Anal. Cnem. 1980, 52, 875-881
distribution of the P O E bead size ranging from ca. 35 to 70 M r n (200-400 mesh) and the resulting low efficiency of the packed column, their separation functions could be successfully visualized by high-performance liquid chromatography. This success can be attributed to the large selectivity of the P O E beads, that is, their large separation factors. It should be noted that the best number of theoretical plates attained was 44-62 for the BEOlOMe-aqueous acetone (10 wt 70 H20) system. Other systems exhibited only efficiencies of 9-10 theoretical plates. T h e POE beads studied are very easy to prepare, compared with the resins with crown ethers, cryptands, and POE amide derivatives as anchor groups. However. the development of P O E beads applicable in water eluates and packed columns with high efficiency are required from the practical point of view. Further, application of the P O E beads to separation a n d extraction of other metal salts. and then the comparison with the separation by ion-exchange resin chromatography are of great interest. Further investigation on these is now in progress. ACKNOWLEDGMENT T h e authors express their thanks to Jiro Shiokawa and Toshiyuki Shono, Osaka University, and Daniel Swern, Temple University, for their encouragement and helpful suggestion. They are also grateful to Mitsubishi Chemical IndGstries Co. Ltd. for a giftof cross-linked chloromethylated poly(styrene) sample.
875
LITERATURE CITED Biasius, E.; Janzen, K. P.; Adrian, W.; Klautke, G.; Lorscheider, R.; Maurer. P. G.;Nguyen, V. P.; Nguyen-Tien, T.; Schotten. G.; Stockerner, J. Fresenlus, Z . Anal. Chem. 1977, 284, 337-360, and references cited therein. Blasius, E.; Janzen, K. P.; Luxenburger, H.; Nguyen, V. B.; Klotz, H.; Stockemer, J. J . Chromatogr. 1978, 167,307-320. Okarnura, H.; Aoyarna, S.;Hiraoka, M. Abstracts, 8th Meeting of Chubu Kagaku Kankeigaku Kyokai Shiburengo, Nagoya, Oct. 1977, 1A04. Grossrnann. P.; Simon, W. Anal. Lett. 1977, 10, 949-959. Yanaaida. S.;Takahashi. K.: Okahara. M. Bull. Chem. SOC.J m . 1977. 50. i386-1390. Yanagida, S.; Takahashi, K.; Okahara, M. Bull. Chem. SOC.Jpn. 1978, 51 1294-1299 YanaGda, S.;Takahashi, K.; Okahara, M. Bull. Chem. Soc.Jpn. 1978, 51,3111-3120. Ohornoto, H.; Kai, Y.; Yasuoka, N.; Kasai, N.; Yanagida, S.;Okahara. M. Bull. Chem. SOC. Jpn. 1979, 52, 1209-1210, and private communication. Yanagida, S.;Noji, Y.; Okahara, M. TetrahedronLett. 1977, 2893-2894. Yanagida, S.; Takahashi, K.; Okahara, M. Yukagaku, 1978, 28, 14-19. Yanaaida. S.:Takahashi. K.: Okahara. M. J . Ora. Chem. 1979. 4 4 , 1099-1103. Merrifield, R. B. J . Am. Chem. SOC. 1963, 85, 2149-2154. Farrall. M. Jean; Frechet, Jean M. J. J . Org Chem. 1976, 4 1 , 3877-3882. Treadweil, F. P.; Hall, W. T. "Analytical Chemistry", Vol. 11, 8th ed.;John Wiley & Sons; New York, 1935; p 654. Warshawsky, A.; Patchornik. A.; Kalir, R.; Ehrlich-Rogozinski, S. Hydrometallurgy. 1978. 4 , 93-104. Warshawsky, A.; Kaiir, R.; Deshe, A.; Berkovitz, H.; Patchornik, A. J Am. Chem. SOC. 1979, 101, 4249-4258.
RECEIVED for review September 10,1979. Accepted November 20, 1979.
Delayed Injection-Preconcentration Gas Chromatographic Technique for Parts-per-Billion Determination of Organic Compounds in Air and Water Richard G. Melcher" and Victor J. Caldecourt Michigan Division Analytical Laboratory. Dow Chemical U.S.A., Midland, Michigan 48640
A gas chromatographic injection/concentration apparatus and technique has been developed for the direct determination of trace organic compounds in ah and water. Organic compounds are retained for extended periods on a collection precolumn while a sample of air (1-2 L) or water (10-200 pL) is vented. The concentrated organics are then injected into the analytical column by applying a high current directly across the precolumn causing it to heat In seconds. High sensitivity and resolution are obtained by controlled rapid heating and low dead volume. Sharp peaks are obtained for high boiling compounds elirnlnatlng the need to temperature program. The technique is particularly valuable for the determination of very low levels of Organic compounds and for compounds which are difficult to extract or purge from water. i n addition to increased sensitivity, a wider choice of GC columns can be used which might otherwise be damaged by direct aqueous injections. The system has been used with flame ionization, electron capture, nltrogen/phosphorous, and photoionization detectors.
T h e delayed injection technique (DIT) operates under two basic principles: (1)Organic compounds contained in a matrix, such as gas, air, water, or volatile solvents. are retained in a 0003-2700/80/0352-0875$01 OO/O
tube containing a solid sorbent while the matrix passes through and is vented. Since a large amount of the matrix can be passed through, the trace components are concentrated. (2) When the tube is heated, the organic compounds are thermally desorbed and injected directly into a gas chromatograph by a flow of carrier gas. Since the sample matrix has been eliminated, high sensitivity is obtained with very little column or detector upset. Early use of these principles was for the concentration and determination of trace organic compounds in air (1-5). A quantity of air was first pulled through a tube containing a solid sorbent, and the collection "slug" was subsequently connected to a gas chromatograph and heated to release the collected compounds. The purge-trap technique applied these principles for the determination of purgeable compounds in water, where carrier gas is bubbled through the sample carrying the compounds into the collection tube (6, 7). In recent studies (8, 9), aqueous solutions have been injected into the sampling slug. The water was purged with carrier gas before analysis. Early studies in this laboratory showed that high resolution in addition to high sensitivity for injection of air and water samples could be obtained by connecting a small collection precolumn directly to the analytical column using a heated valve, and by heating the precolumn rapidly with a movable 2 1980 American Chemical Society
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ANALYTICAL CHEMISTRY, VOL. 52, NO. 6, MAY 1980 2 Resistive Heating System
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Flgure 1. General setup for delayed injection technique using resistive heating and (inset) movable oven alternate heating system. (1) Valve oven, (2)valve handle, (3) precoiumn, (4) transformer, (5) analytical column flow control, (6) purge flow control, (7) gas chromatograph, ( 8 ) valve oven temperature control, (9) resistive heating temperature control, (10) alternate moveable oven
oven. T h e apparatus described in this paper is a further development and uses direct resistive heating of the precolumn trap. T h e integrated design of t h e apparatus, in addition t o efficient analysis of air and purge-trap samples, enables large direct injections of aqueous samples. By maintaining t h e collection precolumn at a controlled elevated temperature during sample collection, the matrix vapor is quickly expelled. T h e subsequent rapid desorption of collected compounds by resistive heating of the precolumn produces sharp chromatographic peaks eliminating t h e need t o temperature program t h e analytical column unless necessary for analytical separation. These characteristics of versatility, sensitivity, and high resolution make i t suitable for use as a n ambient air or effluent stream monitor as well as a valuable accessory for the laboratory.
f r o n t view
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EXPERIMENTAL Apparatus. A Hewlett-Packard Model 5710A gas chromatograph, was equipped with a hydrogen flame ionization detector or an electron capture 63Nidetector. The photoionization detector Model HNU PI-51 (HNU Systems, Inc., Newton Upper Falls, Mass. 02164) was installed in a Varian 2700 gas chromatograph. The temperature controllers, LFE Corporation Controller 227, Type J, was obtained from LFE Process Control Div., 1601 Trapelo Rd., Waltham Mass. 02164. Reagents. The chromatographic packings, 10% SPlOOO on 80/100 mesh Chromosorb W-AW, 1%SP1240 DA on 100/120 mesh Supelcoport, 5% QF-1 on SO/lOO mesh Gas Chrom Q and Tenax-GC 60/80mesh were obtained from Supelco, Inc. (Bellefonte, Pa.). Fluorad FC-431, Lot $3, as obtained from the Commercial Chemicals Division, 3-M Company (Saint Paul, Minn.). The special column, 0.5% Carbowax 20M over bonded :20M on SO/lOO Chromosorb W-AW plus 5% micronized Carbopack C, is a development of The Dow Chemical Company and has been licensed to HNU Systems, Inc., for sale and distribution. Valve Oven. T o obtain flexibility and portability, the valve and precolumn connections are mounted in an insulated metal housing. This integrated system provides minimum dead volume in the critical areas and provides elevated temperature control for all connections and lines once the sample has entered the system. It can be connected (or disconnected) t o most gas chromatographs in 15 min. The design also allows use of either of the rapid heating techniques (Figure 1) and could also be used as a conventional gas sampling valve by replacing the precolumn with a fixed volume sample loop. For dedicated applications, the valve can be mounted inside the gas chromatographic oven with the precolumn trap mounted outside the oven wall. The components of the DIT valve oven, Figure 2, are mounted on a 3/4-inchaluminum block [g]. Holes are drilled to accept the
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Figure 2. Construction of the resistive heating system. (a) Septum nut, (b) septum, (c) glass injection sleeve, (d) injection port, (e)glass wool injection fitter, (f) wire connections to transformer, (9)metal housing, (h) insulation, (i) purge gas/air sample inlet line, (j) precolumn (packed with sorbert), (k) cartridge heaters, (I) precolumn electrical insulation assembly, (m) standoff bolts, (n) Swagelok '/,6-in. fitting welded to injection port, (0)valve oven control thermocouple, (p) purge gas outlet, (9) metal heater block, (r) 1/18-in,tube, (s) precolumn temperature conbol thermocouple, (t) glass wool spacer, (u) six-port high temperature valve, (v) carrier gas supply line, (w) carrier gas to GC column, (x) block of insulation, (y) brass plate, (z) automatic vatve actuator c a n be connected to shaft. The valve is shown in the analyze mode
six-port valve [u], the cartridge heaters [k], and the '/,-inch injection port [d]. The oven control thermocouple [ o ]is placed near the middle of the block. The block is fastened in the metal housing with "standoff' bolts and the system is insulated with glasswoo! and Kaowool (Babcock and Wilcox) [h]. The precolumn [j] packed with the collection sorbent, is attached t o the valve with a 1/8- to '/l,-inch Swagelok reducing union which has been silver-soldered to the brass plate [y]. The upper precolumn
ANALYTICAL CHEMISTRY, VOL. 52, NO. 6, MAY 1980
connection assembly [i] is electronically isolated from the system by using spacers and washers of insulating material and by using a '/*-inch Swagelok union with a to '/,,-inch Vespel reducing ferrule. Heavy gauge wire [fl for connections to the transformer are silver-soldered or bolted to the two fittings. If high valve temperatures are to be used, a small block of insulation [XIshould be attached or the fitting mounted inside the insulated valve oven. All the tubing in the valve system is inch and connected with "zero volume" fittings. Glass-lined tubing should be used for lines which come in contact with the sample to reduce adsorption or reaction of compounds. The precolumn thermocouple [SI is either silver-soldered or clamped tightly t o the precolumn with a thin copper wire. The septum nut [a] and septum [b] can be attached directly to the injection port [d]; however, for lower background from septum bleed, a septum isolation device can be attached. The injection port is drilled through so that a 6-mm glass liner can be inserted [c]. A small amount of glass wool is placed in the end to keep particulates out of the valve. Fitting [n] is a Swagelok union which has been silver-soldered to the injection port and connects tube [r] to the valve. The 1/16-inchline [w] passes through the injection port of the gas chromatograph and is connected directly to the analytical column using a Swagelok union and a Vespel reducing ferrule. Carrier gas is supplied through [VI and purge gas through ti] using separate flow controllers. The valve oven is heated by two 100-W cartridge heaters controlled with a LFE Corporation Controller 227, Type J proportional controller. Valve. The valve [u] is a six-port Valco value (CV-6-HTa, '/,,-inch Zero Volume Fittings, High Temperature) initially conditioned by heating rapidly to 300 "C with carrier gas flowing through all ports. The valve has been used a t 200 to 250 "C for over three years with little difficulty. An air actuated valve can be used if automatic valve switching is desired. Precolumn. The precolumn ti] is made from standard '/,-inch stainless steel tubing. For the experiments described here, a 16-cm length was filled with Tenax-GC, 60/80 mesh packing. The type of sorbent and length of packing depends on the sample matrix and the components to be collected. The sorbent must efficiently collect the desired components while passing the matrix, and it must have good temperature stability with little bleed or decomposition a t high desorption temperatures. Tenax-GC porous polymer gas chromatographic packing has excellent properties. It retains a wide range of organic compounds while passing air, water and volatile solvents, and can be heated to 200 "C to rapidly desorb the compounds with little decomposition background. For volatile compounds in air, such as acrylonitrile and chlorinated solvents, Porapak Q or R desorbed a t 125-150 "C works well. Highly volatile compounds, such as vinyl chloride in air, are efficiently collected using Carbosieve B or S, and desorbed a t 15G200 "C. No packing should be placed within 1 cm of the tube ends since this section may not reach the maximum desorption temperature and peak resolution will be affected. Thin-wall and glass-lined tubing heated slightly faster than standard '/,-inch tubing but no differences were observed in the chromatograms produced. Transformer. A high-current, low-voltage transformer is needed to supply the power necessary to heat the precolumn at a sufficient rate. Transformers $1 and $2 used in this study were made using primary coils from Powerstat 10B voltage regulators (115 V, 175 W) by wrapping a secondary coil of either 11 or 22 turns of 48 insulated wire. Transformer r3, which gave the optimum heating rate, was made using a primary coil from a Powerstat 21 voltage regulator (120 V, 600 W) by wrapping a secondary coil of 10 turns of 4'8 insulated wire. The temperature was controlled with a second LFE controller and a thermocouple attached to the precolumn ti]. The proportional band was widened on the LFE controller by changing the proportional band resistor R 28 from 560 to 3300 9, so that the temperature overshoot was less than 10 "C. The precolumn is wrapped with glass fiber ribbon to eliminate drafts and uneven heating. The cooling curves should be similar for all transformers and are dependent on the amount of insulation and cooling draft. Moveable Desorption Oven. An alternate technique for rapid desorption is a small moveable oven (Figure 1). Basically the oven consists of a metal block drilled slightly larger than inch so that it will slide easily over the precolumn. The block should also
877
be drilled to take an injection port cartridge heater and temperature sensor compatible with the auxiliary or GC injection port control unit. An extra or salvaged injection port block may be suitable. The block should be insulated so that it can maintain a temperature of 200 to 250 "C. The precolumn needs to be twice as long, but only half is packed with sorbent. The oven is positioned back on the empty portion of the precolumn during sample collection and moved forward to rapidly heat the sorbent during the desorption mode. DIT Operation. While the valve is in the sample mode, liquids are injected through the septum [b] with purge gas flowing through [i]. Air samples are either injected through the septum or enter through inlet line [i] or directly to the valve through a modified line [r] by positive pressure or a vacuum pump on line [p]. When the sample and carrier matrix enter the precolumn at [t], organic compounds are adsorbed and collected while the carrier gas, air, water vapor, and the nonadsorbed organics pass through the precolumn and exit through [p]. After sufficient sampling and/or purging time, the valve is switched to the injection mode. The carrier gas [VIwhich has been flowing into the analytical column through [w] now backflushes the precolumn. When the heating transformer is turned on, the collected compounds are desorbed and flow into the analytical column through [w]. Calibration a n d Optimization. Simulated air samples for calibration and optimization of parameters were prepared in several ways. Standards of volatile compounds were prepared in Saran film air bags. The bags were filled with a known amount of air using a wet test meter, and microliter amounts of the test compound, or a solution of the compound, were injected into the bag. After mixing, a known volume of sample was pulled through the precolumn using a calibrated pump. High boiling compounds were tested by injection into a U-tube ( I O ) or directly into the injection port [d] while a known volume of charcoal-filtered laboratory air was being pulled through the system. Aqueous standards were prepared by serial dilution of water-soluble compounds or by injection of methanol or acetone solutions of the compound into a known volume of water or water containing 10% methanol. When analyzing actual samples, a series of low level spikes were made in the sample and matrix effects on recovery were examined by a standard additions plot. Optimization of the system was accomplished by observing variations in background, sensitivity, resolution, and quantitative recovery as a function of a number of parameters. For a particular precolumn and compound, the effect of valve temperature, sample or purge time and rate, precolumn collection temperature, desorption rate, desorption temperature, and flow rate were studied. The optimum size of aqueous injections was determined by injecting various volumes of a standard and plotting peak height (or area) as a function of volume injected. A leveling trend in the plot indicated the limit of injection volume.
DISCUSSION Background. T h e background (various peaks observed during a blank run) appears t o be the biggest difficulty when using t h e DIT for high sensitivity analysis. T h e precolumn will collect and concentrate many impurities from the gas lines, valve, septum, etc., and inject them in t h e desorption mode. The background can be minimized in a number of ways. The lowest temperature which will produce good peaks should be selected for the valve and precolumn. T h e valve and precolumn should be conditioned for several days a little above the temperature a t which they will be used. A low-bleed septum or other septum purge device should be used, and t h e glass liner and glass wool filter in t h e injection port should be replaced periodically. A carrier and purge flow of high purity gas should be maintained through the valve while it is heated. Large injections of high boiling solvents should be avoided, and an elevated collection temperature (50-100 "C) should be used whenever possible t o limit the background caused by very volatile compounds. O p t i m u m Desorption Temperature. A study was made t o determine t h e optimum desorption temperature. T h e different desorption temperatures were attained by turning temperature control t o 300 "C (max.) and then to "off' so that
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ANALYTICAL CHEMISTRY, VOL. 52, NO. 6, MAY 1980
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Flgure 3. Optimum desorption rate ("CIS) for Carrier gas flow 40 mL/mln N,
a 1 k m Tenax precolumn.
the desired temperature was just reached. In this way a rapid, linear heating rate (12.5 "C/s, with transformer $2) was obtained for each temperature. Biphenyl and diphenyl oxide were selected for test compounds because of their relatively high boiling points (254 and 259 "C) and because the separation was almost base-line with the analytical column and conditions used. Identical peak heights were obtained for all desorption temperatures above 150 "C. At desorption temperatures below 150 "C, peak heights, precision, and resolution decreased due t o peak broadening. This indicated that by the time the temperature reached 150 "C, desorption was complete and heating to higher temperatures or for longer periods did not improve desorption of these compounds from Tenax. Other precolumns and compounds can be optimized in the same manner. Since higher temperature increases the desorption background, a desorption temperature of no more than 25-50 "C above the critical temperature should produce the optimum results. Use of the optimum temperature is more critical when elution times of the desired compounds are relatively short and when elution times are under isothermal operation of the analytical column. If the analytical column is temperature programmed, a lower desorption temperature could be used. O p t i m u m Desorption Rate. Different heating rates were obtained using three different transformers. Transformer 21 reached 150 "C in 15 s (8.3 "C/s), transformer $2 took 10 s (12.5 "C/s) and transformer 23 took 6 s (20.8 "C/s). Figure 3 shows there was some difference between transformers z2 and $3 but a 20% decrease in peak height when fl was used. A slower heating rate was obtained by manually programming the precolumn at 1.3 "C/s and a 55% reduction in peak height was obtained. This indicated an optimum heating rate of approximately 20 OC/s. Because the Tenax packing at the axis of the precolumn heats more slowly, faster heating rates will probably not improve desorption and may cause higher background due to decomposition of packing in contact with the metal tube. The resolution is related to the cross-sectional heating rate of the precolumn packing and the carrier flow rate through the precolumn. S a m p l e Collection a n d P u r g e Rate. Air samples can be collected a t 10-100 mL/min. The collection time depends on the retention of the compound on the precolumn and is typically 5-15 min. After the sample is collected, it is not always necessary to Nzpurge before desorption; however, a 1-min purge at 50 mL/min produces a much lower background by eliminating water vapor and oxygen which may cause oxidation of the packing or the collected compounds. T h e technique for making 10-25 pL aqueous injections is not critical; b u t if large injections, 50-200 p L are made too rapidly, the temperature of the precolumn is increased due to steam from the heated valve. The exact conditions for injection of large samples depend on the valve temperature. type and length of precolumn, purge flowrate, and the com-
pound being collected. The technique for higher boiling compounds is less critical. Usually larger volumes of sample can be injected a t a faster rate without loss. T h e optimum technique determined in this study for a wide range of compounds was to adjust the purge flow to 50 mLd/min and allow the precolumn to cool from the previous injection down to 3C-40 "C. Aqueous samples were then injected at a rate which did not increase the precolumn temperature above 75 "C (2-5 pL/s). Since the thermocouple is near the inlet end, the temperature of the rest of the precolumn is somewhat lower. After injection, the temperature of the precolumn was increased with the controller to 7 5 "C for the remainder of the purge (4-6 rnin). Six to eight minutes are necessary to purge 200-fiL injections when the precolumn remains at room temperature. D e s o r p t i o n Mode. High boiling compounds are readily desorbed from the Tenax precolumn even though they may not elute from a Tenax analytical column. These compounds collect at the very front end of the precolumn, and during the desorption mode they are backflushed into the analytical column. Desorption of most compounds is complete in 1 min or less, and power to the transformer can be turned off and the valve can be switched a t any time after this. This flexibility allows the desorption mode to be adjusted according to the length of the chromatogram. For short chromatograms of 2 to 4 min, the precolumn can be turned off after 1 min and allowed to cool for 3 min. T h e valve is then switched to the collect mode for the next sample. For long chromatograms, it is better to keep the precolumn hot up to the last 3 min to reduce collection of background peaks. T h e lowest background for aqueous injections, when long, temperature programmed chromatograms are involved, is obtained by switching the valve back to the collect mode (N, purge) after 1-2 min desorption. T h e precolumn is kept a t 200 "C and the high Nz purge flow rate of 50 mL/min reconditions the precolumn. At the end of the temperature program, the precolumn is cooled, while the analytical column is returned to its initial temperature. Sensitivity. In addition to the increase in detection capability resulting from larger samples, peak heights are generally increased since the chromatograms can be run faster when the matrix effect is eliminated. Although the highest sensitivity attenuation cannot always be used owing to background, extension of the detection limit is usually in direct proportion to the sample size increase. T h e increased resolution also adds an increase in sensitivity. T h e exact detection limit depends greatly on the type of compound and the specific inst,rument. When a hydrogen flame ionization detector is used, a ppm detection limit is extended into the ppb range. When a specific, highly sensitive detector such as an electron capture, photoionization, nitrogen/phosphorus, or GC/mass spec (selective ion monitoring) is used, the detection limit is extended into the low and sub-ppb range. C o m p a r i s o n of Resistive H e a t i n g a n d Moving Oven. Rapid heating with either the resistive heating or moving oven technique produces similar sensitivity and resolution. T h e moving oven is a simpler and less expensive system, and by mounting the valve in the gas chromatographic oven and using the auxiliary or injection heater controls, very little additional equipment is necessary. The moving oven technique works well for air samples but it is not as efficient as the resistive heating technique for large aqueous samples since the collection temperature cannot be easily controlled. It would also be more difficult to automate. APPLICATIONS The delayed injection technique can be used for almost any compound which can be gas chromatographed. In addition
ANALYTICAL CHEMISTRY, VOL. 52, NO. 6, MAY 1980
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Figure 4. Delayed injection determination of hexachlorobutadiene (HCBD) in air. Concentration of 20 ppb (v/v) in air sampled for 5 min at 200 mL/min
to the increased sensitivity obtained, it is useful for samples which contain high amounts of residue. T h e glass injection port insert, which contains a glass wool filter, can be changed after each injection when samples such as muddy water, brine solutions, blood, urine, etc. are analyzed. Some solvents pass through the Tenax precolumn very rapidly a n d can be used for extracting aqueous samples or desorbing compounds from charcoal or silica gel industrial hygiene samples. Using the delayed injection technique for carbon disulfide, methylene chloride, methanol, or other volatile solvents, can increase sensitivity 10- to 100-fold. When solvents are used, care must be taken to vent the purge flow into a hood. A few examples are discussed below. Only one example is given for air analysis, since the chromatograms for air samples would be almost identical to those obtained for aqueous injections when the actual weight of each compound is considered. Example 1. Determination of hexachlorobutadiene in air is shown in Figure 4. Air was pulled through a precolumn, containing 5 cm of Tenax, a t 200 mL/min for 5 min. T h e valve oven was 250 "C and the sample was desorbed for 1 min at 200 "C. The analytical column, 6 f t X in. stainless steel column packed with 10% SP 1000 on 80/100 Chromosorb W-AW, was run isothermally at 130 "C with a flow rate of 40 m L N2/min. T h e detection limit using a hydrogen flame ionization detector (FID) was 1-2 ppb (v/v) for a 1-L sample, with a signal-to-noise ratio of 3:l. A plant monitor has been designed using these data and is being tested for continuous operation. Example 2. T h e analysis of a mixture of glycol ethers in water is shown in Figure 5 . Glycol ethers are nonpurgeable a n d highly soluble in water. The boiling point range for the compounds shown is 170 to 230 "C, and a temperature program was necessary to analyze the full range. T h e detection limit was aproximately 50 ppb with this program; however, if only one component was to be analyzed, isothermal con-
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Figure 5. Relayed injection determination of glycol ethers in water. Sample injected was 200 WLof 500 ppb of each compound: (1) ethylene glycol-isobutyl ether, (2) ethylene glycol-n-butyl ether, (3) dipropylene glycol-methyl ether, (4) diethylene glycol-ethyl ether, ( 5 ) diethylene glycol-isobutyl ether, (6)diethylene glycol--n-butyl ether
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Figure 6. Delayed injection determination of chlorobenzenes in water, 200-pL sample injection: (1) 1,2,4-trichlorobenzene (3.7 ppb), (2) 1,2,3-trichlorobenzene (3.3 ppb), (3) 1,2,4,5-tetrachlorobenzene (3.6 ppb), (4) 1,2,3,4-tetrachlorobenzene (8.0 ppb). (5) pentachlorobenzene (3.6 ppb), (6) hexachlorobenzene (3.0 ppb)
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ANALYTICAL CHEMISTRY, VOL. 52, NO. 6, MAY 1980
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Flgure 7. Delayed injection determination of Chlorobenzenes in methylene chloride, 50-pL injection: (1) monochlorobenzene (110 ppb), (2) 1,2dichlorobenzene (104 ppb), (3) 1,3dichlorobenzene (103 ppb), (4) 1,4-dichiorobenzene (108 ppb), (5)1,2,3-trichlorobenzene (123 ppb), (6) 1,2,44richiorobenzene (1 16 ppb), (7) 1,3,5-trichIorobenzene (123 ppb). ( 8 ) 1,2,3,4-tetrachlorobenzene (87 ppb), (9) 1,2,4,54etrachlorobenzene (98 ppb), (10) pentachiorobenzene (89 ppb), (11) hexachlorobenzene (92 ppb)
ditions were used and a detection limit of 10 ppb was obtained with FID. A sample volume of 200 pL was injected slowly with the injection port (valve oven) held a t 200 "C. T h e water was purged from the 14-cm Tenax precolumn in 4 min with a N2 purge flow of 60 mL/min and the precolumn a t 7 5 "C. The sample was desorbed at 225 "C for 1 min, and the analytical column, a 2 m X 2 mm glass column packed with 10% Fluorad FC-431 on 100/200 mesh Chromosorb W-HP ( I I ) , was temperature programmed from 80 to 170 "C a t 16 "C/min. Higher molecular weight glycol ethers such as tetrapropylene glycol methyl ether, and pentapropylene glycol methyl ether can be determined in water at the 100-ppb level in a similar manner using a 2 M, 10% S P lo00 on 80/100 mesh Chromosorb W-AW column a t 240 "C and a 40 mL/min N2 carrier flow. This system has been used for determining these compounds in clear pond water. Example 3. T h e chromatogram shown in Figure 6 is an example of the sensitivity that can be obtained when a selective highly sensitive electron capture detector is used. The mixture of six chlorobenzenes was prepared as a concentrate in methanol and then diluted into water. Standards and samples were run immediately after preparation to reduce errors due to adsorption on the container walls. This is reduced by adding 10%-20% methanol. Samples stored for extended periods may have to be extracted (including the container walls) to obtain accurate results. This becomes less of a problem for compounds which are more water-soluble, and when on-site analyses are made. The chromatogram was obtained by injecting 100 FL of an aqueous (10% methanol) solution slowly into the injection port
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'r
tr\
Delayed injection determination of phenols and soluble aromatic compounds in water, 100-pL injection: (1) 2-chlorophenol (230 ppb), (2) 2-nitrophenol (350 ppb), (3) phenol (280 ppb), (4) phenyl interference, (5)2,6-dichlorophenol (380 ppb), (6) ether (300 ppb) 2,4dichlorophenoi (290 ppb), (7) 2-chloro-4-tert-butyI phenol (300 ppb), ( 8 ) 2-sec-butylphenol (270 ppb), (9) 4-tert-butyl phenol (260 ppb), (10) dimethylphthalate (300 ppb), ( 11) 4-chloro-m-cresol (600 ppb), (12) 2,4-dinitrotoluene (390 ppb)
+
(valve oven) held a t 225 "C. The water was purged from the 12-cm Tenax precolumn with a 50 mL/min N2 flow while holding the precolumn a t room temperature for 5 min and then by holding the precolumn a t 100 "C for 5 min. T h e sample was desorbed a t 200 "C for 1 min, and the analytical in. stainless steel column packed with 5% column, 6 f t X QF-1 on SO/lOO Gas Chrom Q, was run isothermally a t 130 "C with Ar + 5% methane carrier gas. Flow rate was 40 mL/min. T h e gas chromatograph used was a HewlettPackard 5710 equipped with a 63Nilinear electron capture detector. Neither the silicone column nor the electron capture detector, which are both sensitive to aqueous injections, showed any upset. Example 4. T h e chromatogram shown in Figure 7 was obtained using a HNU photoionization detector (PID), 10.2 eV. Unlike the electron capture detector, the PID has nearly equal molar response to all the chlorobenzenes (12)and at least 50 times the sensitivity for aromatics than the FID (13). The chromatogram was obtained by a 50-pL injection of a chlorobenzene mixture in methylene chloride. If this solution had been a 1O:l extract of a water sample, detection at the sub-ppb level would be possible. The chromatogram of a 50-pL water injection was identical except for a negative solvent peak and indicated possible detection limits of 2-10 ppb. This system was also shown to greatly extend the detection limit for the determination of chlorobenzenes in collected air samples using a procedure described by Langhorst (12). The 50-pL injection was made slowly with the injection port (valve oven) a t 225 "C. The 14-cm Tenax precolumn was purged for 4 min a t room temperature with 50 mL/min Nz flow. T h e sample was desorbed a t 200 "C for 1 min, and the
Anal. Chem. 1980, 52, 881-885
analytical column, 8 f t X in. stainless steel column packed with 0.5% Carbowax 20M over bonded E 20M on 80/100 Chromosorb W-AW plus 5% micronized Carbopack C, was temperature programmed from 50 to 190 "C a t 8 "C/min. Example 5. Figure 8 shows the determination of a mixture of phenols and other water soluble aromatic compounds at the 300-ppb level. T h e chromatogram was obtained by injecting 100 ILLof an aqueous solution slowly with the injection port (valve open) held a t 200 "C. The water was purged from the 14-cm Tenax precolumn in 8 min with a N2 flow of 50 mL/min, while holding the precolumn a t 75 "C. The sample was desorbed for 1min, and the analytical column, 2 mm x 4.5 m glass column packed with 1% SP 1240DA on 100/120 Supelcoport, was temperature programmed from 50 to 180 "C-at 4 OCImin. A hydrogen flame ionization detector was used.
881
LITERATURE CITED (1) Urone, P.; Smith, J. E. Am. Ind. Hyg. ASSOC.J . 1981, 22, 36. (2) Novak, J.; Vasak, V.; Janak, J. Anal. Chem. 1985, 3 7 , 660. (3) GalbicovaRuzickove, J.; Novak, J.; Janak, J. J. Chromatogr. 1972, 64. 15. (4) Shadoff, L.; Kallos, G.; Woods, J. Anal. Chem. 1973, 45, 2341. (5) Russell. J. Environ. Sci. Techno/. 1975, 9 , 1175. (6) Zbtkis, A.; Bertsch, W.; Lichtenstein, H.; Tishbee, A,; Shunbo, F. Anal. Chem. 1973, 45, 763. (7) Bellar, T.; Lichtenberg, J. J. Am. Water Works Assoc. 1974, 66, 739. (8) Bowen, 0.E. Anal. Chem. 1976, 48, 1584. (9) Ryan, J. P.: Fritz, J. S. J. Chromatogr. Sci. 1978, 16, 488. (10) Severs, L. W.; Melcher, R. G.; Kocsis. M. J. Am. Ind. Hyg. Assoc. J.
1978, 39,321. Blaser, W. W.; Kracht, W. R. J. Chromatogr. Sci. 1978, 16, 111. (12) Langhorst, M. L.; Nestrick. T. J. Anal. Chem. 1978, 51, 2018. (13) Driscoll, J. N. J. Chromatogr. 1977, 734, 49-55.
RECEIVED for review November 21,1979. Accepted February 21, 1980.
Microparticulate Bonded Pyrrolidone for High Performance Liquid Chromatographic Separation of Estrogenic Steroids in Urine Thomas H. Mourey' and Sidney Siggia' Department of Chemistty, GRC Tower
I, University of
Massachusetts, Arnherst, Massachusetts
A chemically bonded pyrroiidone phase is used to separate estrogenic steroids by reversed phase liquid chromatography. The amide substrate exhibits unique interactions with the estrogens through a combination of hydrogen bonding, partitioning, and electronic phenomena, and thus provides selective isolation of the phenolic steroids. The selective interactions result in the reduction of interferences In the analysls of urine samples, and hence an improvement over hydrocarbon substrate reversed phase separations. Hydrogen bonding effects are presented in detail through the separation of a chlorophenol isomer model system. The amide phase is best applied to the analysis of free estrogenic steroids in nonpregnancy urine via a simple ether extraction, although a shorter and more efficient hydrolysis and extraction procedure of estrogen conjugates can be applied using the amide column because of a minimization of interferences resulting from the selective interactions.
T h e determination of estrogenic steroid concentrations in urine provides useful information in several distinct areas. Estrogen concentration plays an essential role in the regulation of the female reproductive cycle and, under normal circumstances, large amounts of estrogens are produced in the body a n d excreted through the urine in the latter months of pregnancy. T h e analysis of urine for estrogen content is an accepted method for monitoring pregnancy, and unusual steroid levels are strong indicators of possibly damaging malfunctions, typically in the placenta. Nonpregnancy estrogen levels are equally as important, especially in diagnosing problems in patients using oral conPresent address: The Eastman Kodak Co., Research Labora-
tories, Rochester, N.Y. 14650.
0003-2700/80/0352-0861$01 .OO/O
0 1003
traceptives and the so-called "fertility drugs" which alter levels of estrogens in the body. Fishman (1) has recently reported a statistical link between low estrogen concentrations in urine and women categorized as high risks for familial breast cancer. In severe cases, fluctuations or irregularities in estrogen levels of nonpregnancy urine can also indicate ovarian disorders and can affect secondary sex characteristics. Total estrogen concentration in pregnancy urine is normally 3-5 mg/L of urine. This concentration is high enough to enable the estrogens to be isolated and quantit.ated by chromatographic methods quite readily. Nonpregnancy urine is somewhat more troublesome because of its total estrogen concentration being more than 100-fold lower than that found in pregnancy urine. Liquid chromatographic methods tend to fail a t these concentration levels because of phenolic or other ether extractable interferences in the ether soluble fraction of the sample. There has been considerable effort to improve the separation of estrogens in urine by high performance liquid chromatography, and to eliminate interferences which become especially troublesome in nonpregnancy samples. Synthetic mixtures have been separated on chemically bonded stationary phases by Butterfield e t al. (2) and by Majors and Hopper ( 3 ) . Tscherne and Capitano ( 4 ) have reported improved separations on a CI8 phase obtained by the addition of 2% silver nitrate to the mobile phase. Additional hydrocarbon phase separations have most recently been reported by Fukuchi et al. ( 5 ) . Application of this separations technology to the analysis of estrogenic steroids in urine is somewhat more involved. At high concentrations, the estrogens are usually present as their glucuronide or sulfate conjugates, and require hydrolysis by either chemical or enzymatic means before the high performance separation can be run. Direct separation of the conjugates on a cellulose anion-exchange resin has been reported (6). This method eliminates the need for hydrolysis 0 1980 American Chemical Society
