too applicable on SE-30, since the specific hydrogen bonding between the phenolic group and the stationary phase is weak. It can also be noted in this table that the correlation coefficient for anilines on EGSS-X at 168 “C is rather high at 0.89, even though pis small in this case. The standard deviations in p are also shown in Table IV. In most cases, sp is considerably smaller in this table in comparison to the similar standard deviation values shown in Table 111. As a result, whereas EGSS-X, Reoplex 400, and PDEAS could not be differentiated in regard to selectivity when Hammett substituent constants were used, a definite difference in selectivity can be established for these three phases when chromatographic substituent constants are employed. It may also be seen that the trends in p values previously discerned in Table 111 occur again in Table IV. Thus, for example, lowering the temperature on the EGSS-X column increases P . It is apparent that a number of the causes of scatter of the results in Table I11 are corrected by application of uc. The choice of EGSS-X as the standard phase in this work for the determination of uc was somewhat arbitrary; however, our purpose was largely to illustrate the method. Nevertheless, these uc values have been shown to be quite useful in the ap-
plication of the linear free energy relationship to GLC. It may eventually turn out that another liquid phase may be more appropriate for the determination of uc, but for the present, the values tabulated in this paper can be useful, especially for polyester liquid phases. CONCLUSION
The Hammett Equation has been shown to be applicable to log of the activity coefficientratio (phenol :substituted phenol) for a series of meta and para isomers of phenol. Work is continuing in this laboratory in the study of substituent effects in gas-liquid chromatography. We are currently examining Hammett relationships using non-polymeric liquid phases for a better understanding of solute-solvent interactions. Also linear free energy studies are being extended to other aromatic systems. The results of these studies will be reported at a later date. RECEIVED for review October 12, 1967. Accepted March 27, 1968. Paper presented at the 154th National Meeting, ACS, Chicago, September 1967. Work supported by NSF grant GP5742.
Detection of Subnanogram Quantities of Hexachlorophene by Electron Capture Gas Chromatography Peter J. Porcaro and Peter Shubiak Research Department, Gicaudan Corp., Clifton, N . J . 07014 The widespread use of hexachlorophene has posed a need for its detection and estimation at levels heretofore unattainable. A method is described for de. tection in the subnanogram region by gas chroma. tography. An electron capture detector is employed which utilizes no radioactive source. The chemical activity of the phenol posed chromatographic problems which were suitably solved by the use of special column parameters and silylating techniques. An illustrative application of the method is made to the quantitative recovery of hexachlorophene from skin. Other ve. hicles may also be investigated at these levels, after suitable isolation.
BECAUSEOF THE APPEARANCE of a gas chromatographic method for the detection of halogenated bisphenols ( I ) , interest has been aroused as to how this useful technique could be extended to investigate further the commercially most important member, hexachlorophene [2,2’-methylenebis(3,4,6-trichlorophenol)]. The usual methods to date employ spectrophotometric techniques whereby the phenol is converted to colored derivatives or to its alkali salts which are detected in the ultraviolet. These and other methods are mentioned earlier (2-4). (1) P. J. Porcaro, ANAL.CHEM., 36, 1664 (1964). (2) V. D. Johnston and P. J. Porcaro, ibid., p 124.
(3) Sindar Corp., G-11 (Hexachlorophene USP) an annotated bibliography, Tech. Bull. €1-1, New York (1962). (4) Sindar Corp,, Methods of Analysis for the quantitative determination of G-ll, Tech. Bull. €1-7, New York, (1961).
1232
ANALYTICAL CHEMISTRY
With the gas chromatographic technique, approximately 5 pg of hexachlorophene, as an example of the halogenated
bisphenol class, was detected as the lower limit using a flame ionization detector (1). The routine detection of chlorinated materials such as pesticides in the nanogram and picogram region employing electron capture detectors is well known. The highly selective and extremely sensitive electron capture phenomenon as described (3, offers an increase in selectivity for hexachlorophene also. The detectors commonly used employ a radioactive electron source which would deteriorate rapidly at the temperatures necessary to chromatograph hexachlorophene, which are in excess of 225 “C. However, an attempt was made by Gudzinowicz (6) to determine if hexachlorophene could be detected similarly to a pesticide using a tritium cell. A linear response was obtained in the nanogram region for hexachlorophene. No further attempts were made to extend the range or determine the reproducibility or precision of these results with repeated experiments (7). Private communications with suppliers of the more recent e3Nidetector Pracor Co., Union, N. J . ; Varian Aerograph, Union, N. J.; Barber-Colman, ( 5 ) J. E. Lovelock and S. R. Lipsky, J . Am. Chem. SOC.,83, 431 (1960). (6) B. J. Gudzinowicz, “Analysis of Pesticides, Herbicides, and
Related Compounds Using the Electron Affinity Detector,” Jarrel-Ash Co., Waltham, Mass., 1965. (7) B. J. Gudzinowicz, Cambridge, Mass., personal communication, 1967.
collector electrode located in the effluent line at near ground potential, generates the background current (le). Carbon dioxide acts as a scavenger or absorber of stray low energy photons which are produced at the electrode area. As the effluent passes through the cell, colliding with the active helium, any capturing species present will cause a decrease in ZB in the usual manner.
+ He He* + hv He* + Y + H e + Ye-
+
The principle is analogous to other absorption phenomena which obey the Beer-Lambert Law. I = I B e -act where Z is measured current after absorption
le is the background current (or base line) a = constant or “capturability” of the species c =
k
0 40
50
60
70
- Vp
80
9 0 100 110
120
130 140 I50
POLARIZING VOLTAGE
Figure 1. Typical Vp US. ZB at two VB values Hoboken, N. J. ; Hewlett-Packard, Avondale, Pa.) indicate a sensitive but erratic response is also observed for hexachlorophene with this detector, which is thermally stable at 300 “C. These observations were also encountered in our studies over the past year employing a novel electron capture detector having no radioactive source. The solutions of these problems, as presented here, may be applicable to those encountered using radioactive sources. EXPERIMENTAL
Apparatus. A Beckman GC-5 gas chromatograph with a helium discharge electron capture detector was used in this work. The cell is essentially a photoionization source utilizing a helium discharge as the supply of capturable electrons (8). Its mode of operation depends on the configuration of the electrodes used. In operation, a source current (Is) of usually 4 or 7 mA is generated across an electrode gap which is at a potential of from 165-200 V. As the discharge helium flow passes through this gap, activated metastable helium atoms are produced, among other things, which are attracted to the collector electrode. The potential of a polarizer, which is analogous to a grid in a triode, is regulated by a duodial. The polarizing potential ( V P ) varies from +2 to -150 V. A duodial bias voltage ( V B ) control varies the anode value from 0 to -150 V. The continuous flow of activated helium through the orifice in the discharge chamber to the (8) Beckman Instruments, Inc., Fullerton, Calif., Tech. Bull. 1567-A.
=
concentration cell geometry constant
Optimum operation is obtained when there is a steep gradient from discharge electrodes to the polarizer terminal in combination with a lesser gradient from the polarizer to the collector in much the same fashion as a conventional triode tube. Peaking of V pand VBshould be made at column temperature with carbon dioxide off. The value for V p is usually at midscale duodial settings. Increasing the VB duodial from zero will usually increase ZB. Finally, carbon dioxide is opened and adjusted for an increase in ZB. Proper choice of Is is indicated by the best SIN ratio, usually 7 mA is used, Figure 1 illustrates a typical VP us. ZB response at two VB settings. Proper operation is indicated by constant VP us. ZB values before and after column connection at the desired column temperature. The VP duodial should not be on either extreme of center, and the detector should show rapid response to slight changes in carbon dioxide flows. These are usually 1 to 3 ml/min. DETECTOR PARAMETERS. VP = -100 V (6.22 duodial), VB = -20 V (2.02 duodial), IS = 7 mA, ZB = 0.84 x 10-8 AFS operated at a 2-fold expansion on the electrometer, Only the column flow and temperature are lowered overnight. The electrical settings need little, if any, adjustment to maintain peaking values. COLUMN. 20% UC W-98 silicone on 60/100 Chromosorb Q was packed in IS”, 2.5 mm i.d. glass. This was conditioned at 275 “C for 72 hours prior to use. Injections were made on-column with conventional Hamilton syringes, Metal columns (stainless steel, aluminum, and copper) were also suitable. TEMPERATURES. Column 225 “C; detector 275 “C; detector lines 275 “C. FLOWS.Column valve wide open at 40 psi cylinder pressure causes flows in excess of 500 ml/min helium; discharge helium 40 ml, min. ; carbon dioxide 2 ml/min. Procedure. CALIBRATION CURVE. A stock solution of hexachlorophene is made by weighing 5 mg into a 5-ml volumetric flask followed immediately by 30 p1 of Silyl-8 (Pierce Chemical Co.), added directly to the solid. Then hexane is added as solvent, diluting to mark. Aliquots are taken and diluted with hexane to contain from 0.1 to 1.0 ng/pl The response is linear to approximately 4 ng. The calibration range is extended to 10.0 ng as shown in Figure 2 to illustrate departure from linearity. RECOVERY FROM SKIN. Known amounts of hexachlorophene were deposited in alcoholic solution to forearms which were previously flushed with alcohol. The deposition was then flushed with a liter of fresh alcohol and the recovery was determined by EC-GC and the colorimetric method used VOL. 40, NO. 8, JULY 1968
1233
yK4. I K
00
0 0 - W K
"3
-
fld -.
.:
I,O
2.0
3.0
5.0 6.0 7.0 CONCENTRATION ( ~ f & ?
4.0
8.0
rqo
9.0
Figure 2. Response range trimethylsilyl derivative extended to 10.0 nanograms by Manowitz and Johnston (9). To detect colorimetrically, the liter of ethanol is subsequently concentrated, from which an aliquot is made for the usual colorimetric procedure employing 4-aminoantipyrine (IO, 11). In the proposed EC-GC method, an arbitrary 40-ml portion of the liter is used, from which an aliquot is taken. It is evaporated to near dryness to remove solvent. Then acetone is added to wash the sides of the container and finally it is evaporated to dryness carefully. A slight vacuum is used in the final stages. (Oxygenated solvents and water seriously reduce detector sensitivity.) Thirty microliters of Silyl-8 are added and then the mixture is diluted to 10 ml of hexane. If further dilutions are necessary, more Silyl-8 need not be added. The quantity of 2,2'-methylenebis-(3,4,6-trichlorophenyl)-tr~methylsilyl ether, hexachlorophene-TMS, is read off the calibration curve after chromatographing and checking two points on the calibration curve. The amount recovered is calculated back to milligrams for comparison. RESULTS AND DISCUSSION
A comparison of data obtained by EC-GC and colorimetry on a series of skin recoveries is given in Table 1. It should be noted from Table I that 50 ml of 0.008 mg/l ~
~ ~ ~ _ _ _ _ _ _
(9) M. Manowitz and V. D. Johnston, J. SOC.Cosmetic Chemists, 18, 527 (1967). (10) S.Gottlieb and P. B. Marsh, ANAL.CHEM., 18, 16 (1946). (11) C. A. Johnson and R. A. Savidge, J. Pharm. Pharmacal. 10, 171T (1958).
Subject A
Table I. Comparison of Detection Ranges and Skin Recoveries Amount recovered, mg Amount added, mg Detected by EC-GC Detected by colorimetry Right arm Left arm Right arm Left arm Right arm Left arm
B
C D
E * Not detectable. a Skin oil interference. 1234
concentration used for deposition contains 400 ng of hexachlorophene. In order to detect this final concentration (0.040 ng/pl), a peak height comparison is made at the 20, 40, and 60 picogram levels. This level would represent the lowest level detectable with any degree of reliability (0.04 ppm). Picogram quantities of hexachlorophene-TMS can be detected using the EC-GC procedure. As little as 20 picograms is seen over the noise level in Figure 3. However, a reliable calibration made in the region below 100 picograms has not been achieved as yet. The reliability of the colorimetric method suffers by the amount of obvious insoluble skin extracts present after concentrating. These have a pronounced effect on the color formation. The mean deviation of five 0.400-mg depositions estimated by EC-GC is 0.0086 mg, and the standard deviation is 0.0104 mg. Error is less than 3 at this level. The method affords a reliable degree of precision heretofore unattainable. Known aliquots of hexachloropheneTMS can not only be reproduced on successive injections, but the slope of the calibration curve remains constant during the day and from day to day, providing the detector remains _ on without interruption. Chromatographing hexachlorophene has been no serious problem at low levels as depicted in a typical chromatogram shown in Figure 4 for 0.1 ng. Studies were initially made on hexachlorophene underivatized, but recovering it consis-
2.000 0.400 0.400 0.040 0.040
ANALYTICAL CHEMISTRY
0.400 0.400 0.400
0.080 0.0080
2.000 0.400 0.400 0.040
0.036
0.407 0.420 0.420 0.078 0.0072
2. 100 0.440 0.423
0.430 0.423
m
m
*
*a
m
W
fn
2 0
W
a
fn 2
fn W
0
a
a
P
a W 0
a
a
0
W
V W
n
a
a 0 V
w
a
IN
izM/-? 0
5
'I0
TIME MIN.
Figure 3. Detection of 20 picograms of trimethylsiiyl derivative over the noise level
5
IO TIME MIN.
15
20
Figure 4. Detection of 0.1 nanogram hexachlorophene tently in the nanogram region was not successful. It is an active phenol and, consequently, retained on column substrates. The effect is more pronounced with increasing polarity. The phenol is also retained on solid supports, even the specialty silylated grades, as well as glass beads whether in metal or glass columns. It was found that hexachlorophene had a tendency to hold up on longer low loaded columns, more so than on short ones (1 to 2 feet) with heavier substrate loads (15-2Oz). These shorter columns are used for the TMS derivative also. The current methods in use for forming TMS derivatives (12-14)) have also yielded anomalous responses with hexachlorophene and are more suitable for qualitive rather than quantitative studies. Instead of using the usual procedures employing solvents, followed by silylating agents with or without catalysts or heating, a modified method is used. An excess of Silyl-8, which is a mixture of three agents without solvent, is added directly to a weighed quantity of hexachlorophene. Then without additional treatment, dilution is made with a suitable solvent. To check the speed of derivatization, Silyl-8 was dissolved (12) C. C. Sweeley, R. Bentley, M. Makita, and W. W. Wells, J. Am. Chem. SOC.,85,2597 (1963).
(13) J. F. Klebe, H. Finkbeiner, and D. M. White, J. Am. Chem. Soc., 88, 3390 (1966). (14) L. Birkofer, W. Konkol, and A. fitter, Angew. Chem., 71, 101 (1959).
475 UC W-98 on 60/80 Diatoport S,8', '/a'' 0.d. copper
in chloroform-d containing 1 tetramethylsilane as an internal reference, and its NMR was run on a Varian A-60A. Figure 5 depicts the trimethylsilyl protons of the reagent at 3 cps downfield from the reference. An amount of solid hexachlorophene, not in excess of Silyl-8, was added to the sample tube and dissolved. Another singlet peak at 20 cps as shown in Figure 6 appears for the silyl ether protons, more downfield because of the paramagnetic influence of oxygen. The height of this peak remained constant until more hexachlorophene was added. Then an increase in intensity was noted simultaneously with a decrease in Silyl-8 intensity. Similar instant reactivity is noted with hexamethyldisilazane (HMDS), N-0-bis(trimethylsily1)-acetamide (BSA), and trimethylsilyldiethylamine (TMSDEA). These are the active components of Silyl-8. The infrared spectrum of the silyl derivative prepared (mp 96-97 "C) indicates both phenolic groups are silylated. Quantitative removal of the TMS hexachlorophene derivative can be shown by first injecting Silyl-8 alone to ensure a flat base line, then the derivative is injected. After elution, another injection is made of Silyl-8. No response is noted as in the case of 1 ng, depicted in Figure 7. An interesting observation is the close retention times (t?)of hexachlorophene and its TMS derivative. The derivative actually elutes after hexachlorophene but in excellent VOL. 40, NO. 8, JULY 1968
1235
4 Figure 5. NMR spectrum of Silyl-8, 10 chloroform-d with 1 tetramethylsilane
x
I , . , .. , I I . . . , I . . . . l , . . . I . . . . I . . 3 .O 2.0 1.o
,
. .
x V/V in
I ! I I , I I I I > I
.
.
.
. I . - . . 0
b Figure 6. Hexachlorophene added to Silyl-8
1236
ANALYTICAL CHEMISTRY
....
I . , , . 1 , . , I . , . , I . . . . I . . . . I . . . . I . . . . I . . . . I 3.0 2.0 1.o I
.
,
0
8.0.
7.0
6.0
-
~
5.0.
w
m
2 0
n
m Y
a a W 0
a 0 Y 0
K
4.0.
'7
I
-
3.0-
t 1.0~4' : 90 100
-
.
'
119
:
:
120
!
'
130
!
'
140
'
:
150
Vp POLARIZING VOLTAGE
Figure 8. Shift of maximum ZB peaking into the photoionization region TIME MIN.
Figure 7. Quantitative recovery of 1 nanogram of hexachlorophene-TMS (before and after Silyl-8 injections) symmetry, whereas, the phenol has a tendency to tail slightly at these fast flows. Of course, with the short column lengths used in the method, the difference in t7 is measured in seconds (30 sec) under the condition of the analysis. The usual f7 for each is in the order of less than 5 minutes which will be noted from the chromatograms, Therefore, errors in incomplete silylation would cancel, if the analysis and calibrations were made precisely the same way. Usual radioactive sources would exhibit an accelerated rate of volatility at the temperatures used. The helium detector is particularly suitable for high temperature investigations since its high temperature limit is given at 400 "C. It can be used indefinitely at 215 "C with no serious loss in sensitivity. Figure 1, as mentioned, illustrates a typical V pus. ZB curve, from which the parameters are chosen for a calibration curve or analysis. The shape of these curves may vary from detector to detector, but the general procedure for optimizing the parameters is the same. The operating V p , VB, and Is values are critical for maximum sensitivity and precision. Equally important are the flow rates of carbon dioxide, discharge helium, and column. A thorough familiarity with the variation that each of these parameters has on the ZB is essential for proper sensitivity. A serious drawback to the use of electron capture detectors is their ease of contamination with subsequent sensitivity loss. The helium discharge cell is no exception. Extreme care must be exercised to avoid substrate bleed as well as residue condensation in the cell and atmospheric oxygen. UC W-98 was found to be best suited, of the many silicones tried, to the continuous 225-50 "C ranges required for the analysis, These included SE-30, SF-96, XE-60, OV-1, DC710, QF-1 among others.
As the detector is used, the possibility of contamination becomes apparent in the shifting of maximum peaking into the photoionization region as shown in Figure 8, with a loss of sensitivity and appearance of anomalous responses. It then becomes necessary to either bake out the detector at elevated temperature with a continued helium purge, disconnect the column and purge, raise the Is across the electrodes or check for gas leaks, which are usually indicated by abnormal electrode potential readings. The remarkable advantage of increased sensitivity with this method opens up new areas of investigation. It was illustrated how hexachlorophene could potentially be detected on any portion of the skin. Further, its use as an anthelmintic for animals or in drugs and cosmetics which are applied topically opens up possibilities of its detection even in body fluids and tissues, if indeed it is present at all. This work will be the subject of further investigations. CONCLUSIONS
Electron capture detection of hexachlorophene offers at least a 5 x lo4 fold increase in sensitivity over conventional flame detection. The TMS derivative offers a more reliable means of quantitative recovery from the chromatographic column. The proposed method can reproducibly detect hexachlorophene-TMS at the 0.1 ng/pl (0.1 ppm level) to 1 ng/pl level with linearity. ACKNOWLEDGMENT
The authors appreciate the advice and technical assistance of L. Clougherty and T. Johns of Beckman Instruments, as well as the aid of M. Manowitz who prepared the skin test solutions and participated in the colorimetric analyses. In addition, the council of H. Daeniker, our Research Director, is gratefully acknowledged.
RECEIVED for review January 29, 1968. Accepted April 9, 1968. VOL. 40, NO, 8, JULY 1968
1237
