Practical Methods for Derivatizing and Analyzing Bacterial Metabolites with a Modified Automatic Injector and Gas Chromatograph John B. Brooks, C. C. Alley, J. W. Weaver, V.
E. Green, and A. M. Harkness
Center for Disease Control, Health Services and Mental Health Administration, Public Health Service, U.S. Department of Health, Education, and Welfare, Atlanta, Ga. 30333
A Perkin-Elmer Model 900 gas chromatograph, equipped with an electron capture detector and a Hamilton Autosampler, has been modified to permit automatic injection and analysis of a variety of important bacterial metabolic products. The Autosampler was modified to correct leak problems connected with the inlet system, and a microswitch was installed to permit temperature programming. The gas chromatograph was modified by adding a Beckman switching valve that permits use of two columns (polar and nonpolar) at different time intervals through the same detector or the venting of undesirable compounds. In order to convert the Model 900 gas chromatograph equipped with an autosampler into a two-column system, an injector block through which samples could be injected manually was added to the system. The modifications are described, and representative analyses of standard mixtures and bacterial products are presented.
The gas-liquid chromatograph is an excellent instrument for analyzing spent culture media for bacterial metabolites. When these analyses are used in conjunction with other laboratory data, microorganisms often can be identified at the species level (1-7). In addition, progress has been made toward the gas-liquid chromatographic analysis (GLC) of body fluids for bacterial metabolites and other volatile compounds ( I ) . If a reliable automatic injector system were available, the gas chromatograph could be used during off-duty hours, when the instrument would otherwise be idle, Thus, both the cost and the time required for routine analysis of cultures and body fluids could be reduced. Furthermore, a successful approach to automatic sample injection would undoubtedly lead to further automation of the procedure and open an avenue for reduction of the data by a moderately priced computer. EXPERIMENTAL Apparatus. A modified Perkin-Elmer Model 900 gas chromatograph equipped with a 63Ni (10 mCi) detector, a modified Hamilton Autosampler, and a Beckman 10-in. potentiometric recorder connected through a latching relay system (6) was used. The instrument was fitted with dual coiled glass columns (0.3 cm x 7.3 m ) . One of the columns (nonpolar) was connected to the auto(1)
J. B. Brooks, W. B. Cherry, L. Thacker, and C. C. Alley, J. Inf. Dis.,
126, 143 (1972). (2) J . B. Brooks, C. C. Alley, and R. Jones, Anal. Chem., 44, 1881 (1972). (3) J . B. Brooks and W. E. C. Moore, Can. J. Microbioi., 15, 1433 (1969). (4) J. 8. Brooks, V. R . Dowell, D. C. Farshy, and A. Y . Armfield, Can. J. Microbioi.. 16, 1071 (1970) (5) J. 6 . Brooks, D. S. Kellogg, L. Thacker. and E. M . Turner, Can. J. Microbioi.,17, 531 (1971). (6) J . B. Brooks. D. S. Kellogg, L. Thacker, and E. M. Turner, Can. J. Microbiol, 18, 157 (1972). (7) L. V . Holdeman and J. 6. Brooks, Proc. U . S.-Jap. Conf. Toxic Micro-Organisms, 1st 7968, 278 (1970).
matic injector and was packed with 3% OV-1 on Chromosorb W 80/100 mesh (AW-DMCS H.P.) (Applied Science Laboratories, Inc.). The second column (polar) was attached to the manual injector port and was packed with Tabsorb (Regis Chemical Co.). For analyses of amines, alcohols, and hydroxy acids, the instrument was operated isothermally for 4 min at 70 "C. Then it was programmed for a linear increase of 4 "C/min to 225 "C and was held at this temperature for 32 min. For the analysis of bromomethyldimethylchlorosilane(BMDCS) derivatives of acids, the instrument was programmed for a linear increase of 3 "C/min from 125 to 225 "C, and this temperature was held for 48 min. The detector temperature was 250 "C and the injector temperature was 225 "C. Nitrogen was used as the carrier gas at a flow rate of 50 cm3/min. The modified autosampler was set for sample intervals of 88 min, a delay time of 0.5 sec, and a flush time of 2 sec. Either a 1- or 2 . 2 4 aliquot of each sample was automatically injected. When the 2.2-4 injection was used, the derivatized sample was diluted 1 : 2 . 2 in xylene to give the same concentration of sample as the 1-pl injection. Modification of the Perkin-Elmer Model 900 Gas Chromatograph. In order to convert the Model 900 fitted with a Hamilton autosampler to a dual column system, we installed a manual injector block to the right of the autosampler (Figure 1. No. 4). In addition, the end of one of the columns was modified to fit the new injector block. The temperature of the manual injector block was controlled by a rheostat and monitored by a thermocouple connected to the "hot wire detector" position of the selector switch (Figure 2, Schematic A). A four-way valve (Beckman Instruments) was installed in the manifold section to permit effluent from either column to be fed to the detector. Modification of the Hamilton Autosampler. A microswitch was installed on the pneumatic valve portion of the autosampler to permit temperature programming. Upon injection of the sample, the microswitch energizes a relay circuit which starts the programmed run (Figure 2, Schematic B ) . Longer sampling time intervals were obtained by installing an additional l.5-MQ resistor in series with the existing potentiometer in the autosampler control unit. A switch which permits use of the control module in the normal mode also was installed. The inlet system was extensively modified to correct serious leak problems. The existing round front end of the inlet block was removed flush with the square section (Figure 3A), and the modified inlet end (Figure 3B) was installed so that the center hole was in perfect alignment with the hole in the inlet block. The existing septum retainer was turned to 0.194 in. diameter on the small end and pressed into the new septum retainer block (Figure 3, C and D ) . The hole in the septum retainer was in alignment with the hole in the glass liner in the inlet block. The modified septum retainer is shown installed on the gas chromatograph (Figure 1, No. 6). A hole slightly larger than the modified septum retainer was cut in an asbestos block ('is X 2% x 3 i n , ) , and the block was placed over the end of the inlet to prevent heat loss. The carrier gas input was modified by drilling and taping the existing hole t o a l/a-in. tapered pipe thread to eliminate the Teflon O-ring. A 39/64-in.hole was drilled through the rear block to allow clearance for a modified column fitting (Figure 3E). Reagents. A useful hydroxy acid standard was prepared as follows: 360 pmol of lactic, 1.44 mmol of a-hydroxybutyric, 1.08 mmol of a-hydroxyisovaleric, 360 pmol of a-hydroxyvaleric, 720 pmol of a-hydroxyisocaproic. and 360 pmol of a-hydroxycaproic acids were added t o 2 ml of ethyl ether. One-tenth of a milliliter of this mixture was used to prepare derivatives. Eight milliliters of xylene were used as the final solvent for the derivative.
A N A L Y T I C A L C H E M I S T R Y , VOL. 45, N O . 12, OCTOBER 1973
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OF MODIFIED BLOCK
OF FRONT OF MODIFIED BLOCK
EN0 VlEW MODlFlED SEPTUM RETAINER
SIDE V l E W MODIFIED SEPTUM RETAINER
Figure 1. Modified iniet end and manual lnlector block NO. 4, manual injector block: NO. 6, modified iniet end: NO. 7, sample
viais
@
,,eS Y L O E L O I FlTTlNlj
THERMOCOUPLE
ELECTRICAL DIAGRAM
OF M A N U A L INJECTiON BLOCK
MODIFICATION TO COLUMN FlTTlNG
0
Figure 3. Diagram of modifications to the GLC inlet system
POWER SUPPLY
MICROSWITCH
RELAY
PROGRAMMER
MICROSWITCH
IPVOC RELA"
ELECTRICAL DiAGRAM OF A U T O S T A R T CIRCUIT ON P R O G R A M M E R
8
Figure 2. Electrical diagram of the manual injector block ( A ) and autostart programmer circuit (6)
'
A standard mixture of alcohols was prepared as fallows: 1.95 mmol of I-propanol, 0.716 mmol of I-butanol, 0,183 mmol of isaamyl alcohol. 0.276 mmol of I-pentanal, 2.4 mmol of 2-hexanol. 0.279 mmol of I-hexanol, 0.212 mmol of 1 - h e p t a d , 0.0253 mmol ofp-phenylethyl alcohol, and 0.115 mmol of 1-nonanal. The mixture was diluted to a total volume of 6 ml with chloroform. Onetenth of a milliliter of the standard was used to prepare the derivatives. T h e final solvent for the derivative was 1.5 ml of xylene. A useful standard mixture of acids was prepared as follows: 1.06 mmol of formic, 0.499 mmol of acetic, 0.383 mmol of propionic, 0.108 mmol of isobutyric, 0.137 mmal of butyric, 0.101 mmal of isovaleric, 0.115 mmol of valeric, 0.114 mmol of isocaproic, 0.115 mmol of caproic, 0.101 mmol of heptanoic, and 0.085 mmol ofoetanoie acid. The total volume was brought to 1.2 ml, and 0.08 ml was used to prepare the derivative. Three-tenths of a milliliter of xylene was added as a final solvent. Organisms. The strain of Clostridium bifermentans 3789 and the strain of C. sordellii 2210 were obtained from the Center for Disease Control stock culture maintained in brain storage medium (8) a t -20 "C. The source and biochemical characteristics of the two species have been described (4). The strain of C. bifermentans was urease negative, and the strain of C. sordellii was and T. M. Hawkins. "Laboratory Methods in Anaerobic Bacteriaiogy." Public Health Service Publication No. 1803.
(81 V. R. Dowell. Jr..
1968.
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1. The existing round front end was removed flush with the square section. and the piece Shown in B was installed so that the canter hole was in perfect alignment. 2. The existing Septum retainer was turned to 0.194 in. diameter on the small end and then,pressed into the seplum retainer block C
urease positive. The cultures were inoculated (ea.O.1 ml) from brain medium into 8 ml of cooked meat medium with glucose (CMG) (8)and incubated for 20 hr a t 37 'C. Procedure. For the preparation of hydroxy acid derivatives, 8 ml of CMG was acidified t o about p H 2 with 50% H&OI (vjv) and extracted with 20 ml of pesticide quality diethyl ether (Mathemn Coleman and Bell). The samples were concentrated, and double derivatives were prepared by a modification of the proeedure described previously (6). Instead of trifluoroacetic anhydride, heptafluorobutyric anhydride (HFBA) was used as follows: one drop (ca. 0.014 ml) of a 1:4 pyridine-chloroform mixture was added to the butyl ester sample, and then four drops (eo. 14 pl) of HFBA. This mixture was allowed to react for 10 min a t mom temperature and then acidic (4% HCI) and basic ( I N NaOH) washes were used as described (6) to remove reagents. The chloroform layer was carefully removed and placed in a second test tube. The chloroform was then evaporated by air to about 0.01 ml, and 0.3 ml of xylene was added as the final solvent. For the analysis of acids, the aqueous solution was acidified t o about pH 2 with 50% HsSO4, extracted with 20 ml of diethyl ether, and evaporated to about 0.05 ml. One drop (ca. 0.01 ml) of a 1:fi diethylamine-chloroform mixture and one drop (ea. 0.014 ml) of a 1:fi bromomethyldimethylchlarasilane-chlorofo~msolution were added. The test tube was sealed and the sample was heated for 1 hr a t 80 "C. The sample was cooled with water, and the chloroform was evaporated by air to near dryness to remove most of the chloroform and reagent. Three-tenths of a milliliter of xylene was added as the final solvent. For determination of alcohols, amines, and N-nitrosodimethylamine, the analyses were conducted as described (1, 2). Three-tenths of a milliliter of x y lene was substituted for ether a6 the final solvent.
RESULTS AND DISCUSSION T h e prototype 1-fil injector n e e d l e u s e d in the s t u d y d e livered reproducible injections of s a m p l e s wiithin limits of 0.9 to 1 @I. T h e use of t h e standard mixture5i proved to he of immense v a l u e for checking the performaiice and sensi-
* ANALYTICAL CHEMISTRY, VOL. 45, NO. 12. OCTOBER 1973
tivity of the GLC-autosampler system. An excellent check to determine whether or not the injector needle consistently introduced the proper amount (1 or 3 pl) of samples was to mix 0.1 ml of chloroform with 16 ml of xylene and fill a series of sample vials. For the sample injection check, we used an isothermal temperature of 70 "C and sampling intervals of 20 min. The results were compared with those obtained with samples injected manually. T o obtain reproducible injections, the system must be leak free, the Teflon tip on the plunger must be properly fitted and in good condition, and the stroke of the plunger must be set correctly. Adjustment of the plunger stroke was accomplished in our laboratory by means of a brass spacer with a finger grip 0.308 in. high with a groove 0.153 in. deep and 0.045 in. wide cut to fit on the needle shaft. Also, the depth the injector needle goes into the glass vaporizer tube must be regulated. The desired depth was obtained by gently pressing the injector needle into the inlet until it bottomed out against the restriction in the vaporizing tube. We then adjusted the cylinder rod until there was just clearance for a 1.2-mm gauge between the end of the rod and the bridge. As originally purchased, the autosampler was equipped with a solenoid which retracted the needle plunger and a spring to return the plunger to its seat in the injector needle. The original injector needle had only one flush hole (ca. 1.3 cm above the needle tip), which was opened and closed by the Teflon tip on the plunger. The spring that returned the needle plunger worked adequately when the original injector needle was used, but with a modified injector needle having a ring of holes to improve flushing, the Teflon-tipped plunger tended to stick after a few injections even though it was well fitted. The Hamilton Co. supplied, a t their own expense, an air-driven plunger retractor that permitted positive retraction and the use of a stronger spring for better return and seating of the plunger. At the concentration of components analyzed for in this study, there was no problem associated with carryover or inadequate flushing with any of the injector needles tested. At carrier pressures used in this study (52-54 psi), a perfect seal between the needle and plunger was essential. We found that metal rods, when lapped to provide a good seal, were superior to Teflon-tipped plungers. However, the return spring of the plunger had to be strong enough for a positive return of a well-fitted rod. The air-driven plunger lift was strong enough to permit the use of a strong return plunger spring, and we added a spring externally on the air-driven plunger lift to further increase the force of the return. A short plunger stroke of about 1 mm gave best results. It was possible to moderately increase the strength of the spring on the solenoid plunger lift, but the air-driven plunger lift has a much stronger lifting force. In later work, we reduced the carrier gas flow to 40 cm3/min, which permitted a reduction of carrier pressure to 38 psi. We also started our temperature program a t 80 "C instead of 70 "C. Reducing the pressure of the carrier gas by about 33% somewhat relieved the pressure on the needle. The increased starting temperature (80 "C) produced GLC profiles similar in resolution and appearance to the profile obtained by using the fast flow (50 cm3/ min) with 70 "C starting temperature. In addition to the 1-pl needles, we also evaluated two 3-pl needles. The 3-pl injection needle was more satisfactory since it permitted analysis of dilute samples which might be encountered when a type of spent culture media other than that used with C. sordellii and C bifermentans
was analyzed. In addition, with the 3-pl needle, more concentrated samples could be analyzed by making a simple dilution with xylene. One of these needles was fitted with a Teflon-tipped plunger rod. It functioned well for 3 to 4 days until the plunger was removed, but then the plunger no longer retained its seal. The second 3-111 needle was prepared in our shop by enlarging with a No. 70 drill bit a tip removed from a 1-01 needle and then silver soldering the enlarged tip back into the needle shaft. The needle shaft was fitted with a straight snug-fitting 16-gauge steel needle cleaning rod which was lapped to a fit. The first four samples injected from the needle were reduced in volume, but the plunger rod then seated itself and began to inject 2.2 to 2.4 p1 of sample. After the plunger has been seated, it often will rotate in the needle if it is removed and returned. This rotation results in an improper seal between the rod and needle. The correct position of the rod and needle can be maintained by marking the rod and needle so they may be realigned after removal from the injector block. The possibility exists that when needles with crimpedon tips are used, the tip may be dislodged from the needle shaft by the strong stroke return of the steel plunger recommended above. Further study is needed to establish how much of a problem this might be. With a small file, we fashioned a smooth-tapered shoulder between the needle tip and shaft on the 3-pl needle assembled in our laboratory and finally smoothed the surface with crocus cloth. Whether or not the smooth-tapered shoulder between the tip and needle shaft (essential for silver soldered tips to prevent septum tear) is necessary for crimped fastened tips remains to be investigated. In the needles tested, the two fill holes were no closer than about 1.5 mm above the needle tip. The tips were almost sealed, and all burrs were removed from the two fill holes. Otherwise the needle will cut the injector septum, and pieces of septum will get into the needle and obstruct the filling of the needle. A wellmade needle with a smooth tip does not have bits of septum coming out of the flush holes after injection of the sample. Other factors that affected filling of the needle were the volume of solvent containing the derivatized sample and the fit of the piston in the sample vial. When less than 300 p1 of solvent was in the vial, the amount of sample entering the needle was reduced. Most of the pistons fit the vials well enough to permit filling of the needle, but sometimes either the piston or the sample vial had to be discarded. The autosampler vials shown in Figure 1. No. 7, did not form a satisfactory seal for retaining solvents as volatile as ethyl ether and hexane. Therefore, derivatizing methods had to be devised in which a solvent, such as xylene, with a relatively high boiling point could be used. Advantages of the nonpermanent!y sealing vials were that they were disposable and easily loaded. The inlet system was modified to correct leak problems. As originally purchased, the inlet was equipped with a spring tension septum holding device. The heat of the injector (225 "C) reduced the tension of the spring, and a new septum often leaked after only two or three injections with the autosampler. The frequent changing of the septum posed serious problems. because removing pressure from the column to correct for a leaky septum often resulted in base-line bleeding that could only be corrected by baking out the column. The modified septum retainer which screws on (Figures 1 and 3) permitted as many as 150 runs or more to be made before septum change. To obtain the necessary tension with the modified septum retainer, we heated the inlet to 225 "C with the septum
A N A L Y T I C A L C H E M I S T R Y , VOL. 45, NO. 12, OCTOBER 1973
2085
E
L--+t--t 5 10
L 5
'25.:
+
.-
IO
---------------
t~
15
t5
20 TIME
~---+-.--* 30 35
--
225
c
Figure 4. Gas chromatograms of standard mixtures of acids and alcohols automatically injected on a 3% OV-1 column Curve A, brornomethyldimethylchlorosilane derivatives of a standard mixture of acids: curve B, HFBA derivatives of a standard mixture of alcohols: curve C , butyl esters-HFBA derivatives of a standard mixture of hydroxy acids: hyd. = hydroxy: I.V. = isovaleric, V = valeric, I.C. = isocaproic, C6-2 = 2-hexanol, C6-1 = 1-hexanol, and C7 = 1-heptanol
loose and then tightened the retainer by hand (but not enough to break the glass liner of the inlet). A protective cover was used to avoid burning the hands. At daily intervals for a few days and then a t longer intervals, the septum retainer was tested by hand for correct tension. Often leaks were corrected by applying increased tension on the septum retainer. Because of its durability, a Perkin-Elmer Model 900 injector septum was substituted for the Hamilton septum in the modified inlet system. As purchased, the septa were too large for the inlet system, but the correct fit was obtained by trimming the septa with a No. 6 cork bore. In general, the methods used here permitted detection of the compounds in the standard mixtures a t the low nanomole and picomole levels. The standard curve obtained (Figure 4) is typical of the high resolution and efficiency of the OV-1 column for the derivatized compounds. The Tabsorb column is also a very efficient column for these compounds (1). In most instances, we used the Tabsorb column to verify tentative identifications of compounds made on the OV-1 column. Frequent use was made of the standard mixtures to test the performance and sensitivity of the GLC-autosampler system. In addition, a standard mixture of chloroform in xylene, described above, was included a t the beginning and end of each series of analyses to test for sample application and for possible carry-over. Figure 5 demonstrates the use of the modified GLC-autosampler to study amine products produced by a strain 2086
c
10
~
15
t
+
t
t
+ -+-
20
25
10
35
LO
TIME , m , n ,
225
45
--t-+* 50
55
60
65
c
Figure 5. Gas chromatograms of automatically injected samples of HFBA derivatives prepared from pH 10 chloroform extracts of cultures which were incubated 20 hr on cooked meat medium supplemented with glucose Samples were analyzed on a 3% OV-1 column: curve A, control medium: curve B, Clostridium sordellii; curve C , Clostridium bifermentans; M, compounds detected in the control medium
of C. bifermentans 3789 and of C. sordellii 2210. The organisms were incubated for 20 hr in CMG medium. The amine profile produced by the strain of C. bifermentans (Figure 5 , curve C) was different from that produced by the strain of C. sordellii (Figure 5 , curve B). GLC profiles of both organisms differed from that of the control CMG medium (Figure 5 , curves A, B, and C). Repeated analyses of the same two organisms showed that reproducibility was good. Since these organisms have previously been studied extensively for amines and other compounds ( 5 ) , we made no attempt to study additional strains of the species. Production of P-phenylethylamine and tryptamine by C. bifermentans and lack of the production of these compounds by C. sordellii were the main differentiating criteria between these species. Our data show that C. sordellii 2210 produced isoamylamine, putrescine, and peaks 6 and 7, whereas C. bifermentans 3789 produced (3-phenylethylamine, tryptamine, and peaks 9, 10, 12, and 14. C. sordellii also produced some P-phenylethylamine which was not detected by less sensitive flame detectors ( 5 ) .Production of putrescine and isoamylamine by C. sordellii has not been previously reported. Because of their highly polar nature, putrescine and cadaverine are difficult to extract from aqueous solutions; however, when they are reacted with HFBA, they form highly electron capturing derivatives which make them detectable a t concentrations too small for flame detectors. The modified GLC-autosampler system performs well with the electron capture (EC) detector. The selective sensitivity offered by the EC makes its use highly desirable for analysis of very complex mixtures. When new col-
ANALYTICAL CHEMISTRY, VOL. 45, NO. 12, OCTOBER 1973
umns are properly conditioned by overnight baking (or longer if necessary) a t 245 "C before use, a good base line is maintained. The number of analyses that could be made by fulltime use of one gas chromatograph with the modified automatic injector system (for a 7-day period) would be equivalent to the number that can be made by four gas chromatographs (on an 8-hr, 5 days per week basis). An additional advantage of the automatic injector system is that it eliminates the need for washing the injector needle manually after each analysis. The incorporation of the ideas presented here into the design and manufacture of the autosampler would be highly desirable. The manufacturer of the autosampler agrees that basic changes in design are needed and plans to make changes along the lines described above. A reliable system for automatic injection of samples constitutes a significant step toward the systematic analysis of bacterial metabolites and volatile components in body fluids by gas chromatography.
ACKNOWLEDGMENTS The authors thank Charles E. Smith and James A. Moore for their helpful suggestions and skills in designing and installing the modifications made to the autosampler and injector needles. We would also like to acknowledge the cooperation of the Hamilton Co. for supplying us with prototype injector needles and the air-driven needle rod retractor for evaluation, and we thank Frances S. Thompson of the Anaerobe Laboratory for supplying the strains of C. bifermentans and C. sordellii used in the study.
Received for review October 3, 1972. Accepted April 27, 1973. Use of trade names is for identification only and does not constitute endorsement by the Public Health Service or by the U.S. Department of Health, Education, and Welfare.
Thermodynamics of Molecular Association by Gas-Liquid Chromatography A Comparison of Two Experimental Approaches Hsueh-Liang Liao, Daniel E. Martire, and James
P. Sheridan
Department of Chemistry, Georgetown University, Washington, D.C. 20007
Two approaches have been developed and used for the gas-liquid chromatographic measurement of organic complex formation constants, one in our laboratory and the other in Purnell's. The former method requires far less time, but utilizes additional assumptions. In order to compare the two approaches, three acceptor-donor test systems were studied at 40.0 "C. The electron acceptors were CHC13, CH2C12, and CH2Br2, and the electron donor was di-n-octylmethylamine. The association constants obtained through the two different approaches were in excellent agreement, thus supporting the validity of our additional assumptions (including that of 1 :1 complex formation).
It is now generally recognized that gas-liquid chromatography (GLC) is an effective and advantageous method for studying the thermodynamics of nonelectrolytic solutions. The most recent application is to the accurate and rapid measurement of association constants of organic complexes in nonaqueous solution. The many advantages of the GLC method over the commonly used spectroscopic ones have been described (1-3). Off-setting these, no obvious disadvantages which are not common to other methods can be discerned, except the requirement that of the electron acceptor-electron donor pair, one must be volatile and the other nonvolatile. A classification scheme has been suggested ( I ) for the various more probable GLC ap(1) J. H . Purnell. ' G a s Chromatography," A. B. Littlewood, Ed., Elsevier, Amsterdam, 1966, p 3.
proaches. Recently, two such approaches were independently developed and utilized. A comparison of these is the subject of the present paper. Martire and Riedl (2) proposed a quantitative GLC method.[class B(ii) according to Purnell ( I ) ] for the study of hydrogen bonding. I t employs two columns-one containing the electron donor liquid phase and the other a "reference" liquid phase of approximately the same molecular size, shape, and polarizability as the electron donor. Association constants, enthalpies, and entropies of hydrogen-bond formation were obtained for eight alcohols with di-n-octyl ether and di-n-octyl ketone; the reference liquid was n-heptadecane. More recently, this method was used to study the association (hydrogen-bond and chargetransfer) of various haloalkanes with di-n-octyl ether and di-n-octyl thioether ( 3 ) and with di-n-octyl methylamine and tri-n-hexylamine ( 4 ) ; the reference liquid phase was n-octadecane. Cadogan and Purnell ( 5 ) measured the formation constants of, and thermodynamic parameters for, complexes formed between aromatic electron donors and di-n-propyl tetrachloro phthalate. Their method [class A( ii) according to Purnell ( I ) ] requires the use of several (5 or 6) columns containing different concentrations of additive (electron acceptor in this case) in an inert solvent. Recently, they (2) D. E. Martire and P. Riedl, J . Phys. Chem.. 72, 3478 (1968). (3) J. P. Sheridan, D. E. Martire, and Y. B. Tewari, J. Amer. Chem. SOC.. 94, 3294 (1 972), (4) J. P. Sheridan, D. E. Martire, and F. P. Banda, J. Amer. Chem. SOC., 95, 4788 (1973). ( 5 ) D. F. Cadogan and J. H. Purnell, J. Chem. SOC.A . 1968, 2133.
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