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. ACKNOWLEDGMEW
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,
The Pennsylvania State University, September 1971 ; the helpful suggestions of Rosemary Schraer, Department of Biochemistry are also gratefully acknowledged.
RECEIVED for review May 22, 1972. Accepted August 23, 1972. The experiments reported herein were conducted according 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.
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
89
Table I. Extraction Extractor efficiency, cy ___ 1 Solute Solvent 2 3 4 Sum Valueb Toluene Hexane 22.02 20.72 21.99 7.71 72.44 3.3 0.34 1.04 1.75 1.67 2-Pentanol Benzene 4.80 2.9 10.72 7.61 6.41 Hexane 13.53 2-Pentanol 38.27 4.03 14.53 3.09 84.04 ... Toluene Trichlorotrifluoroethane 101.66 6.6 73.17 Toluene Carbon tetrachloride 14.11 4.34 ... 91.62 2.58 76.36 Chloroform 34.11 7.6 ... 118.07 Toluene 4.10 70.49 110.35 2-Pentanol Trichlorotrifluoroethane 28.25 11.61 ... 8.20 9.79 42.90 30.53 ... 83.22 2-Pentanol Chloroform 3. 1 Extraction efficiency is the amount of sclute recovered from each extractor divided by the total amount of solute in the test solution. Each extractor was part of a serially coupled extraction train. Extractor 1 was the first extractor to contact the feed water. The effluent from extractor 1 was the influent for extractor 2 and so forth.
7
Ln I J
~
G
A - m
L
C
ciency of each of 6 extraction solvents. Both the toluene and the 2-pentanol were reagent grade. Purity was verifiec' on a Varian Model 1800 gas chromatograph employing s flame-ionization detector (FID). GASCHROMATOGRAPHY. A Varian Aerograph Model 1800 dual column gas chromatograph with FID detector wa! used for all analyses. Helium was the carrier gas; thc flow rate was 20 ml per minute. The flame was fed by air and hydrogen with an air flow rate of 200 ml/niinute and s hydrogen flow rate of 20 ml per minute. The helium carriei gas was scrubbed in liquid nitrogen through a 20-foot, inch stainless steel (304) tube. Standing current on thc chromatograph held steady during the period of analysis at 25 when measured on a scale of 100 equals 8 X 10-12 A. Analysis of peak areas was accomplished by recording the signal on a Hewlett-Packard strip chart recorder witf disc integrator. All data were calibrated in disc integratec units. A ten-inch chart was used; chart speed was 0.: inch per minute. Procedure. Three solvent heavier-than-water extractor: were set up in one series and four solvent lighter-than-wate: extractors were set up in a second series. Each extracto. was charged with 500 in1 of solvent. A continuous feed o test water was put through each set of extractors. As thic occurred, sufficient analytical samples were taken a t varioui times to make a solute-mass balance. Analytical samplc points were: the influent water, the effluent water, the solvent in each extractor, and a water blank. RESULTS AND DISCUSSION
Figure 1. Extractor design for solvent lighter than water Organic solutes extracted from water then concentrate in bulb Q . Reagents. EXPERIMENTAL SOLVENTS.Several immiscible solvents were tested; they were : hexane, trichlorotrifluoroethane, chloroform, carbon tetrachloride, and benzene. STANDARD SOLUTION.Approximately 950 mg of solute was dissolved in 4 liters of water. This was effected by overnight stirring. The concentrated standard was diluted to 37.8 liters with distilled water. Final concentration was approximately 25 mg/liter. This solution was run through the extractors a t a flow rate of 7.56 liters per hour. EXTRACTEDSOLUTES.Toluene and 2-pentanol were chosen as solutes. This allowed a measurerpent of the extraction efficiency of typical aromatic and aliphatic molecules as well as molecules with large dipole moment differences. These solutes were tested individually to evaluate extraction effi90
Table I lists the solute recovery value for each extractor ir the test series and the summation of solute extracted by thc series sampled extractor train. In all of the five experiment that employed heavier-than-water solvents: the solutes wen recovered in amounts greater than 50% by the first extract01 in the extractor train, except 2-pentanol in chloroform. Thc summed or total recovery o f the three extractors was greate' than 80%. This is not the case for the lighter-than-wate extractors, in which the first extractor in the train of extractor: recovered 22% or less of the total solute. Concentration. The concentration of the solute is the prime sensitivity parameter of gas chromatographic analysis In light of this fact and the fact that the nonaqueous extractani recovered after extractor operation is composed mainly 0: pure solvent with small amounts of dissolved solutes, it i: necessary to remove excess solvent. The analyst has severa techniques a t his disposal to accomplish this; they includt distillation, freeze concentration, and freeze drying among others. Distillation was chosen for this work and was im. plemented by use of a Kuderna-Danish concentrator. The
ANALYTICAL CHEMISTRY, VOL. 45, NO. 1, JANUARY 1973
3fficiency Effluent Flow rate analysis, of extractor, (37.8 liter basis) I./hr 26.52 71.90 56.64
7.90 7.90 7.41 7.79 7.18 7.79 8.39 7.90
Extraction efficiency after concentration, 1
2
3
4
Sum
8.90 0.34 1.22 27 70 26.95 51.46 42.86 9.60
5.47 0.14 0.52 4.65 1.49 24.42 16.07 36.58
9.59 0.36 0.35 1.04 0.87 5.87 6.50 26.41
2.10 0.12 0.13
26.06 0.96 2.22 33.39 29.31 81.75 65.43 72.59
Av concn factor" 1 . 8 X 106 3 . 2 X 10* 7 . 8 X loa
3 . 3 x 105 ... d 1 . 5 X 106 ... 3 . 5 x 106 ... ... d 3 . 4 x 106 ... 1 . 9 x 106 ... 16.66 * Alpha values are calculated by Equation 1, a large alpha value indicates a large difference between the volatility of the solute and solvent. c The concentration factor is the increase in concentration of the test solute. Thus, a factor of 106 means an original concentration of 25 ng/l. has been changed to 25 X lo5 mg/l. Below detectable limit. d
I
:uderna-Danish apparatus employs a three-ball Snyder slumn that furnishes about 2.7 plates. It is an evaporative, mcentration technique. Extraction is accomplished by use of the extractors dexibed. Concentration of the extracted solutes takes place L two steps. First, separation from water and storage in the ilvent; second, reduction of solvent volume. The extracon apparatus concentrates the solute, while separating it from ater. As an example, a 1 pg/l. concentration of any given )lute, if extracted 100% from 100 liters of water would yield 2 accumulated weight of 100 pg. Following this example, sing 100liters of water at 1 pg/L solute concentration and in a ractical case, our solvent volume is about 500m1,then the conmtration changes from 1 pg/1. to 200 pg/l. or increases by a ictor of 200; a second concentration step is reduction of the {tractive-solvent volume. If the 500 ml of solvent containig the 200 pg of solute is reduced in volume to 5 ml, and assum'g no loss of solute in the process, then the concentration has ianged to 40000 pg/L or was concentrated by a factor of I000 from the original. In actual fact there are losses of ,lute as follows:
1. The extraction is less than 100 efficient. 2 . Some of the solute is lost in the second step, which is solvent evaporation (Kuderna-Danish). ctual data, taken during step one and step two of this pro:ss are listed in Table I. The extraction efficiencies are also jted in Table I. An important fact discerned from these ita is that the solute concentration increases even though ibstantial amounts of solvent are lost during the solvent reduc3n process. Thus the data in Table I indicate a reduction in :traction efficiency after concentration even though the effecve concentration has increased. During the evaporative Incentration process (Kuderna-Danish), more than one-half i the absolute amount of the originally extracted solute is 1st; however, the solute concentration is increased from two I four orders of magnitude. (See Table I1 and the right ilf of Table I.) The series process of extracting the solute from water to an rganic solvent, then evaporating (Kuderna-Danish) that )lvent to a small volume, results in a total concentration crease of five orders of magnitude. Flame ionization detector sensitivity for organic species iries according to the molecular composition of the ionizing iolecule. If the detector sensitivity was normalized at 1 g/1. (a median figure), then these extraction and concentraon procedures would extend the detection limit to 10 pg/pl.
Q Y
i
I
p
Figure 2. Extractor design for solvent heavier than water Concentration efficiencies are presented in Table XI. These tests were made on pure solvent and pure solutes. About 50% loss of solute is common for the evaporation process. A possible explanation is that slight amounts of water remain in the extracted solvent and this water allows a co-distillation effect to occur resulting in a loss of solute. Concentration Index. To evaluate the significance of the difference in vapor pressures between solute and solvent as a factor in concentration efficiency, a calculation was made of the vapor pressure ratios (a values) for each solvent-solute
ANALYTICAL CHEMISTRY, VOL. 45, NO. 1, JANUARY 1973
91
Table 11. Kuderna-Danish Concentration Recovery Studies Final Original volume, ml Recovery, % volume, ml Solute, 0.1 ml 2.0 65.34 200 Toluene 2.4 200 72.88 Toluene 60.01 1.6 200 Toluene 66.08 Av ... 2.8 200
Solvent Chloroform No. 1 Chloroform No. 2 Chloroform No. 3 Chloroform blank Carbon tetrachloride No. 1 Carbon tetrachloride No. 2
Concn factor 6 . 5 X 10 6 . 1 X 10 7.5 x 10 6.7 X 10 Av
a
Values 4.10 4.10 4.10
Toluene
200
2.2
26.64
2.4 X 10
2.58
Toluene
200
2.1
32.87 29.8 Av
3.1 X 10 2.8 X 10Av
2.58
Carbon tetrachloride blank = Below detectable limit.
1.8
As the difference increases, the mutual solubility lessens. This indicates that factors describing the intermolecular forces can be correlated with solubility, thus it is likely that dipole moment would also serve as a mutual solubility index. Terms in the dipole moment equation, Equation 2, are derived from the Poisson-Boltzman equation.
‘“1 i
1, I
02
.
0.4
I
, 0.6
.
,
,
0.0
, 1.0
,
, 1.2
,
,
1.4
I
,
f
1.6
Prolvent- Psolute
Figure 3. Dipole moment difference as a function of extraction efficiency couple listed in Table I. The following rationale was followed : The CY value is defined by (4) where CY = vi/vz, u1 = volatility of the higher boiling component, and u2 = volatility of the lower boiling component, using Troutons rule applied to the Clapeyron equation, Equation 1 results.
where P = dipole moment, E = dielectric constant, p = density, and M = molar weight. These variables relate ta the van der Waal’s a term through the equation of state for an ideal gas (An obvious step for the reader). Comparing the extraction results listed in Table I with tht absolute difference in dipole moment between individua! solute and solvent couples, confirms the above hypothesis This relationship is graphically illustrated in Figure 3. Ont notes in Figure 3 that as the difference between the dipolt moment of solute and solvent approaches zero, the extractior efficiency for that couple approaches an optimum. Extrac. tion efficiency varies between 70 and 100% in the cases wherc the dipole moment difference is less than 0.6 debye but thc extraction efficiency drops to below 5 in cases where the di pole moment difference exceeds 1.49 debye. Hence, dipolc moment values can be used to estimate gross degree of extrac tion. The experimental evidence presented in this paper sug gests the use of dipole moment as an index to select the solven best suited for extraction of a given solute or the solvent bes suited to extract a group of materials with similar dipoll moments. A large amount of experimental dipole moment data is available (6, 7), and as a supplement one can use the vectoi addition method for calculation of the dipole moment througl general group-moment addition (4).
where T = temperature in degrees Kelvin. Equation 1 was used to calculate the alpha values listed in Table I and Table 11. Comparison of alpha values with the concentration data in Table I and Table I1 leads to the conclusions that relative volatility of the solute-solvent couple is not a sensitive index of concentration efficiency and relative volatility may not be the dominant factor in the overall extraction efficiency. Thus it should be possible to disregard the differencein boiling point between the solute and solvent. Solvent Index. A qualitative tool to correlate the solubility of one material in another is listed by Gilman (5) as the internal pressure; defined as the term ala2in the van der Waal’s equation. If the difference between the internal pressure of the solute and solvent is small, then mutual solubility is high.
An organic extraction and concentration apparatus is de scribed that will separate and concentrate organic material from water. It employs any given organic solvent as long 8 that solvent is immiscible in water, and it will concentrate i given aqueous organic solute up to a factor of lo5 with thi
(4) H. A. Laitinen, “Chemical Analysis,” McGraw-Hill Book Co., New York, N.Y., 1960, p 477. (5) H. Gilman, “Organic Chemistry and Advanced Treatise,” Vol. 11, John Wiley and Sons, New York, N.Y., 1943, p 1755.
(6) A. L. McClellan, “Tables of Experimental Dipole Moments,’ W. H. Freeman and Company, San Francisco, Calif., 1963. (7) R. C. Weast and S. M. Selby, “Handbook of Physics ant Chemistry,” The Chemical Rubber Co., Cleveland, Ohio, 1966.
92
CONCLUSIONS
ANALYTICAL CHEMISTRY, VOL. 45, NO. 1, JANUARY 1973
stipulation that the solute partitions between water and the organic solvent. The differential in vapor pressure between solute and solvent does not seem to be a factor that regulates concentration efficiency. The dipole moment difference between solute and solvent will indicate the extraction efficiency and can be used as an index to select the extraction solvent. For broad spectrum extraction, several solvents can be used either in a series or parallel extractor trains; and with adequate dipole moment
differences between solvents, the extractor train will selectively concentrate on the basis of solute-solvent dipole moment match.
RECEIVED for review May 25, 1972. Accepted July 31, 1972. The USGS does not necessarily endorse any manufacturers product discussed in this text. Approved for publication by the Director of the U.S. Geological Survey.
Factors Affecting Radioimmunoassays: Specifically That of Human Luteinizing Hormone Using Labeled Human Chorionic Gonadotropin Eugene Cerceo Thomas Jefferson University Hospital, Philadelphia, Pa. 19107
Human chorionic gonadotropin (HCG) labeled with iodine425 was investigated as a tracer for the radioimmunoassay of human luteinizing hormone (HLH) using both the double antibody and a solid phase technique. Iodinations were conducted at three specific activities, namely 11.1, 3.93, and 1.57 I*51/HCG. The extent of degradation was followed at -20, 22, and 37 OC by ion exchange and gel filtration chromatography. Standard curves were determined using freshly prepared tracer at the three specified specific activities and utilizing four selected antibody titer values. The results indicated that HCG labeled with iodine-125 to a specific activity of approximately 1.0 lZI/HCG gave the best standard curves. Ideally, the tracer should be freshly prepared for each assay; practically, at the specified specific activity good doseresponse relationships were obtained for a period of approximately two weeks. For radioimmunoassay, the modified solid phase method developed in this investigation gave results comparable to the double antibody technique. I n either case, the parameter most affecting accuracy and precision was the stability of the tracer hormone.
THE RADIOIMMUNOASSAY TECHNIQUE has allowed the estimation of protein hormones in the picomolar range. It has also been applied to steroids, globulins, and viruses. In general, it can be applied to anything for which an antibody can be produced. Essentially, the basic technique involves the measurement of the competitive binding of labeled and unlabeled antigen for antibody, but naturally anything which inhibits this can be measured as apparent antigen if the system in question is not carefully examined. Therefore, as radioimmunoassays are determined over a period of time, the immunological integrity of the system should be followed by noting the position of the standard curve, its point of inflection, and the per cent tracer bound in the “zero” standard. The specificity, sensitivity, and precision of the radioimmunoassay method ( I , 2) is a direct function of the nature and properties of the radioiodinated hormone. In the case of gonadotrophins not only is the identity of the labeled
(1) W. D. Odell, G. Abraham, H. R. Raud, R. S. Swerdloff, and D. A. Fisher, Acta E i i d o c r i d . (CopcnAagcn), Suppl., 139-142, 54 ( 1969). (2) S. A. Berson and R. S. Yalow,.J. Cliri. Imesi., 38,1996 (1959).
species at times questioned, but also its immunological activity. The sensitivity and specificity of the assay also depends primarily on the avidity and specificity of the interaction between the antigen and the specific binding sites of the antibody. This becomes the most important single factor in establishing a satisfactory radioimmunoassay, and thus requires close observation. Generally, in the antigen-antibody interaction, the energy of binding is determined by the complementary relationship between the antigenic determinant, those parts of the antigen molecule which combine with the binding sites of the antibody, and the combining sites of the antibody molecule. Here a number of forces are involved, including ionic interactions between charged polar groups, hydrogen bonding, van der Waal‘s forces, hydrophobic interactions, and London forces operating between non-polar groups, It is likely that no single type of bonding predominates, although the relatively long range electrostatic attraction is probably responsible for initial binding between the molecule, followed by secondary interactions when the distance is of the order of a few Angstroms. The close fit of molecular structures which is necessary for a high binding energy may also explain the great specificity of the antigen-antibody reaction. Since the stereochemistry of antibodies assumes such a prominent role, the general structure of these macromolecules merits a closer look. Thus, antibodies are gamma-globulins belonging to one of a number of classes of which the principal members are IgG, IgM, and IgA. All three consist of both light and heavy chains containing some 200 to 450 amino acids, respectively. In most mammalian species, the IgG group is the most important and is the only one involved in the radioimmunoassay reaction. Their molecular weights are in the area of 160,000 and they circulate at a concentration of 10 to 15 mg/ml. The most important factors in antibody production are the ability of the antigen to provoke an antibody response in the chosen animals and its availability in adequate amounts. After it is finally obtained, it is found that the antisera with the greatest avidity usually yields the most sensitive assays, and such an antiserum is produced with the aid of Freunds complete adjuvant.
ANALYTICAL CHEMISTRY, VOL. 45, NO. 1, JANUARY 1973
93
