Apparatus and Procedures for Rapid Extraction and Identification of Pesticides by Single and Multiple Distribution in Binary Solvent Systems MORTON BEROZA Entomology Research Division, Agricultural Research Service, U. S. Department o f Agriculture, Beltsville, Md. MALCOLM C. BOWMAN Agricultural Research Service, Tifton, Ga.
b A new apparatus capable of performing many simultaneous extractions rapidly extracts small volumes of one liquid phase with another. Equilibration of a solute between the phases i s usually complete after 20 seconds of mechanical agitation because of the large interfacial contact between the small volumes. Because new, highly sensitive instrumental methods of analysis require only very small samples and use highly purified solvents, extractions with small volumes are desirable to avoid wasting these expensive solvents. The time-consuming evaporation of extracts to small volumes i s also minimized. The value of extractions with unequal volumes o f the two solvent phases, especially for identification purposes, was demonstrated. Mathematical equations and graphs relating the distribution of a solute between equal and unequal phase volumes to its p-value are presented.
A
for the rapid extraction of one liquid phase with another iinniiscible one, especially in small volumes, was devised and applied to the analysis of pesticides. The apparatus permits the extraction of many samples simultaneously. The ultility of the p-value [fraction of solute partitioning into upper phase ( I , @ ]for the identification of pesticides, as derived from a single extraction, has been extendedthrough the use of unequal volumes of the two pha+es-to give more accurate and useful extraction data for pesticides having low or high p-values. These results have been compared with those obtained by multiple extraction with equal phase volumes. llatheinatical formulas are given for calculating the amount of solute in each layer from the p-value, or vice versa. The p-values, which can be determined within 0.02, are sufficiently different to allow their use in identifying pesti.ides, even if only nanogram amounts are being analyzed, as in electron-capture gas chromatography PPARATUS
U
Figure 1 . A. E.
C.
Positions of multiple extraction tube
Equilibration Transfer of upper phose to reservoir (decant) Transfer of reservoir contents to flask
(1). T o aid such identifications the p-values of 131 pesticides in six solvent systems have been reported (3). For identification purposes the amount of pesticide need not be known, only its ratio in the two phases. At the high dilutions employed in gas chromatographic analysis the p-value is rarely concentration-dependent ( 2 ) . These advantages make the p-value method easy to use. p-Values have also been utilized for determining the number of extractions needed to extract a given amount of pesticide (2,s). EXPERIMENTAL
All extractions were carried out with solvents equilibrated at 25' i. 0.5' C. Solvents were distilled reagent-grade liquids purified as described previously
( 2 , 3 ) . Analyses were made by electroncapture gas chromatography, as previously reported ( 2 ) . Pesticides are identified in a previous publication (3). Apparatus for Multiple Extraction. The extraction unit is Craig's glass countercurrent distribution cell (4) modified to deliver upper phase (usually 10 ml.) to a 5O-ni1. reservoir and equipped with a stopcock made of Teflon (Du Pont) to permit recovery of the lower phase (10 ml.). The unit is shown in Figure 1 in the three basic positions of operation. Figure 2 shows a battery of five such units [mounted on a rack that is fastened to a shaker (W. H. Curtin Co., Houston, Texas #19013H)] in the equilibration (horizontal) and decant positions. Each unit can be quickly and easily removed while it is held in the equilibration position by lifting off its spring-loaded retainer ring A (Figure 3) from the stopcock delivery tube and pulling the cell VOL. 38, NO. 7, JUNE 1966
a
837
Figure 2.
Battery of five extraction units o n shaker A. B.
Equiiibrotion position Decant position
forward. The long tube of each cell must be level during the equilibration process. Ring A not only holds the tube level, but, by holding the addition tube (11 cm. from end of long tube, Figure 1A) in the vertical 3/rinch diameter
are added by means of volumetric pipets. Multiple Extractions of Lower P h a s e with Upper Phase. With the cell in the horizontal position (Figure 1 A or 2 A ) the 10-ml. portions of each WPOOVP (Pienre ? .\ .. it, mn.int,n.ina the. nhase I~" r n n t , a i n i n s t,he neqt,ipide) ~.."..~. .,, .... cell .-__ .... ~-~ .:-.-., R.FP upright, as shown in Figure 2A. Figure added through each addition tube. 3 gives a front and rear view of the rack The shaker, set to oscillate about 175 with dimensions; none of these are times a minute, is turned on for ahout critical but the volume of the lower 20 seconds to equilibrate the phases. phase should be 10 ml. after transfer of After the phases separate (usually the upper phase. after about 30 seconds), the rack of A shaker may be constructed with as tubes is tilted forward to the 45' many extraction units as desired. position (rack rests on turnbuckle F as Apparatus for Single Extraction. shown in Figure 3, rear view); then A 10- or 15-1111. glass-stoppered graduwhen the layers appear well separated, ated centrifuge tube is used, as apthe rack is brought to the decant propriate. The equilibrated phases position (Figure 1B or ZB) to transfer ~
~
Figure 3. Front ond rear view of rack mounted on shaker. solvent-resistant epoxy paint
the upper phases to the reservoirs. To bring the rack to the decant position, it is released by removing spring D fmm hook G (Figure 3 rear view), tilted forward as far &s it will go, and held there for 10 seconds. The rack is then returned to the original horizontal position, and spring D is replaced. Fresh 10-ml. portions of upper phase are added, and the equilibration, transfers, and solvent additions are repeated for as many as five extractions of lower phase with upper phase. The lower phases are removed via the stopcocks after the last transfer of upper phases to the reservoirs. The combined upper phase is recovered by removing each tube from the rack, rotating it to position C of Figure 1, and emptying the reservoir contents into the desired vessel. The tubes may be rinsed with acetone or other solvent and dried before the next extrastion. Although the volume of the upper phase may he varied, that of the lower phase is fixed at 10 ml. with the cell described. Multiple Extraction of Upper P h a s e with Lower Phase. After the phases are equilibrated and separated as described above, the rack is brought to thc 45' position (no flow of upper phase to the reservoir occurs); the lower layer of each tube is then drawn off into a flask set under each stopcock. The tubes are returned to the horizontal position, fresh lower phase is added, and the procedure is repeated for the desired number of extractions. The lower phases are accumulated in the flasks, and eventually the uliiier phase is rvithdrawn separately. The reservoirs are not used when the upper phase is extracted, and the volume of upper and
Rack is made of %-inch plywood and finished with two coats of
A.
0.5-inch i.d. ploslie rings 0.25-inch diom. X I-inch spring C. Hinge D. 0.25-inch diom. X 3.5-inch spring E. Leveling device 10.25 inch lhickl is held light with wing nuh. Either and is adiurlabis l o maintain tunes ievei. when spring D i s fonened to hook G F. Turnbuckle lobovt 6.5 inches long1 is adjusted to hold rack 01 45" angle
8.
838
.
ANALYTICAL CHEMISTRY
wan n o m
WDCI
~n nonzonrai p o m m
lower phasps may be varied since one does not depend on decantation to a set height.
Procedure for Single Extraction with Unequal Phase Volumes. A 5~ 1 aliquot . of upper phase containing the pesticide is analyzed. The upper phase is then extracted with the appropiiate volume of lower phase in a 10- 01 15-ml. glass-stol)pered centrifuge tube by shakiqg the phases vigorously for 1 minute. A 5-pl. aliquot of the upper layer is aoain analyzed in exactly the same way. %he ratio of the second analysis (amount of pesticide in upper phase) to the first (total amount) is the fractional amount in the upper phase (or E u ) . (This ratio becomes the pvalue when the phase volumes are equal.) Should an emulsion occur, the tube is centrifuged to separate the layers. RESULTS
The multiple extraction apparatus was used to determine the fractional quantities (E,) of 10 insecticides in the combined upper phase after making two and four extractions of lower phase with equal-volume portions of the upper one. The results obtained with hexaneacetonitrile and isooctane-dimethylforiiiamide are given in Table I. Table I1 presents E , values for seven insecticides obtained similarly, except that the upper phase was extracted with
Table 1.
EPN
Heptachlor Liiidaiie Methyl Trithion Parathion Telodriii
Included in parentheses in the four tables are the fractions of solute in the upper phase (EY)calculated from the experimentally determined p-values. Calculations were made as follows: For multiple extractions with phases of equal volume, the following equations were used to calculate the fractional amount of solute in each layer from the p-value, or vice versa. Lower phase extracted with upper phase n times
1 - p n or p
=
=
(1 -
(4)
where
E,
fractional amount of solute in upper phase E1 = fractional amount of solute in lower phase p = p-value n = number of extractions =
For a single distribution between unequal phase volumes, the following equations were used to calculate the fractional amount of solute in each phase from the p-value, or vice versa:
E,
“’
=
or p
CYp-p+I
=
E, CY
- E,(a
“P
E1=1-
- 1)
(5)
or p =
ap-p+fl
E , = 1 - (1 - p)nor
p = 1 - (1
El
=
(1
- p)“ or p
=
- E,)l’n (1) 1 - (El)”. (2)
Upper phase extracted with lower phase n times
where
CY =
volume of upper phase volume of lower phase
p-Value 0.42 0.40 0.29 0 35 0 042 0.57 0.13 0.081 0.048 0.49
Alultiple extraction apparatus was used Hexane-acetonitrile Isooctane-dime thylformamide n = 2 n = 4 p-Value n = 2 n = 4 0.64 (0.66) 0.63 (0.64) 0.50 (0.50) 0 57 (0 58) 0 088(0 082) 0.80 (0.81)’ 0.25 (0.24) 0.15 (0.16) 0.10 (0.094) 0.74 (0.74)
O.SS(0.89) 0.89(0.87) 0.74(0.74) 0 82 (0 82) 0 16 10 16) 0.95 (0.97j 0.44(0.43) 0.28(0.29) 0.19(0.18) 0.94(0.93)
0.15 0.080 0.14 0 13 0 015 0.22 0.049 0.022 0,031 0.18
0.29 (0.28) 0.14 (0.15) 0.23 (0.26) 0 23 (0 24) 0 027 10 029) 0.40 (0.39)’ 0.091(0.096) 0.040(0.044) 0.058(0.061) 0.31 (0.32)
E , values in parentheses are calcd. from experimentally determined p-values shown in table. n
Table II.
=
0.45 (0.48) 0.25 (0.28) 0.41 (0.44) 0 40 (0 42) 0 061 10 060) 0.64-(0.63)’ 0.16 (0.18) 0.083(0.084) 0.11 (0.12) 0.52 (0.54)
number of extractions.
Fractional Quantities of Insecticides in Combined Upper Phase (E,) after Two and Four Extractions of 10 ml. of Upper Phase with 1 0-ml. Portions of Lower Phase with Two Solvent Systems”
Insecticide Aldrin ?-Chlordane Dieldrin p,p’-DI)T Heptachlor Heptachlor epoxide
TDE a
CALCULATIONS
El
Fractional Quantities of Insecticides in Combined Upper Phase (E,) after Two and Four Extractions of 10 ml. of Lower Phase with 1 0-ml. Portions of Upper Phase with Two Solvent Systems.
Insecticide ?-Chlordane P,P’-PDT IXazinon Dieldrin
a
equal-volume portions of the lower one, and isooctane-80% acetone and heptane-90% ethanol were the solvent systems. Data on the fractional amount of insecticide in the upper layer after a single extraction with unequal phase volumes when CY = 5 and 10 (CY = ratio of upper to lower phase volumes) are given in Table 111 for 10 insecticides in two solvent systems; d a t a are given in Table IV for seven insecticides in two other solvent systems when CY = 0.2 and 0.1.
p-Value 0.97 0.95 0.91 0.93 0.96 0.90 0.91
Multiple extraction apparatus was used Isooctane-80~o acetone n = 2 n = 4 pValue 0.91(0.93) O.SS(0.90) 0.81(0.83) O.SS(0.86) 0.93(0.92) 0.81(0.81) 0.85 (0.83)
0.87 (0.88) 0.80(0.81) 0.68(0.68) 0.76(0.75) 0.83(0.85) 0.65(0.66) 0.70(0.69)
0.77 0.59 0.58 0.61 0.73 0.58 0.48
Heptane-907, ethanol n = 2 0.58(0.59) 0.33(0.35) 0.34 (0.34) 0.39(0.37) 0.53(0.53) 0.33(0.34) 0.23(0.23)
n = 4 0.37 (0.35) 0.12 (0.12) 0.12 (0.11) 0.15 (0.14) 0.28 (0.28) 0.12 (0.11) 0.057(0.053)
Same as in Table I.
VOL. 38, NO. 7, JUNE 1966
839
DISCUSSION
The introduction and widespread use of new instrumentation have markedly altered the extraction requirements for analytical purposes. Previous reliance on comparatively insensitive colorimetric and other procedures in pesticide residue analysis made i t necessary to extract large amounts of starting material, usually with conventional separatory funnels and large volumes of solvent. The new methods, many of which rely on electronic measuring devices that are extremely sensitive, may require for an analysis only a few microliters of a very dilute solution (or a few nanograms of solute). They also usually require solvents of extremely high purity, which are expensive. To meet present analytical needs we have devised a multiple extraction apparatus capable of extracting small volumes rapidly. Extraction takes place by mild rotatory oscillation of the tubes in a horizontal position; equilibration of the solute between phases is achieved rapidly (usually within 20 seconds) because the comparatively large interface and small volume facilitate rapid exchange of solute. Inasmuch as only 10-ml. phases are employed, the use of excessive amounts of expensive solvent is avoided. Time is saved because many extractions may
Accordingly, knowledge of the pvalue enables the analyst to calculate the distribution of solute in the upper and lower phase in multiple extractions with equal phase volumes or in single distributions of unequal phase volumes. One major objective of this study was t’o overcome the difficulty in distinguishing pesticides having high or low p-values that are close together. This problem arose because many pesticides have very low p-values in some of the binary solvent systems that are most widely used in pesticide analyses ( 3 ) . For example, the p-values of parathion (0.031) and Methyl Trithion (0.022) in the isooctane-dimethylfornianiide system are not distinguishable with any degree of certainty, since we can determine p-values only within 0.02. An examination of the E, values in Table I shows that the E,, values of parathion and Methyl Trithion are not distinguishable after two and four extractions with phases of equal volume (0.058 us. 0.040 and 0.11 us. 0.083), nor after an extraction employing unequal volumes with CY = 5 (0.12 us. 0.10). However, the E , values are sufficiently different in an extraction with a = 10 (0.22 os. 0.17). These data show t,hat both multiple extractions and extractions with unequal phases can spread or magnify small differences in p-value to the point that these differences may be-
be run simultaneously and because the need t o evaporate down large volumes of solvent is eliminated. Extractions are accomplished with a minimum of manipulations. Another advantage of the new apparatus is that emulsions are frequently avoided because shaker oscillation is much less vigorous than the shaking required to equilibrate phases in a separatory funnel. Shaker action may be slowed to any degree to avoid emulsification, but a longer shaking period is then required to attain equi librium. Tables I and I1 present data obtained with the multiple extraction apparatus after two and four extractions. The values in parentheses, calculated from experimentally determined p-values by using Equations 1 and 3, are in excellent agreement with E , values determined experimentally with the multiple extraction apparatus, deviations usually being 0.02 or less. The achievement of practically theoretical results indicates that equilibration of the solute between the phases was attained and that the apparatus was performing satisfactorily. The data of Tables I11 and IV, obtained by single extraction with unequal volumes, likewise show excellent agreement between the E , values calculated from experimentally determined p-values by using Equation 5 and those obtained directly by trial.
Fractional Quantities of Insecticides in Upper Phase (E,) after a Single Distribution between Unequal Phase Volumes When a = 5 and 104
Table 111.
Hexane-acetoni trile Insecticide ?-Chlordane P, P’-DDT Diazinon l>iplrlrin I _
EPN
Heptachlor Lindane Meth 1 Trithion Par atXion
Telodrin
p-Value 0.42 0.40 0.29 0.35 0.042 0.57 0.13 0.081 0.048 0.49
a = 5 0.80 (0.79) 0.76(0.77) 0.67(0.67) 0.73(0.73) 0.19 io. 18j 0.90( 0 . 8 7 ) 0.43 (0.43) 0.32(0.31) 0.21 (0.20) 0.84 (0.83)
Isooc tane-dimethylf ormamide a =
10
0.90(0.88) 0.89(0.87) 0.80(0.80) 0.86 (0.84) 0.33(0.31j 0.95(0.93) 0.61 (0.60) 0.49(0.47) 0.35 (0.34) 0.93 (0.91)
p-Value 0.15 0.080 0.14 0.13 0.015 0.22 0.049 0,022 0.031 0.18
a = 5 0.47 (0.47) 0.30 (0.31) 0.43 (0.45) 0.45 (0.43) 0.076 (0.075) 0.61 (0.59) 0.19 (0.21) 0.10 (0.10) 0.12 (0.14) 0.54 (0.53)
a E , values in parentheses are calcd. from the experimentally determined p-values shown in table. volumes; 10:2 and 1O:l phase volumes in milliliters were used.
a
CY
=
10
O.SS(0.64) 0.48(0.47) 0.62(0.62) 0.63( 0 . 6 0 ) 0.11 (o.ii5) 0.76(0.74) 0.35(0.35) 0.17(0.19) 0.22(0.245) 0.71 (0.69)’
is ratio of upper to lower phase
Table IV. Fractional Quantities of Insecticides in Upper Phase (E,) after a Single Distribution between Unequal Phase Volumes When a = 0.2 and 0.1.
Isooctane-80% acetone
a
Insecticide
p-Value
Aldrin ?-Chlordane Dieldrin p,p‘-DDT HeDtachlor Heptachlor epoxide TDE
0.97 0.95 0.91 0.93 0.96 0.90 0.91
a =
0.2
0.84(0.87) 0.76(0.79) 0.65(0.67) 0.75(0.73) 0.81(0.83) 0.66 (0.64j 0.68 (0.67)
Heptane--90% ethanol a =
0.1
0.75(0.76) 0.65 (0.66) 0.51 (0.50) 0.58(0.57) 0.68(0.70) 0.50 (0.48j 0.53 (0.51)
p-Value 0.77 0.59 0.58 0.61 0.73 0.58 0.48
a =
Same as in Table I11 except that upper to lower phase volumes in milliliters were 2:10 and 1:10.
840
ANALYTICAL CHEMISTRY
0.2
0.39(0.40) 0.22(0.22) 0.23(0.23) 0.25(0.24) 0.34(0.35) 0.22(0.22j 0.16(0.16)
a =
0.1
0.25 (0.25) 0.13 (0.13) 0.13 (0.13) 0.14 (0.14) 0.20 (0.21) 0.13 (0.12j 0.087(0.085)
B
A ..
0
,I 3 w
W
,2
W
.3 a r n 4 [y.
e
,5
3
6 2' -
p-VALUE Figure 4. Extraction with unequal phase volumes: fractional amount in upper phase (E,) vs. p-value CY
is ratio of upper to lower phose volumes
come useful. Since i t is quicker and easier to run a single extraction than multiple ext,ractions, the single extraction with unequal phase volumes appears to be preferable for identification. For single extractions with phases of unequal volume (Tables I11 and I V ) , the difference in E , values becomes greater for solutes with lorn p-values when a is increased; for solutes with high p-values, the difference becomes greater when CY is decreased. These relationships are further demonstrated in Figure 4 which relates p-value, E,, and CY. K i t h such a graph tedious calculations of these values may be eliminated. For multiple extractions with phases of equal volume, the difference in E, values becomes greater for solutes with low p-values as the number of extractions of lower phase with upper one increases; for solutes with high p-values the difference becomes greater as the number of extractions of upper phase with lower one increases. This effect can be readily seen in Figure 5 , part of n.hich was preiiously reported ( 2 ) . The graph allows p-values and E , values to be approximated from each ot'her without tedious calculation. For an exact value, t'he appropriate equation (1-4) is used. Figure 5 may be useful for identification when mult'iple extractions are used, but it will probably find greater application in guiding residue extract'ion processes and cleanup operations. Several extractions of one phase with another are used routinely in pesticide and other analyses to separate the unwanted from the wanted material.
z *7 0
c.8
0
a
.9 100
I
I
I
2
3
4
NO. OF EXTRACTIONS (n) Figure 5.
Extraction with phases of equal volume
Scale A: number of extractions VI. froction of solute extracted into upper phase after lower phase i s extrocted with upper phase. Scale 6 : number of extractions vs. fraction remoining in upper phase after One extraction equals pupper phase i s extrocted with lower phose. value
With knowledge of the p-value the number of extractions necessary to separate a given fraction of solute may be read off the graph. I n this connection the 771 pesticide p-values in six solvent systems, available from a previous study ( S ) , may be helpful to the pesticide analyst. It is apparent that modifications of the basic apparatus described here may be made readily. JJ7e made a useful modification by attaching a reservoir to units of t n o , three, or four Craig cells (cells available from H. 0. Post Instrument Co., LIiddle Village, Long Island, S . Y., reservoir is attached to last cell). This arrangement is meant for cleanup of pesticide reyidues which require a distribution of several plates to achieve separation of the residue from interferences. [Details of this general approach are given in (S).] The multiple-plate arrangement serves the same purpose as a very different and more complex apparatus descrised by L. Martin (5) for removal of fat in the analysis of chlorinated pesticide residues in food.
Although operation of the multiple extraction unit is illustrated for pesticide residue analysis, it may be equally useful for other analyses that require extraction with separatory funnels. ACKNOWLEDGMENT
The technical assijtance of F. G. Crumley is gratefully acknowledged. LITERATURE CITED
(1) Beroza, >I., BoJvman, 31. C., AR 4 ~ CHEM.37, 291 (1965). ( 2 ) Beroza, AI., B o ~ m a n11. , C., J . .lssoc. O$Lc. d g r . Chemasts 48, 358 (1965). (3) Bowman, AI. C., Beroza, II.,Ibzd., 48,
943 (1965).
(4) Craig, L. C., A X ~ L CHEX 22, 1346 i19.501 (5j-!&kin, L., Food Tech. in Australia 16,
456 (1964). RECEIVED for review January 19, 1966. Accepted April 11, 1066. Presented in part at the 79th Annual Meeting of the Association of Official Agricultural Chemists on October 12, 1963 at iVashington, 13. C. Ment,ion of a proprietary product is for identification only and does not constitiite an endorsement by the U. S. Department of Agriculture. VOL. 38, NO. 7, JUNE 1966
841
.
