Determination of Catechol-ContainingCompounds in Tissue Samples by Gas-Liquid Chromatography E. L. Arnold1 and Roddey Ford 6570th Aerospace Medical Research Laboratory, Aerospace Medical Dioision AFSC, Wright-Patterson Air Force Base, Ohio 54433 A method has been developed for the extraction, separation, and measurement of 3,4-dihydroxyphenylalanine (dopa) and several of its metabolites in brain tissue. These compounds are extracted from tissue homogenates by adsorption on activated aluminum oxide. Lyophylization of the alumina prior to elution with acidic methanol reduced loss and provided anhydrous conditions for subsequent derivative formation. Pentafluoropropionyl derivatives were measured by gas-liquid chromatography employing an electron capture detector. Relative recovery of dopa, dopamine, norepinephrine, and 3,4-dihydroxyphenylacetic acid added to tissue samples averaged in excess of 90%; relative standard deviation = 15.43%. Dopamine concentrations as low as 0.05 pg/g tissue could be accurately determined using 500-mg tissue samples. Analysis time for six samples averaged 4-5 hours.
SINCEITS INCEPTION in 1952, gas-liquid chromatography (GLC) has been employed for the analysis of a wide variety of compounds of biological importance. Moreover, the applications of GLC have been greatly expanded in the past decade by the use of derivatives which now allow the GLC of an increased number of different compounds (1-5). While the use of GLC has been incorporated into numerous analytical procedures, practical application of this powerful tool in the area of biogenic amine analysis has been relatively neglected. Several investigators have, however, formulated procedures for the formation and chromatography of a variety of derivatives of the biogenic amines, particularly the catecholamines, and have separated model solutions of these compounds (6-9). Recently an assay has been devised employing gas chromatography-mass spectrometry of volatile catecholamine derivatives (10). While many derivatives have been described which will impart sufficient volatility to allow separation and measurement of 3,4-dihydroxyphenylalanine (dopa) and its metabolites, preliminary procedures for sample purification have not been developed which will allow full use of the potential sensitivity of GLC analysis using electron capture detection (ECD). Present address, Department of Life and Behavioral Sciences, USAF Academy, Colo. 80840.
(1) W. J. A. Van den Heuvel, J. Cliromatogr., 27,85 (1967). (2) Y . Sasaki and T. Hasizume, Ami. Bioclitnz., 16,1 (1966). (3) C. W. Gehrke and D. L. Stalling, Srpur. Sci.,2, 101 (1967). (4) P. I. Jaakonmaki and J. E. Stouffer, J . Gas Chromatogr., 5 , 303 (1967). ( 5 ) M. L. Taylor and E. L. Arnold, ANAL.CHEM., 43, 1328 (1971). (6) M. G. Horning, A. M. Moss, E. A. Roucher, and E. C. Horning, A w l . Lett., 1, 311 (1968). (7) D. D. Clarke, S. Wilk, and S. E. Gitlow, J. Gas Cliromatogr., 4,310(1966).
(8) S. Kawai, T. Nogatsu, T. Imanari, and Z. Tamura. Clirm. Pliurm. Bull. Jap., 14,618 (1966). (9) A. C. Moffat and E. C. Horning, Bioclwm. Biopliys. Acta, 222, 248 (1970). (10) S. H. Koslow. F. Cattabeni, and E. Costa, Screricc, 176, 177 (1972).
The objective of this study was to investigate, evaluate, and select procedures which could be integrated into a general method for the measurement of dopa and its metabolites in biological materials by GLC. EXPERIMENTAL
Apparatus. A Varian Model 2100 Gas Chromatograph equipped with an electron capture detector (250 mCi tritium, dc mode) was employed. Columns were glass U-tubes ( 2 mm i.d. X 12 ft) which had been silanized with dimethyldichlorosilane prior to use. All column packings were prepared using a solvent evaporation technique in an all-glass rotary flash evaporator. Column packings were sieved prior to use to obtain a narrow particle size range. The solid support was 60/80 mesh Gas-Chrom Q coated with either 3 OV-l,3 SE-52, or a 1 :1 w/w mixture of these two phases (Applied Science Labs). Conditions for the chromatography were: temperature: injector, 165 "C; column, 145 "C; detector, 180 "C; carrier gas (prepurified nitrogen, J. T. Baker Chemical Co.) flow = 60 cm3/min. Reagents. Methanol, acetonitrile, and benzene were chromato-quality reagents (Matheson, Coleman and Bell). Aluminum oxide, Woelm neutral activity grade 1, was activated by the procedure of Anton and Sayre (11). Perchloric acid, 70 (Mallinckrodt), disodium EDTA (Cambridge Chemical Products), and sodium metabisulfate (Sigma) were CP grade. Trifluoroacetic anhydride, pentafluoropropionic anhydride, and heptafluorobutyric anhydride (Pierce Chemical Co.) and anhydrous hydrogen chloride gas (Matheson Gas Products) were used as received from the supplier. DLNorepinephrine HC1 salt, B grade; dopamine HCI, A grade, L-epinephrine, USP, (all Calbiochem); ~-3,4-dihydroxyphenylalanine (Hoffmann-LaRoche, Inc.); 3,4.-dihydroxyphenylacetic acid (K & K Chemicals); 3,4-dihydroxyhydrocinnamic acid (Aldrich Chemicals) were used without additional purification. Procedures. For the initial characterization, derivatives were prepared by dissolving 1-10 mg of each of the catechol compounds in 0.01N HCI; aliquots of these solutions were then lyophylized prior to derivative formation. Acidic compounds, dopa and 3,4-dihydroxyphenylaceticacid (dopac) were dissolved in 2N anhydrous HCI in methanol and stirred with a small glass-encased, magnetic stirrer for 30 minutes at room temperature to effect the esterification of the carboxylic acid groups contained in these compounds. The HCI: methanol was removed under vacuum. Fluoroacyl derivatives were formed by dissolving the residues in 1 ml of a 20z v/v solution of the appropriate fluoroacyl anhydride in acetonitrile and allowing this solution to stand at room temperature for 10 minutes. The acylating agent was removed under a stream of dry nitrogen and the residues were dissolved in an appropriate volume of benzene for chromatography. Structures of the various derivatives were ascertained by mass spectrometry of the purified solids or oils utilizing a CEC Model 21-491 Mass Spectrometer equipped with a solid sample probe. Fluoroacyl derivatives of catechol compounds obtained from biological samples were prepared in a similar manner.
z
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(11) A. H. Anton and D. F. Sayre, J . Pl7armacol.. 138, 360 (1462).
ANALYTICAL CHEMISTRY, VOL. 45, NO. 1, J A N U A R Y 1973
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100
Table I. Retention Data for Fluoroacyl Derivatives of Dopa Metabolites Retention Index (M.U.) on 3
Compound Epinephrine Norepinephrine Epinephrine (aliphatic methyl ether) Norepinephrine (aliphatic methyl ether) Dopamine Dopa (methyl ester) Dopac (methyl ester)
a
Derivative TFA PFP HFB TFA PFP HFB TFA PFP HFB TFA PFP HFB TFA PFP HFB TFA PFP HFB TFA PFP HFB
3z SE-52 15.64 15.80 16.80 15.56 15.60 16.60
15.62 15.92 16.88 15.38 15.62 16.84
16.32
16:24
l6:24
16.20
15:96 15.80 16.80 16.52 16.84 18.00 14.19 14.78 15.60
16108 15.96 17.02 16.50 16.84 17.51 14.30 14.74 15.72
. . .a ...
3 z
ov-1
... ...
zSE-52 ov-1;
1:1 w/w 15.66 15.88 16.76 15.50 15.66 16.58 16.10 16.56 17.92 16.00 16.30 17.30 15.76 16.12 16.74 16.56 16.86 17.89 14.28 14.78 15.66
. . . Not determined.
Since in these samples the catechol compounds had been extracted into an HC1 :methanol solution, all carboxylic acid groups were esterified during the extraction procedure. After the solvent (HCI :methanol) had been removed under vacuum, fluoroacyl derivatives were prepared as previously described. Tissue samples were prepared by homogenizing 500 mg of frozen brain tissue (Macaca mdatta) in 2 ml cold 0.4N perchloric acid in a Tenbroek homogenizer. The homogenizer was washed twice with additional cold 0.4N perchloric acid to obtain a final volume of 4.5 ml of homogenate. One-half milliliter of a solution containing 50 mg/ml sodium metabisulfate was added as an antioxidant, and the homogenate was centrifuged at 1570 X g for 30 minutes. The supernatant liquid was then used as the sample. For extraction, 200 mg of activated aluminum oxide (alumina) were placed in a 10-ml freeze-drying vial. Four milliliters of sample (tissue homogenate supernatant) were added to the alumina along with 1 ml of a solution containing 100 mg/ml disodium ethylenediamine tetraacetate (Na2EDTA). The pH of the mixture was quickly adjusted to 8.6 with 0 . 5 N sodium hydroxide and maintained at this pH for 5 minutes while stirring with a magnetic stirrer. The alumina was allowed to settle by gravity, and the supernatant liquid was removed with a pipet and discarded. The alumina was washed four times using 3-ml aliquots of doubly distilled water. A 0.2-ml volume of 0.1N HCI was added to the moist alumina, the mixture was frozen in a Dry Ice-acetone bath and all water removed by lyophylizing with a Virtis Model 10-010BA freeze-dryer. The tops of the individual drying vials were covered with circles of Whatman No. 1 filter paper to prevent loss of alumina during freeze-drying. Following freeze-drying, the catechols were removed from the alumina by stirring with 3 ml of 2 N anhydrous HCI in methanol for 30 minutes. A 2-ml aliquot of this HC1:methanol solution was then dried under vacuum and fluoroacylated. When tissue homogenates containing known amounts of catechol compounds were desired, these compounds were added as aqueous solutions to the 0 . 4 N perchloric acid prior to homogenizing the tissue. An internal standard, 3,4dihydroxyhydrocinnammic acid [(HO)2C6H3CH2CH2COOHl 86
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Figure 1. Mass spectra of the pentafluoropropionyl deriva tives of dopamine and the methyl ester of L-dopa (DHC) was added as an aqueous solution to the samples prio to extracting with alumina. Calibration standards were prepared by combining mea sured volumes of aqueous stock solutions of the catecho compounds with a constant volume of a stock solution of thi internal standard. This provided solutions in which thc concentration of the catechol compounds varied in relation to i constant concentration of the internal standard. Thesc calibration solutions were lyophylized and derivatives wen formed in the same manner as outlined previously. Cali bration curves were drawn by plotting the ratio: GLC peal area of the individual catechol compound/GLC peak are: of the internal standard, against the concentration of thr catechol compound in the calibration solution. The con centrations of internal standard were identical in both tht samples to be analyzed and the calibration solution. RESULTS AND DISCUSSION
Chromatographic Characteristics of Fluoroacyl Derivative! of Catechol Compounds. The trifluoroacetyl (TFA), penta. fluoropropionyl (PFP), and heptafluorobutyryl (HFB) deriv. atives of dopa and four of its metabolites were formed anc chromatographed. Mass spectra were also obtained for the PFP derivatives of all compounds studied. Figure 1 shows the mass spectra of PFP derivatives of dopamine and thc methyl ester of dopa. Both spectra show minor moleculai ion peaks and a base peak arising from cleavage to give a fluorinated amide fragment. Other species (i.e., fragments arising from incompletely fluoroacylated molecules) were undetected in the spectra. Mass spectra of the PFP derivatives of norepinephrine, epinephrine and dopac were consistent with results obtained with dopamine and dopa; methyl ester. The only exception to this behavior was discovered in the latter portion of this study. When a solution containing all five of the catechol compounds was esterified using 2 N anhydrous HCI :methanol prior to fluoroacylation, epinephrine, and norepinephrine, showed increased chromatographic retention times. Since these two compounds contain aliphatic hydroxyl groups, the formation of a methyl ether was suspected. The formation of this ether would prevent the fluoroacylation of this group and would be expected to increase the retention time of the resulting compound.
ANALYTJCAL CHEMISTRY, VOL. 45, NO. 1, JANUARY 1973
Subsequent mass spectral analysis has confirmed the existence of these aliphatic methyl ethers. No evidence was obtained which would indicate the methylation of aromatic hydroxyl groups. Conversion of norepinephrine to a fully fluoroacylated derivative of its methyl ether was quantitative by the procedures employed. Methylation of epinephrine appeared to be less than quantitative since a small peak with retention time corresponding to the tetra-fluoroacylated derivative appeared in chromatograms of this compound. Retention indices for the derivatives employed are shown in Table I ; these retention indices are expressed in methylene unit (M.U.) values (12). On columns employing either OV-1 or SE-52 as a single liquid phase, a complete separation of all five of the compounds was not possible, regardless of the procedure used to form the derivatives. However, when a 1 :1 mixture of these two liquid phases was used, an acceptable separation of all compounds as either PFP or TFA derivatives was obtained. An unacceptable overlap of at least two of the compounds occurred when HFB derivatives were used. Several very polar liquid phases, EGSP-Z (ethylene glycol succinate silicon gum), and OV-210 (Fluorosilicone oil) were also tested. While separation efficiencies were superior to either OV-1 or SE-52, hydrolysis of the fluoroacyl derivative on the columns was unacceptable. Table I1 illustrates the sensitivity of the electron capture detector to these fluoroacyl derivatives in benzene solution. Solvents other than benzene were also employed during the course of this study; however, other solvents such as hexane or acetonitrile led to increased degradation of the derivatives. As had been indicated by work in other laboratories (13), TFA derivatives were much more susceptible to hydrolysis than either PFP or HFB derivatives. The ECD response to the TFA derivatives was also considerably less than to the PFP or HFB derivatives. For instance, the minimum amount of dopamine which could be detected as the PFP derivative was 4.0 X gram; but if the TFA derivative of dopamine was used, the minimum detectable was 3.0 X 10-l1 gram. It should be noted that when an aliphatic methyl ether of norepinephrine was formed prior to fluoracylation, a gain in sensitivity was recorded. This unexpected development is not fully understood but may be due to a favorable change in the geometry of the molecule. The aliphatic methyl ether of epinephrine showed only a slight increase in sensitivity over the fully fluoroacylated form. Analysis of Tissue Samples. To fully exploit the high sensitivity of the ECD to fluoroacyl derivatives of the catecholcontaining compounds, a highly selective means for their removal from biological material must be employed. Activated aluminum oxide has a large adsorption affinity for compounds containing a catechol nucleus and has been employed extensively in analytical methods for the measurement of certain catecholamines (11). In fluorimetric procedures used for catecholamine measurement, these adsorbed compounds are subsequently eluted from the alumina with acid and measured by fluorimetry in this acid eluate. Since the formation of fluoroacyl derivatives for GLC analysis required anhydrous conditions, a modification to the above procedure was required. Two procedures were investigated in this study in an attempt to use alumina extraction in conjunction with GLC measurement of fluoroacyl derivatives. The first simply in_--_____
(12) E. C. Horning, M. G. Horning, N. Ikekawa, E. M. Chambaz, A. Jaakonmaki, and C. J. W. Brooks, J . Gcls Chrornatogr., 5, 283 (1967). (13) E. Anggard and G. Sedvall, ANAL.CHEM., 41, 1250 (1969).
Table 11. ECD Response of Fluoroacyl Derivatives of DOPA and Its Metabolites' Detector response, Compound Derivative cm2/ng* TFA 0.271 Epinephrine PFP 0.961 HFB 0.912 TFA 0.169 Norepinephrine PFP 0.831 HFB 0.878 0.508 TFA Dopamine PFP 1.423 HFB 1.755 TFA 0.377 Norepinephrine PFP (aliphatic methyl 1.114 HFB 1.341 ether) TFA 0,209 Epinephrine PFP 0.981 (aliphatic HFB 0,957 methyl ether) TFA 0.210 Dopa (methyl ester) PFP 0,446 HFB 0.475 Dopac (methyl ester) TFA 0.942 PFP 1.460 HFB 1.692 OV-1: SE-52 (1 : 1 w/w) column. Determined using 3 b Chromatograph electrometer attenuation = 16 X a.f.s. Table 111. Recoveries of Dopa and Three of Its Metabolites Added to Brain Homogenates Amount Average amount added, pg/g recovered, Percentage Compound tissue pg/g tissue" recovery 13.63 13.41 i 0 . 4 0 98.4 Dopamine 13.45 12.94 i 0.46 96.2 Norepinephrine 12.00 10.92 i 0 . 4 6 91 .O Dopa 8.36 7.72 i 0 . 3 4 92.3 Dopac 5.45 5.25 f 0 . 2 0 96.3 Dopamine 5.38 5 . 1 9 I. 0 . 2 2 96.4 Norepinephrine Dopa 4.18 4.32 i 0 . 1 6 89.7 Dopac 4.10 4.94 f 0.24 96.1 Dopamine 1.09 1.06 f 0.03 97.6 Norepinephrine 1.08 1.02 i0.05 94.5 Dopa 0.96 0 . 8 9 i 0.08 93.0 Dopac 0.91 0.82 f 0 . 0 4 90.0 Dopamine 0.55 0.51 i 0.03 94.3 Norepinephrine 0.54 0.52 i0.02 97.0 Dopa 0.48 0.43 i 0.04 90.0 Dopac 0.42 0.38 f 0.02 89.1 Dopamine 0.22 0.21 i 0.01 97.2 Norepinephrine 0.22 0 . 2 2 =t0 . 0 2 99.5 Dopa 0.19 0.17 i0.02 87.5 Dopac 0.23 0 . 2 3 i 0.01 100.8 a Mean value of 6 analyses f standard deviation.
volved the removal of water from the acid eluate by lyophylization prior to derivative formation; the second involved the lyophylization of the alumina prior to elution of the adsorbed compounds, followed by elution with 2N anhydrous HCl :methanol. The second procedure was much more satisfactory than the first for several reasons. One advantage is derived from the apparent tendency of alumina to retain a degree of acidity even after drying, which prevents the oxidation of the compounds during the freeze-drying step. A second advantage was a more complete and, therefore, more reproducible elution of the catechol compounds. Recoveries of catechol compounds from aqueous solutions ranged from 79-90z in the latter procedure, while recoveries of as low as 43% were experienced in the former. An added advantage
ANALYTICAL CHEMISTRY, VOL. 45, NO. 1, JANUARY 1973
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TIME (MIN)
Figure 2. Chromatogram of a brain tissue homogenate sample containing added dopamine (DM), norepinephrine (NE), dopa, and dopac Column: 3 OV-1: SE-52on Gas-Chrom Q; column temp., 145 C; N2flow, 60 cc/min
C AUJ ATE (1)
I N 1 CAP (21
PUTAMEN
131
Figure 3. Distribution of norepinephrine and dopamine in discrete brain regions of untreated animals of the second procedure is the simultaneous esterification of carboxylic acid groups during the elution step, thereby reducing the time necessary for a total analysis. Pentafluoropropionyl (PFP) derivatives were chosen for tissue analysis due to their stability, ECD sensitivity, and chromatographic separation efficiency. For quantitative analysis of tissue catechols, 3,4-dihydroxyhydrocinnamic acid (DHC) was used as an internal standard. DHC is quantitatively adsorbed onto alumina and can be added to the sample prior to adsorption and carried through the entire procedure. The PFP derivative of the methyl ester of this compound is completely separated from the other compounds of interest under the conditions employed. The sensitivity of the ECD to the internal standard is approximately one-fourth of that of PFP-dopamine. Table I11 illustrates the recoveries of four catechol compounds which had been added to monkey brain homogenates. 88
The concentration ranges employed were chosen so that the naturally-occurring levels of these compounds in various portions of the brain would be encompassed. Brain sections from the cortex region were utilized for this experiment because the catechol content of this region is normally very low. The relative amount recovered and percentage recovery were obtained by calculating the ratio of the peak areas of each of the compounds to the peak area of a known amount of internal standard and comparing this ratio with previously prepared calibration curves. When calculated in this manner, relative recovery of catechol compounds added to these tissues averaged in excess of 90% with a mean relative standard deviation of +5.43x for all four compounds. Absolute recoveries averaged 74% for the same four compounds. Of the four compounds, the catechol amino acid, dopa, produced the lowest percentage recoveries. This may be due in part to difficulties encountered in accurately measuring the broader chromatogram peaks obtained for this compound. When 500-mg samples of brain tissue were used, dopamine levels as low as 0.05 pg/gram wet tissue could be accurately determined (&20%). The time required for the analysis of six samples was 4-5 hours or less than 1 hr/sample. Figure 2 is a chromatogram of one of the analyses shown in Table 111. Mass spectral analysis of the derivatives of the compounds shown in this chromatogram confirmed that both of the aromatic hydroxyl groups on each compound had been quantitatively converted to pentafluoropropionates. The amine moieties of norepinephrine, dopamine and dopa, were also quantitatively converted to pentafluoropropionylamides. The use of anhydrous HCl :methanol as an eluent resulted in the conversion of the carboxylic acid groups of dopa and dopac to their respective methyl esters and the formation of a methyl ether from the aliphatic hydroxyl group of norepinephrine. No other derivatives were detected when samples of these four peaks were collected and analyzed by mass spectrometry. In addition to the analysis of tissue samples containing added catechol compounds, tissue samples obtained from areas of the brain normally rich in certain catechol compounds were analyzed by the GLC procedure. Figure 3 illustrates the results of these analyses. Samples were obtained by carefully sectioning the frozen brains and removing small sections from three discrete regions of different tissue typecaudate nucleus, internal capsul, and putamen. Analysis was performed on samples weighing from 250-500 mg each. In these tissues, only two catechol compounds, dopamine and norepinephrine, were of sufficient concentration to allow accurate measurement when relatively small samples were used. The use of larger sample sizes would likely allow the measurement of all four compounds but would also result in overlap between different anatomical regions of the brain. These results, however, serve to illustrate the applicability of the technique for the measurement of brain tissue levels of these catechol-compounds in normal animals. While this study was limited to tissue samples, it suggests the feasibility of a GLC method for the analysis of these compounds in other biological material. Preliminary studies in this laboratory have indicated that measurement of norepinephrine, epinephrine, dopamine, dopac, and dopa in normal blood plasma, urine, and spinal fluid is possible with minimum modification of this technique. Further investigations are currently in progress. The precision, accuracy, and rapidity of this GLC methodology provide an efficient means for the measurement of certain catechols in tissue samples. The sensitivity of the
ANALYTICAL CHEMISTRY, VOL. 45, NO. 1, JANUARY 1973
method compares favorably with other procedures currently in use, particularly when required sample size is taken into consideration. Further refinement and amplification of the basic methodology should increase both its clinical and research applications.
The Pennsylvania State University, September 1971 ; the helpful suggestions of Rosemary Schraer, Department of Biochemistry are also gratefully acknowledged.
ACKNOWLEDGMEW
cording to the “Guide for Laboratory Animals Facilities and Care,” 1965, prepared by the Committee on the Guide for Laboratory Animal Resources, National Academy of Sciences-National Research Council. Further reproduction is authorized to satisfy needs of the U. S. Government.
The authors gratefully acknowledge M. L. Taylor for his support in the area of mass spectrometry. A portion of this research is contained in the Ph.D. dissertation of E. L. Arnold,
RECEIVED for review May 22, 1972. Accepted August 23, 1972. The experiments reported herein were conducted ac-
Extraction and Concentration of Organic Solutes from Water Marvin C. Goldberg and Lewis DeLong
US.Geological Survey, Water Resources Division, Denver Federal Center, Denver, Colo. 80225 Mark Sinclair 4dolph Coors Company, Denver, Colo. 80401 A continuous extraction apparatus is described. I t extracts and simultaneously concentrates organic solutes from water. Any immiscible solvent can be used in this apparatus if the solute will partition between the solvent and water. A concentration factor >f up to lo5 is obtained with this technique. The dipole moment difference between the solute and solvent is iemonstrated to be an index of the extraction effiiency. Optimum extraction of a given molecular species may be obtained by use of this index. SEVERAL DESIGNS of continuous solvent extraction apparatus ire discussed in the literature (I). An in-depth evaluation of ;olvent extraction efficiencies and a rationale for controlling he rate and amount of extraction has not been clearly stated. rhis discussion evaluates the continuous extraction apparatus jescribed in ( 2 ) , gives extraction efficiencies for two solutes n water, and describes an index method for the selection of iptimum solvent-solute combinations. According to recent studies (3), organic materials containng many types of reactive groups are present in environnental waters. Because of their relatively low concentraions, it is experimentally difficult to analyze for these maerials. The analyst has two alternatives; first, to extend the imits of analytical sensitivity or second, to concentrate the naterials to be analyzed. The latter course is ofttimes :asier and can often be conveniently accomplished by coninuous liquid-liquid extraction. In this study, the latter :ourse is pursued.
EXPERIMENTAL Apparatus. EXTRACTOR OPERATION.An extraction unit :onsisting of modified liquid-liquid extractors is described 2elow (see Figure 1). 1) G. H. Morrison and H. F. Frieser, “Solvent Extraction in Analytical Chemistry,” John Wiley and Sons. New York, N.Y., 1966, pp 86-105. 2) M. C . Goldberg, L. DeLong and L. Kahn, Emiron. Sci. Tec/7~v~/., 5, 161-2 (1971). 3) A. K . Burnham, G. V. Calder, J. S. Fritz, G. A. Jurnk, H. J. Svec, and R. Willis, ANAL.CHEM., 44, 139 (1972).
The extractor design is a two-cycle system. The water cycle is continuous flow. Water enters at A and exits at B. In so doing it passes through chamber C which is half-filled with solvent. A stopcock, D can be provided to regulate the water flow rate. The second cycle is a solvent cycle. This system is closed in that the solvent cycles exclusively in the extractor. The 500-ml bulb E contains pure, nonmiscible, organic solvent. This solvent is gently boiled and vapor rises in area F up through the upper extractor tube G into reflux condensor H . At this point it is liquified and falls off of drip tang I into funnel J . The long funnel stem sets up a hydraulic head sufficient to drive the solvent through a porous glass frit at K. The frit homogenizes the solvent resulting in fine beadlike particles which form as an emulsion as they rise through the water in chamber C. This emulsion extracts organic solutes during the period of water-solvent contact. The emulsion separates in the extractors’ lower neck L and the solvent-solute mixture spills over connection tube F into boiling flask E. The closed solvent system cycles fresh solvent from the boiling flask into the extractor. After extracting the organic solutes, the “loaded” solvent is returned to the boiling flask thus collecting and concentrating the extracted solutes in flask E but always supplying fresh solvent to the extraction chamber at C . Figure 2 depicts the solvent-heavier-than-water extractor which is similar in principle of operation to the solvent lighter-than-water extractor but somewhat different in design. It operates as follows : Water enters at M and exits at N . The stopcock 0 (optional) regulates flow rate. While in the lower neck of the extractor P, the water flows through the extraction solvent. The solvent cycle, which is closed to the extractor, starts by the solvent being vaporized in bulb Q. The vapor rises in arm R to condensor S where the vapor liquifies and drops from drip tang T to funnel U. The solvent under a hydraulic head, is forced through frit Y where it emulsifies and drops through the upper extractor neck W . Extraction of the water takes place at the interface between the emulsified solvent and the water. A stirring bar at X (optional) stirs the solvent-water mix. The solvent separates in the lower half of the extractor and flows through tube Y into bulb Q.
ANALYTICAL CHEMISTRY, VOL. 45, NO. 1, JANUARY 1973
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