Electron Capture Spectrometry, an Adjunct to Gas Chromatography Quantitative Study of Operating Parameters and the Qualitative and Quantitative Distinction between Compounds Containing the Same Heteroatom R. A. LANDOWNE and S. R. LIPSKY Department o f Internal Medicine, Yale University School of Medicine, New Haven, Conn.
b The effect of changes in operating parameters on the sensitivity of the electron capture detector was determined quantitatively for sec-butyl bromide, a model monofunctional compound of moderate electron affinity. Using a constant direct current voltage as the source of applied potential relatively high detector temperatures and low gas flows yielded optimum results. With a pulsed applied potential, low flows could not b e used and higher detector temperatures only provided a slight increase in sensitivity. The nature of the pulse was not critical as long as the maximum standing current for the detector used was approached. In addition the sensitivities of isomeric butyl monohalides were determined at near optimum conditions. These ranged from 9.3 X 10-11 mole per second for n-butyl chloride to 3.0 X mole per second for tert-butyl iodide. A comparison of all the data obtained showed that affinity for electrons was also dependent upon the hydrocarbon structure of the molecule in addition to the electronegativity of the heteroatom. The halide atom produced a positive center for electron attachment a t the alpha position. Surprisingly the branched isomers were found to enhance electron attachment because of the stable free radicals they form after cleavage of the molecule-ion. This effect was primarily responsible for the high sensitivity noted for the tert-butyl halide isomers.
S
the method of electron capture spectrometry was first described for use in the identification of the components of a mixture resolved by gas chromatography ( 5 ) the number of applications of this technique has been disappointingly small in light of the potential orginally predicted for it. To date only some organic compounds have been noted to have a particular affinity for free electrons which permits them to INCE
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be detected a t sensitivities comparable to or greater than the maximum noted for the highly sensitive flame or argon ionization devices ( 1 , 4, 6). Specifically, the electron capture detector has been utilized only as a very sensitive instrument for determining the presence of minute quantities of highly chlorinated materials such as insecticides (1, d ) , metal-organic compounds such as the lead alkyls ( 6 ) , certain polycyclic hydrocarbons ( 7 ) , and Krebs cycle intermediates (4). Its capacity to function as either a qualitative detector or a device for the quantitative analysis of compounds which provide responses somewhat less than those noted with conventional ionization detectors has been largely ignored. Moreover, it has not been fully recognized that in instances where the conventionally sensitive devices respond poorly or in an anomolous manner to the presence of certain compounds, the electron capture detector frequently is the detector of choice where the measurement of trace quantities of these substances is required. For these reasons, and because it was felt that more detailed information concerning the basic characteristics and active mechanisms of this type of detector would be valuable for subsequent investigations, a systematic study was instituted to obtain additional pertinent, accurate, quantitative data on the behavior of the detector toward various types of organic compounds whose affiities for electrons differ over a very wide range of values. As a corollary to this study the effect of changes in operating parameters on sensitivity was determined with respect to temperature, flow rate, applied voltage, and the means of applying this potential. I n effect, the latter condition was a comparison of the two modes of operating an electron capture detector, Le., the direct current (d.c.) method and the pulsing technique. With this knowledge it was then possible to compare accurately the affinities for electrons of
very closely related compounds. This phase of the investigation included a study of the aliphatic monohalides which were excellent model compounds for determining the effect of structure on the affiity for electrons of compounds containing a single heteroatom, in this case a halide. The availability of pure samples of all the neceesary isomers of a given family also helped in the selection of these compounds for this initial study. EXPERIMENTAL
Apparatus. The basic equipment consisted of a conventional gas chromatographic instrument (Model 10, Barber-Colman Co., Rockford, Ill.) containing the components essential for the operation of an argon ionization system, A precisely regulated 0- to 100-volt supply (Electronic Measurements Corp., Eatontown, S . J.) was substituted for the regular high voltage supply. When the pulsing technique was utilized, the voltage was obtained from the output of a versatile pulse generator (Model B7 Rutherford Electronic Co., Culver City, Calif.). The characteristics of the pulse were monitored simultaneously on an oscilloscope. The method of measuring and recording current and current changes in the detector was made in the conventional manner with the electrometeramplifier supplied with the gas chromatographic instrument. The gain control was set a t 1 X 10-9 amp. The output of the electrometer was fed into a 0- to 50-mv. strip chart recorder. Detector. For the sake of uniformity and because i t is easily and simply reproduced, the standard parallel plate detector of Lovelock (3) was used in this study. Its internal dimensions were 1 cm. long and 1 cm. in diameter. The anode and cathode were of brass; the body of Teflon. The radioactive source of primary electrons was a I-cm. disk of tritiated titanium foil on stainless steel placed in direct contact with the cathode a t the bottom of the detector. This source contained 250 mc. of tritium (2 curies per sq. in.) and provided a maximum
75
H
-
c
w v)
9 50 K V 0 W
zr 21
5
15
10
IO-'
M/scc
Figure 1 . Response of electron capture detector vs. concentration for secbutyl bromide a t various conditions of temperature and argon flow rate Constant applied voltage at 40 volts d.c. A, 27' C., 100 ml./min.; 8, 2 7 ' C., 2 0 ml./min.; C, 1 0 3 ' C., 100 ml./min.; D, 1 0 3 ' C., 2 0 ml./ min.
standing current of 1.32 X 10-8 amp. in argon. Method of Analysis. All of the compounds studied were diluted with N,N-dimethylformamide to the lowest concentration which still permitted detection of the component upon injection of 0.2 to 0.6 pl. from a 1-p1. Hamilton syringe. All samples were analyzed by the conventional gas chromatographic technique using a U shaped glass column 8 feet by inch i.d. containing 5Oj, of either Carbowax 6000 or silicone oil (M & B) on 70- to 80-mesh Anakrom ABS (Analabs, Inc., Hamden, Conn.). Argon was the carrier gas in most cases. Peak height and peak width were used in calculating minimum sensitivities in the following manner. With the noise level (due mainly to the randomness of emissions from the tritium source) of the system a t 7 X amp. the minimum sensitivity was defined as the amount of component required to give a peak height of 2 X amp. This amount, in moles, was then divided by the peak width, in seconds, to give a value for the sensitivity in moles per second. Chemicals. All compounds were reagent grade and were obtained from Distillation Products Industries, Rochester, ?;. Y. They were used without further purification. RESULTS AND DISCUSSION
Determination of Optimum Operating Conditions. I n an ionization chamber of the type employed in this study, the current produced in the presence of a nonreacting gas such as argon or nitrogen is derived from free
electrons (negative charge carriers) and positive ions. The precise value of the base or standing current in a given dynamic system maintained a t a fixed applied potential is in turn dependent upon the number of ions formed, their relative mobilities, and their rate of recombination. When an organic vapor which has a specific capacity for absorbing free electrons is introduced into such a system, a given number of free electrons is removed per unit time. The extent to which the free electrons are attached to the compound with subsequent formation of negative ions is usually limited, however, by the particular geometry of the detector. These newly formed negative ions have relatively s l o ~mobilities and their relative presence in the chamber gives rise to a space charge effect which in turn tends to decrease the electrical field. DIRECTCURRENTMETHOD.Before beginning the actual study of the effect of structure on the affinity for electrons it was deemed desirable to determine the optimum operating conditions for the detector to be used. For this purpose sec-butyl bromide was used as the model compound. The effects of temperature and flow rate on sensitivity and linearity were determined. The results are summarized in Table I. From these data it is apparent that under the conditions of the experiment, an increase in the temperature of the detector results in an increase in sensitivity while a n increase in gas flow induces a decreased response. These changes are of sufficient magnitude to make it desirable to operate a t the highest temperature and lowest flow rate possible when seeking optimum sensitivity. This is based upon the assumptions that other classes of compounds behave in a similar manner and that the compounds subjected to analysis are not heat labile. At moderate flows, 100 ml. per minute, for instance, a better than tenfold increase in sensitivity is observed with an increase of 75" C. above room temperature. An additional 75" C. increment did not appear necessary since a maximum limit of sensitivity for sec-butyl bromide (about 1X mole per second) was being approached. At a low rate of gas flow where sensitivity is m r its maximum a similar temperatiure increase only re-
Ill
/
I
IO"
80V
M/sec
Figure 2. Response of electron capture detector vs. concentration for secbutyl bromide a t various applied d.c. voltages Temperature, 27' argon
C.;
flow rote, 2 0 ml./min.
sults in a 21/2-fold increase in response. On occasions when a low flow rate mas used to increase sensitivity the necessary column conditions for efficient gas chromatographic analysis were a limiting factor. These circumstances mere circumvented readily by employing */16 inch i.d. packed columns or 0.10 to 0.20 inch i.d. capillary columns. Additional scavenger gas was used only to prevent loss of resolution n ithin the detector. The effects of temperature and flow rate on the measurement of the affinity for electrons over a range of concentrations extending up to that required to saturate the detector is plotted in Figure 1. For those operating conditions yielding the highest sensitivity the decrease in current which is proportional to the degree of electron attachment by the molecule is also a t its maximum. This results in a linear range for sec-butyl bromide of about 370 to 1. In practice this indicates that the response or loss of current in the detector will be proportional to concentration over a concentration range from the minimum detcct-
Table 1. Effect of Temperature and Flow Rate on Response of sec-Butyl Bromide" Temperature, Flow Rate, Sensitivity, Linear to Linear O c. M1. /Min. b Mole/Sec. Mole/Sec. Range X lo2 27 20 4.8 x 1 . 8 x 10-9 3.7 4 . 0 x lo-" 1 . 5 X 10-8 3.8 27 100 7 . 0 X 10-lo 3.7 103 20 1 . 9 x 10-12 103 100 3 . 3 x 10-12 1 . 2 x 10-9 3.6 Applied voltage, 40 volts d.c. * Argon.
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able amount up to 370 times that amount. This value is, for the most part, dependent upon the geometry of the detector and the strength of the radioactive source of electrons. Thus a high linear range combined with a response that results in a substantial decrease in current a t saturation indicate that optimum conditions exkt for a~liievinghighest sensitivity. The effect of the applied voltage on the performance of a n electron capture detector was also determined. The data in Table I1 summarize the changes in sensitivity and linearity with changing voltage for the same model compound, see-butyl bromide. I n the first dcvription of electron capture spectrometry ( 5 ) it was shown that compounds of different electron affinities could be diqtinguished one from the other by rhnnging the applied voltage. By this niocic of operation those compounds n i t h weak affinity would no longer give a detectable signal a t the higher volt~ P S . These phenomena are still true hwiuse under these conditions the probihilitg of combination of the organic
Table II. Effect of Voltage on Response of sec-Butyl Bromide" Volt,Linear
age, Sensitivity, d.c. LZole/Sec. 30
o
o
4 x 18X 10-11 5 2 x 10-l1 1 3 x Temperature, 27" C.;
4 40 4 8 60 1 5 40 3 4
x x x x
Linear to, Rlole/Sec.
10-13
rril /min. Argon.
10-9
Range,
x
102 4 2 3 7 3 5
10-9 lo-* 3 8 flon. rate, 20
Table 111. Effect of Temperature on Response of sec-Butyl Bromide b y Pulser Technique"
Temperatiirr, p. '!? 100 -700 a 25
upec.
Sensitivity, Mole/Sec.
Linear to Mole/Sec.
Linear Range
X 101 3 5 X 10 3 5 X loW9 1 8 2 8 X 3 8 volts applied for 2 psec. every 20
3 6 X lo-" 2 0 x 7 3 X
IV. Effect of Pulsing Conditions on Sensitivity of sec-Butyl Bromide"
'Ibie
Pulse
On,
yL
olt:ige
-
psx.
Cycle, Sensitivity,b psec. Mole/Sec.
20 1 . 1 x 10-11 6 1 . 3 X lo-" .3 i.5 20 1 3 x 10-11 0 2 20 6.4 X ; a-branching \r-ould p ~ o v i d ~ n i o l e c ~ ~tok absorb free electrom. a d w i vase in this capacity ritlier than A c,hromatogrnm of the four iuonic~i~ic~ an mcrease, as was observcxl. Bci x ~ ~ i te il lio ~i ) ~o f f to ZCRJ :ind tlicl cktertor butyl bromiiics is 31.0 shown in Figure, 5 . caiiv, of this, tlic stability of the prodcoulel not lie opcr:tted. Tlicwforc, it Herc tlir 1iighc.r wrisitivity for the t w wtq aftt,r electron capturc n u s t also hc s c ~ i i i c ~~I(~sir:iblo l to use thcl sliortrst tiary isonicr is ohvious since it gii-c,s :i ~~ii~iCit:rrd an important influciice on ~~ossible piilse with thc longing pi.ogi':ini ( ~ I i i i + c\\:is ~ ~ of little3 significanw as l011g :iP :I >tLIlL(l niasiiiiiini for t l i l t i 3
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I n many instances of electron absorption the final product may be the ionradical itself, and its stability may play a cardinal role in the extent of electron absorption. Where cleavage to even more stable products can occur, as was probably the case with tert-butyl bromide and tert-butyl iodide, affinities are more enhanced. Trapping and identification of the products formed in the detector would shed more light on these reactions. PULSCR METHOD.When sensitivities u ere determined by the puleer technique certain differences a ere noted when compared to the results obtained by the d.c. method. From Table VI it can be seen that tert-butyl chloride is now seven times more sensitive than sec-butyl chloride, n hile tert-butyl bromide is 230 times as sensitive as sec-butyl bromide. Sensitivity of the pulser technique depends more heavily on the formation of stable products. This would explain the enhanced affinity of the tertiary halides including the chloride. The rffect of initial electron attack on over-all sensitivity of the system therefore must be small when using pulsed voltage. This is borne out by the inability of n-butyl chloride and isobutyl chloride to give an effective response under the conditions of the experiment. Undoubtedly all four chlorides are attacked to a similar extent by electrons
but only the sec- and tert-isomers furnish the driving force for effective capture by virtue of the more stable radicals they can form. Our knowledge of these mechanisms is still limited and study of more varied types of structurally related compounds is required to support more firmly some of the proposed concepts. This study can act as a model for predicting the relative sensitivities of more complex molecules, regardless of functional group. An extension of present knomledge to compounds containing other electronegative heteroatoms, particularly oxygen, can now be made easily. I n the field of biochemistry a similar study of steroids mould appear to hold great promise in the elucidation of certain structural details. These compounds contain mainly hydroxyl and keto groups but the number of structural variations surrounding these moieties is indeed large. RIoreover, the electron capture detector should have very high sensitivity for some highly oxygenated steroids making this system very useful in this respect as well. Finally, a comparison of the affinity for electrons of certain steroids with their biochemical activity may be possible as has been achieved with Krebs cycle members (4). The present work also indicates that it Kould be desirable to tailor-make certain derivatives that would impart high
electron affinity to compounds which ordinarily have little or no capacity for free electrons thereby greatly augmenting their detection by this technique. The use of polyhalogenated derivatives could conceivably yield sensitivities even higher than the 3.0 X 10-l6 mole per second herein reported for tert-butyl iodide. The use of such high affinity compounds coupled with optimum operating conditions would then make this system the most sensitive mode of detection for organic vapors to date. LITERATURE CITED
(1) Clark, S. J., Division of dgricultural and Food Chemistry, .4CS, 140th
Meeting, Chicago, Ill., September 1961. (2) Goodwin, E. S., Goulden, R., Reynolds, J. G., Analyst 86, 697 (1961). (3) Lovelock, J. E , ANAL. CHEM.33, 162 (1961). (4) Lovelock, J. E., Nature 189, 729 (1961). (5) Lovelock, J. E., Lipsky, S. R., J. Am. Chem. SOC.82, 431 (1960). (6) Lovelock, J. E., Zlatkis, A., ANAL. CHEM.33. 1958 11961). ( 7 ) Lovelock, J. E., Zlatkis, 8., Becker, R. S., Nature 193,540 (1962). RECEIVEDfor review March 5 , 1962. Accepted April 16, 1962. Division of Analytical Chemistry, 141st Meeting, ACS, Washington, D. C., March 1962. Work supported by grants from the National Heart Institute (H-3558) of the U. S. Public Health Service, the National Dairy Council and the Nutrition Foundation.
New Peak-Shift Technique for Gas-Liquid Chromatography Preparation of Derivatives on the Column M. W. ANDERS and G. J. MANNERING Oeparfmenf o f Pharmacology and Toxicology, University o f Wisconsin, Madison 6, Wis.
b A peak-shift technique for gasliquid chromatography is described whereby derivatives are formed directly on the column by following the injection of the parent compound with an injection of either acetic or propionic anhydride. Acetates and propionates can be formed simultaneously by injecting a mixture of the anhydrides. In many cases, derivatives are formed with alcoholic or phenolic hydroxyl groups and with primary and secondary amines. The method is applicable particularly to the characterization of alkaloids and steroids. Trifluoracetates and trimethylsilyi ethers have also been formed on the column.
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ANALYTICAL CHEMISTRY
studying the metabolic fate of codeine in man, an attempt was made to apply gas-liquid chromatography to the quantitative estimation of codeine and its metabolites, norcodeine and morphine, excreted in urine. Codeine and norcodeine could not be separated under conditions similar t'o those described by Lloyd et al. (5). HoFYever, when a mixture of the two alkaloids in ethyl acetate was warmed with equal volumes of acetic anhydride and pyridine and t'hen injected, excellent resolution was observed. The rather improbable idea then presented itself that one might achieve direct esterification of these compounds by immediately follo\\-ing HILE
their injection onto the column with an injection of acetic anhydride. First results employing morphine were sufficiently gratifying to encourage the extension of the study to include a number of other esterifiable compounds, particularly alkaloids and steroids, the latter being of special interest because the gas chromatographic behavior of the acetates of certain of these compounds has already received attention (6, '7). I t was also found that propionic acid esters could be formed on the column in a similar manner and, indeed, that simultaneous formation of acetates and propionates occurs when a mixture of acetic and propionic anhydrides is injected.
