then the resulting complex should be paramagnetic, as are all copper(I1) complexes. If, however, a species CuL results from the interaction of the customary singly charged dithizonate, L-, with copper(I), then the chelate would be diamagnetic. Solutions of approximately 5 X 10-4M keto and enol copper dithizonate in chloroform were prepared by extracting the copper(I1) contained in weakly acid and in pH 10 buffer (NH3-NH4C1) solution, respectively, with a chloroform solution of dithizone. The purple keto solution exhibited a normal copper(I1) electron spin resonance (ESR) spectrum, (a quartet with g = 2.059) but the brown enol gave no signal indicating that it was diamagnetic, and, therefore, was a copper(1) chelate. The spectrum of the normal or keto copper(1) dithizonate in chloroform which was prepared by dithizone extraction from a weakly acid copper solution containing hydroxylamine, consisted of a single broad absorption band with a maximum at 478 nm. The spectrum of the enol chelate was quite similar but its band maximum displaced to 446 nm. Inasmuch as the oxidation state of the copper in the enol complex is unity, it becomes likely that its chloroform solution should contain a stoichiometrically related quantity of diphenylthiocarbadiazone (DTD), the oxidation product of dithizone, according to the reaction 2 Cuz+
+ 3 HDz+
2 Cu Dz
+ DTD
A difference spectrum calculated from the spectra of Cu (1)Dz and the Cu(I1) enol complex resembled closely that of = 425 nm). Equilibrating the CHC13 solution DTD (A, of the Cu(I1) enol complex with aqueous hydroxylamine at pH 4-5, which might be expected to reduce DTD or other oxidation products to dithizone, resulted in the formation of some normal purple Cu(I1) dithizonate. Similarly, the addition of dithizone to a solution of Cu(1) dithizonate resulted in the formation of some purple Cu(I1) chelate, proving that the so-called keto-enol equilibrium ( 4 ) for Cu(I1) dithizonate is really a Cu(II)-Cu(I) redox equilibrium. Because of the unusual coordination number of copper that would be required to explain a 1 :1 dithizone complex, it was decided to measure the molecular weights of both the Cu(I1) enol and Cu(1) keto complexes. Vapor pressure osmometry of solutions of these compounds gave values of 6300 + 300
for the enol and 3100 k 200 for the Cu(1) primary complex. Thus, through intermolecular bonding, copper may achieve a coordination number of four. Although the possibility exists that the diamagnetism observed with the Cu(I1) enol results from metal-metal bonding with consequent electronpairing, it would seem to be improbable because of the need for dithizone to lose a second proton to form an uncharged, CHC13-solublespecies. Other enol dithizone complexes may be formed by mechanisms different from the redox scheme involved in the case of copper. Thus it was recently reported that normal mercury(I1) dithizonate reacted with HgClz to form a mixed ligand complex, ClHgDz (7). Inasmuch as similar complexes formed in which nitrate or bromide replaced chloride, it is likely that under conditions favoring enol formation, namely when Hg(I1) is in excess, mixed ligand chelates form. We found that a CHC13 solution of the pink Hg(I1) enol dithizonate changed to yellow upon equilibration with dilute NHI. That the resulting mercury complex was not the primary or keto (also yellow) was seen from the failure of either KI or NazS 2 0 1 solutions to decompose it, although such treatment readily transformed the keto complex to the green free dithizone in CHC13. The complex, which may well be H*NHgDz,is thus more stable in the presence of the masking agents and may provide the basis for novel analytical applications. To attribute formation of the enol or secondary series of metal dithizonates to the formation of a double charged dithizonate anion, therefore, is no longer possible. BENS. FREISER HENRY FREISER Department of Chemistry University of Arizona Tucson, Ariz. 85721 RECEIVED for review August 11, 1969. Accepted October 31, 1969. Work carried out with the financial assistance of the United States Atomic Energy Commission. ~
~
~~~~
(7) G . B. Briscoe and B. G. Cooksey, J. Chem. SOC.( A ) , 1969, 205.
1
AIDS FOR ANALYTICAL CHEMISTS Improved Glass Frit Interface for Combined Gas Chromatography-Mass Spectrometry Sanford P. Markey Department of Pediatrics, University of Colorado Medical Center, Denver, Colo. 80220
ALTHOUGH several publications have described the coupling of gas chromatographs and mass spectrometers, few systems are suitable for biomedical problems in which high molecular weight and polar compounds are frequently encountered. Laboratories with medium and high resolution mass spectrometers have tried with varying degrees of success to adapt the glass frit pressure-reduction system of WatsonandBiemann (I) or the membrane molecular separator described by
Llewellyn (2). A molecular jet separator developed by Ryhage (3) is the only interface which has won acceptance in biomedical research because of its demonstrated applicability for all types of compounds emerging from the gas chromatograph. However, this interface is available commercially (2) P. M. Llewellyn and D. P. Littlejohn, Pittsburgh Conference on Analytical Chemistry and Applied Spectroscopy. .. _ - _ Pittsburgh. -
(1) J. T. Watson and K. Biemann, ANAL.CHEM., 36, 1135 (1964). 306
Pa., February 1966. (3) R. Ryhage, Arkiu Kemi,26, 305 (1966).
ANALYTICAL CHEMISTRY, VOL. 42, NO. 2, FEBRUARY 1970
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only as an integral part of a gas chromatograph-mass spectrometer combination. The perfection of an alternative separator for use on other mass spectrometers has been of considerable interest. A number of laboratories have modified the glass frit interface by varying its physical size, configuration, and construction without substantially improving performance. Commercially available versions have failed to provide system flexibility and performance expected by investigators doing routine chromatographic analyses. A design for a modified glass frit interface suitable for biomedical applications is presented in this paper, with a discussion of the parameters which must be controlled to incorporate it in any combined system. EXPERIMENTAL
Apparatus. An A.E.I. MS-12 mass spectrometer and a Barber-Colman Series 5000 gas chromatograph were used. Construction of the modified interface is schematically shown in Figure 1. Modifications distinguishing this glass frit interface from the original design are the shortened length of fritted glass used to effect enrichment, combination of the separator, separator oven, and heated source inlet line into a single glass unit, and metal bellows allowing the separator to be moved into and out of contact with the ion source block. The fritted glass tube (8-mm o.d., ultrafine porosity) has been shortened from 8 to 2 inches without a marked change in enrichment factors (4). The fritted tube is sealed to a precision bore capillary (0.2-mm i.d., 2.5 cm-length) at the ion source end of the unit, and to an inlet tube (2-mm i.d.) at the gas chromatograph end. Adjustment of the pressure restrictions prior to and after the glass frit is given under Procedures. Accurate leak sizes can be determined, but can rarely be incorporated into a fixed glass construction because of the variations in conductance of commercially available porous glass and the necessity to adjust restrictions when changing flow rates or pumping capabilities. The center tube of fritted glass and capillary lines is wrapped with Nichrome heating wire and encased in a glass envelope with feedthroughs for heater wires and a side arm for pumping. Silvering the interior surface of the glass envelope reduces the thermal gradients along the center tube, and makes the evacuation envelope serve as its own oven. The need for an exterior oven is eliminated, making possible the combination of separator and ion source housing inlet line. The separator envelope-oven is sealed to an ion source housing flange and bolted onto the source housing. The Watson-Biemann design used a metal envelope and flange combination to achieve a rugged and compact system ( I ) . However, the metal envelope surrounding a glass tube had to be abandoned because of poor heat transfer to the tube, leaking elastomer seals, and arcing from the ion source block to the metal envelope. Sealing the glass separator-oven into an ion source housing flange retains the conceptual elements of the original design and circumvents these problems. The separator is very close to the ion source block, so that chromatographic quality can be lost only prior to enrichment. Compound residence times within the separator are negligible because of a pressure drop acceleration between the interior of the fritted glass and the ion source operating pressures. Glass bellows have been incorporated into the separator envelope and glass-to-metal flanged seal, to make the unit stronger and less prone to thermal shock. A flexible metal bellows and adjusting ring built into the flange construction allow lateral movement and horizontalvertical alignment of the glass separator envelope required to bring the unit into direct contact with the ion source block. Such a retractable system is necessary for intimate contact in mass spectrometers with the ion source mounted on the front flange of the ion source housing. For instruments with the ion source mounted on a rear flange, a fixedlength separator can be connected to the ion source block with a telescoping metal seal before the front cover flange is bolted into place. However, the flexibility and perfor-
U
Figure 1. Side and cross-sectional drawings of retractable glass-frit interface Over-all length 12 inches Nichrome heating wire feedthrough b. Glass bellows C. Kovar glass-to-metal seal d. Seamless, thin-wall flexible stainless steel bellows e. Threaded portion of flange f. Ion source housing flange g. Thermocouple well h. Nichrome heating wire i. Wire restriction in capillary rn i. Glass capillary (2 mm i.d.) connected to chromatographicinlet k . Helium leak testing port in steel driving ring 1. Fritted-glass portion Fn. Pressure restriction capillary a.
mance of the retractable system recommend its usage on either type of instrument. In the construction of this system, connecting lines, valves, and fittings are as important as the units they interface. For biomedical problems, glass connecting lines, glass valves, and low volume glass-to-metal seals are desirable. Heavywall tubing (1 to 2 mm i.d., 8 to 10 mm 0.d.) wrapped with heating wire connects the pressure reaction system to a glass shutoff valve in the detector oven of the gas chromatograph. The chromatograph oven is bolted to the floor to eliminate breakage of this line. The glass shutoff valve (Figure 2) between the gas chromatograph and pressure-reduction system permits isolated operation of either the chromatograph or spectrometer. Several metal valves (bellows metering and bellows shutoff) were tried. While they opened and closed satisfactorily, tailing and decomposition of polar components were observed, with a several-second time delay between flame ionization and beam monitor responses. Consequently, an all-glass, magnetically operated double valve was designed and built. The volume of the valve is a swept path and no holdup or mixing appears to result. Double valve design was necessary for easy opening and closing of the valve magnetically. Because it is mounted in the heated detector oven of the gas chromatograph, the magnets lose some strength. A pressure differential between the valves reduces the force required to open the valves against atmospheric pressure. The valves are on the high pressure side of the pressure reduction system, so that a helium leak-free seal is not required. When tested on a a
I
c
d
Figure 2. Magnetically operated, all-glass double valve a.
Ceramic ring magnets
b. Valve handle (4) R. C . Murphy, Massachusetts Institute of Technology, Cambridge, Mass., personal communication, 1968.
c.
Glass-enclosed ceramic bar magnet
d. Hand-ground glass ball seal
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307
Table I. Operating Parameters for Combined Gas Chromatography-Mass Spectrometry Column. 6 feet X 4 mm i.d. glass column packed with 5 on 100-120-mesh Chromosorb W
OV-22
Flow rates Through column. 60 to 65 ml/min helium To flame detector. 30 to 32 ml/min (valve open to interface) Pressures (uncorrected ion gauge readings) Valve Closed Valve Open Ion source 5 X 10-8torr 0 . 5 to 1.2 X 5 x 10-8 8 x lo-' Ana1y zer 10 microns 500 to 800 microns Forepump Pressure restrictions 8-inch length l/la-incho.d., 0.001-inch i.d. stainless steel capillary between splitter and glass valve 1-inch length 8-mm o.d., 0.008-inch i d . glass capillary reduced with a 0.006-inch 0.d. wire to give a flow of 10 to 15 ml/min through capillary when 60 to 65 ml/min applied at inlet line at 25 "C and atmospheric pressure Temperatures GC oven. 50-300 "C Glass valve (detector oven). 250 "C Glass connecting line. 15G300 "C Separator. 100-125 "C (thermocouple well); 200-350 "C (interior of center tube)
mechanical pump against atmosphere, either part of the double valve seals to the ultimate vacuum of the pump (10 microns of a thermocouple gauge). Low volume and sturdy glass-to-metal seals ( ' 1 4 inch Kovar seals) are used to connect the glass valves to the stainless steel capillary leading to a splitter and to connect the glass chromatographic column to the splitter. These seals avoid the leaks and frequent shutdowns characteristic with silicone rubber septa or elastomer O-ring seals. Short capillary tubing links render the system flexible without sacrificing chromatographic quality. Procedures. Establishment of flow rates and restrictions is critical for the successful operation of a glass frit interface. Table I summarizes the flow rates, restrictions, operating pressures, and temperatures. A pressure drop between chromatographic column and interface is obtained with a stainless steel capillary placed between effluent splitter tee and the magnetically operated glass valve in the chromatograph detector oven. A restriction between the separator and ion source is necessary to control the amount of enriched efRuent allowed into the ion source, a parameter determined by the pumping speed and vacuum requirements of the spectrometer. This restriction is varied by positioning a wire (0.006-inch diameter) within the glass capillary (0.008-inch i.d.) at the ion source end of the separator (Figure 1). The wire must be far enough from to li2 inch) to avoid arcing. the ion source block The lateral position of the separator is adjusted with a threaded driving ring which moves the separator 1 mm for each complete turn. Measurement of the distance from ion source housing flange to ion source block determines the final length required to allow the separator nozzle to seat within the ion source block entry hole. A glass window mounted on the opposite source housing flange allows visual adjustment of the length and alignment. Once this position has been determined and marked on the driving ring, the separator can be moved in or out as many millimeters as are required to allow safe removal of the ion source block (3 to 10 mm). Prior to introducing a sample for mass spectral analysis, proper gas chromatographic conditions are determined (sample concentration, programming rates, etc.) to avoid using the mass spectrometer as a sophisticated chromatographic detector. In the course of a chromatographic analysis, the glass valve can be opened or closed at will, enabling the spectrometer to be used independently if desired. The solvent is vented through the flame detector, sparing the 308
Figure 3. Flame ionization (upper) and beam monitor (lower) recordings of normal hydrocarbons C10,12,14,16.18.20.22,21,28132,36. Column programmed from 50" to 300°C at 10" per minute A . Separator inlet flush with ion source block B. 1-mm gap between separator tip and ion source block C . 3-mm gap between separator tip and ion source block
ion source an unnecessary pressure shock and/or eliminating the need for solid injection systems. Problems associated with the gas chromatograph (column conditioning, column changing, replacement of injector septa, etc.) do not affect spectrometer operation.
RESULTS AND DISCUSSION Evaluation of an interface suitable for biomedical problems requires demonstration that all components emerging from the gas chromatograph enter the mass spectrometer regardless of molecular weight or polarity. Sufficient enrichment to allow mass spectral recording of microgram quantities injected onto the column must be ensured. Figure 3 shows beam monitor and flame ionization recordings resulting from the injection of a mixture of hydrocarbons from n-Clo to n-Ca6. The responses from the two detectors have been recorded simultaneously on a dual pen strip chart recorder, with the pens slightly offset for clarity. In trace A , the separator is in direct contact with the ion source block. Identical responses are evident for all components. The same mixture was recorded at higher sensitivity in Figure 4. Minor components on the tailing edges of higher homologs are evident in flame ionization and beam monitor responses. When the separator is withdrawn 1 mm, trace B (Figure 3) results. A decreased beam monitor response for higher homologs results, although little effect is seen for lower homologs. Because most interfaces are tested with lower molecular weight volatiles (complex petroleum or flavor
ANALYTICAL CHEMISTRY, VOL. 42, NO. 2, FEBRUARY 1970
Figure 4. Flame ionization (upper) and beam monitor (lower) traces at high sensitivities Separator inlet flush with ion source block. Hydrocarbon mixture same as in Figure 5
B
A
I
L\
-------
Figure 5. Flame ionization (upper) and beam monitor (lower) recordings for successive injections of 5.0 pg of cholesterol
Figure 6. Flame ionization (upper) and beam monitor (lower) recordings of cholestane and cholesterol mixtures
Separator inlet flush with ion source block B. 1-mm gap between separator tip and ion source block C . 3-mm gap between separator tip and ion source block A.
samples), most investigators are not aware that such problems exist for less volatile materials. A more dramatic decrease in sensitivity is seen in trace C, where the separator was withdrawn 3 mm (standard for fixed length inlets on A.E.I. instruments). Components emerging from the gas chromatograph with a low velocity due to polarity and high molecular weight are more severely affected by a gap between separator and ion source block. An equivalent series of recordings is shown in Figure 5 for successive injection of 5-pg amounts of cholesterol. A 1-mm gap decreases the beam monitor response to one fifth; a 3-mm gap decreases the sensitivity to one twentieth. Two factors are apparent. A gap between the source inlet line and ion source block functions as the gap in a molecular jet separator [of the order of tenths of millimeters in the separator designed by Ryhage (3)]. The source housing is the counterpart of the evacuation chamber in the jet-type separator. Thus, any small gap will result in a loss of sensitivity, particularly of molecules with low velocities. Second, since the pressure within the ion source block is higher than the pressure in the ion source housing, it is essential that the enriched effluent be forced into the source block for retention of chromatographic quality and elimination of ion source memory effects. The lack of tailing of higher molecular weight homologs in Figure 4 is evidenced by the small components clearly visible after the emergence of major peaks. Traces B and C (Figure 3) show peak broadening (particularly ClSand GO) resulting from the longer pump-out times because the ion source block is less effectively pumped than the surrounding housing. McCloskey found free cholesterol to be a useful standard for determining the performance of interfaces, because it is adsorbed to active or cold surfaces and thermally degraded to cholestene by hot spots (5). Figure 6 shows the recordings resulting from injections of 5-pg (trace A ) and 0.5-pg (trace B) quantities of cholestane and free cholesterol. Flame ionization and beam monitor recordings are identical in both ( 5 ) F.A. J. M. Leemans and J. A. McCloskey, J. Am. Oil Chemists' Soc., 44, 11 (1967).
Separator inlet flush with ion source block pg each of cholestane (first peak) and cholesterol (second peak) B. 0.5 fig each of cholestane and cholesterol
A . 5.0
cases, proving that decreased sensitivity for cholesterol is not a function of the separator, but of decomposition or adsorption prior to the effluent splitter. The M-18 peak recorded from gas chromatographic scans of cholesterol is 45 of the M+ peak, compared to 50z when the sample is run on the probe at identical source temperatures. Thus, the separator interface does not appear to affect thermally labile compounds. So many variables are included in any determination of enrichment factors (flow rates, split ratios, temperatures, compound volatility, and pumping speeds) that attempted quantitation was meaningless. Instead, a minimum usable quantity is defined as the amount required for injection onto the column to produce a flame detector and beam monitor response and a complete mass spectral scan. The amount required for the system described here is 0.05 pg (n-decane)-0.50 pg (cholesterol). The described pressure-reduction system has been in operation for several months in this laboratory. Urinary organic acids (TMS and methyl ester derivatives), steroid derivatives (TMS, methoxylamines), pteridines (TMS derivatives), N-trifluoroacetyl, n-butyl peptide esters, and partially methylated alditol acetates have been analyzed. No compounds have been encountered which did not give identical flame ionization and beam monitor responses. ACKNOWLEDGMENT
The author is indebted to Ray Allen, Landay Scientific Glass, Boulder, Colo., and Sanford Simons and Ivan Frank, of Biomedical Engineering Department, University of Colorado Medical Center, for the technical assistance required to realize the construction of the interface and glass valves.
RECEIVED for review September 29, 1969. Accepted November 10, 1969. Work supported in part by a research grant from the National Institute of Child Health and Human Development ( 5 PO l H D 04024-01) of the National Institutes of Health.
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