weiner, P. H. J . Chromatogf. 1976, 128,65-78. (3) Tanaka, N.; Goodell. H.; Karger, B. L. J . Chromatogr. 1978, 758, 233-248. (4) Bakalyar, S.R.; McIlwrick, R.: Roggendorf, E. J . Chromatogf. 1977, 142,353-365. (5) Hoffman, N. E.; Liao, J. C. Anal. Lett. 1978. 7 7 , 287-306. (6) Schoenmakers, P.; Billiet, H. A. H.; Tijssen, R.; deGalan, L. J . Chromatogr. 1978, 149, 519-537.
Anal. Chem. 1980, 5 2 , 2257-2262 Knox, J. H.; Pryde, A. J. Chromafogr. 1975, 112, 171-188. Scott, R. P. W.; Kucera, P. J. Chromafogr. 1977, 142, 213-232. Scott, R. P. W.; Kucera, P. J . Chromatogr. 1979, 175, 51-63. Westerlund, D.; Theudorsen, A. J. Chromatogr. 1977, 144, 27-37. Tilly-Melin, A,; Askemark, Y.; Wahlund, K. G.; Schill, G. Anal. Chem. 1979, 5 1 , 976-983. McCorrnick, R. M.; Karger, B. L. J , Chromatogr., in press. Knox, J. H., poster session presented at The 3rd International Symposium on Column Liquid Chromatography, Salzburg. Austria, Sept 1977. Berendsen, G. E.; Schoenmakers, P. J.; deGalan, L.; Vigh, G.; VargaPuchony, Z.; InczBdy, J. J. Li9. Chromafogr., in press. HorvBth, C.; Lin, H. J . Chromatogr. 1976, 126, 401-420. Scott, R. P. W.; Kucera, P. J. Chromafogr. 1976, 125, 251-263. Hemetsberger, H.; Kellerman, M.; Ricken, H. Chromafographia 1977, IO, 726-730. Slaats, E. H.; Kraak, J. C.; Brugrnan, W. J. T.; Poppe, H. J. Chromatogr. 1978. 149, 255-270. van de Venne, J. L. M. Ph.D. Dissertatlon, Technische Hogeschool Eindhoven, Eindhoven, The Netherlands, 1979. Karch, K.; Sebestian, I.; Halasz, I.J. Chromatogr. 176, 122,3-16. DeVault, D. J . Am. Chem. SOC.1943, 65, 532-540. Peterson, D. L.; Helfferich, F. J . Phys. Chem. 1965, 69, 1283-1293. Helfferich, F. J. Chem. Hut. 1964, 41, 410-413. Helfferich, F.; Klein, H. "Multicomponent Chromatography"; Marcel Dekker: New York, 1970; Chapter 3. Zhukovitski, A. A. I n "Gas Chromatography 1964"; Goldup. A,, Ed.;
2257
Institute of Petroleum: London, 1965; pp 161-169. (26) Reilley, C. N.; Hiklebrand, G. P.; Ashley, Y. W. Anal. Chem. 1962, 34, 1198-1213. (27) Scott. R. P. W.; Scott, C. G.; Kucera, P. Anal. Chem. 1972, 44, 100-104. (28) hais, K.; Krejd, M. J. Chromatogr. 1974, 91, 161-166. (29) Engelhardt, H. "High Performance Liquid Chromatography"; SpringerVerlag: New York, 1979; p 127. (30) Helfferich, F.; Peterson, D. L. Science 1963, 142. 661-662. (31) Conder, J. R. in "Progress in Gas Chromatography"; Purnell, J. H., Ed.; Interscience: New York, 1968; p 209. (32) Tanaka. N.; Thornton. E. R. J . Am. Chem. Soc. 1976, 98, 1617-1619. (33) Tanaka, N.; Thornton, E. R. J. Am. Chem. Soc.1977, 99, 7300-7307. (34) Unger, K. K. "Porous Silica; Its Properties and Use as Support in Column Liquid Chromatography": Elsevier: Amsterdam, 1979; p 170. (35) Snyder, L. R. "Principles of Absorption Chromatography", Marcel Dekker: New York, 1968; Chapter 8. (36) Parris. N. A. J. Chromafogr. 1978, 149, 615-624.
RECEIVED for review May 12,1980. Accepted September 2, 1980. The authors wish to acknowledge the support of the National Science Foundation. Contribution number 72 from the Institute of Chemical Analysis.
High-Resolution Gas Chromatography of the 22 Tetrachlorodibenzo-p-dioxin Isomers Hans Rudolf Buser * Swiss Federal Research Station, CH-8820 Wadenswil, Switzerland
Christoffer Rappe Department of Organic Chemistry, University of Umea, S-901 8 7 Umea, Sweden
The 22 tetrachlorodibenzo-p-dioxins (TCDDs) were synthesized in microgram quantities by a simple pyrolysis procedure from different potassium chlorophenates. The separation of these TCDD isomers was studied on high-resolution glass capillary columns with different stationary phases (Sllar IOc, OV-17, OV-101) and by use of mass spectrometric detection. Conditions were found that allowed the unambiguous asslgnment of many of these Isomers, including the very toxic 2378-TCDD. The determination of the various TCDD Isomers is illustrated in the analysis of samples from known contaminated areas in Seveso, Italy, and in eastern Mlssourl, and the method is also applied to the analysis of fish from the Tittabawassee River In Michigan and fly ash samples from municipal incinerators in Switzerland.
Polychlorinated dibenzo-p-dioxins (PCDDs) are a group of compounds that have been the subject of much concern in recent years. Some of these compounds have extraordinary toxic properties and are teratogenic, mutagenic, and potentially carcinogenic ( 1 ) . They are known as highly stable contaminants present in a variety of synthetic chemicals like the chlorinated phenols, phenoxy acids, and chlorobenzenes. PCDDs can be formed in substantial quantities in pyrolytic reactions from such common chemicals as the chlorinated phenols and chlorobenzenes (2, 3 ) . Recently, PCDDs have been identified in fly ash and emissions from municipal incinerators and industrial power facilities (4-8). 0003-2700/80/0352-2257$01 .OO/O
In all there are 75 positional PCDD isomers ranging from the mono- up to the octachloro compounds; there are 22 tetrachlorodibenzo-p-dioxins(TCDDs) alone. Positional isomers of the various PCDDs appear to vary greatly in acute toxicity and biological activity ( 9 , I O ) . The most toxic isomer seems to be 2,3,7,8-tetrachlorodibenzo-p-dioxin (2378-TCDD) (11). This compound was involved in several industrial accidents and has caused severe environmental contamination, most recently a t Seveso, Italy (12). Highly sensitive analytical methodology is required to monitor levels of these compounds in environmental samples and to assess possible risks associated with the presence of these compounds. Detection limits in environmental and biological samples must be in the parts-per-trillion ( 10-l2) range (13). Furthermore, the analytical procedures should allow a differentiation between the various PCDD isomers. Many analytical techniques have been described for the analysis of PCDDs, the most specific methods making use of mass spectrometry (MS). In a series of papers we have reported on the application of high-resolution gas chromatography (HRGC) in combination with MS for the analysis of PCDDs and related compounds in various environmental, biological, and industrial samples (14-16). In this paper now, we report on the application of HRGC for the separation and identification of the various TCDD isomers. All 22 TCDD isomers were synthetically prepared from different chlorophenates using a simple pyrolysis procedure. These isomers were also synthesized by Nestrick et al. (17) using a more elaborate ex0 1980 American Chemical Society
