Anal. Chern. 1987, 59, 1716-1720
1716
Determination of Water by Liquid Chromatography Using Conductometric Detection Timothy S. Stevens*’ and Karen M. Chritz The Dow Chemical Company, U.S.A., Michigan Division Analytical Laboratories, 574 Building, Midland, Michigan 48667
Hamish Small 41 76 Oxford Drive, Leland, Michigan 49654
column with a “density detector” to determine water in a fluorination process stream in about 12 min, but with relatively poor column efficiency, i.e., about 30 effective theoretical plates (6). This contribution describes the use of an HPLC system for the determination of water by using an electrical conductivity detector. Considering the extensive use of direct conductometric determination of water ( 7 ) it is surprising that HPLC/conductivity has not been previously described. Examples are shown for the determination of water in samples that would otherwise frustrate or complicate water determination by the frequently used GC and/or Karl Fischer methods. Generally, water is the last peak to elute in the chromatogram and analytical time is less than 5 min with detection limits of about 2.5 ppm. Columns filled with ion-exchange resin were selected due to the known affmity of such resins for water (8). Nevertheless other chromatographic packings were also evaluated.
Most determinations for water are easlly made by a Karl Fischer titration but interferences are known including oxidizing agents, unsaturated compounds, and thio compounds. Gas chromatography (GC) can be both rapid and sensitive but some components of a sample can elute slowly or decompose on-cdumn and interfere. This contribution describes the determination of water by liquid chromatography with a conductivity detector. The inclusion of an acid in the eluent significantly improved sensitivity of detection, which was iimited at about 2.5 ppm. At the 20% water level, precislon of determination was 0.22 %. Examples are shown for the determination of water in samples that interfered with GC and/or Karl Fischer methods.
Most determinations for water are easily made by a Karl Fischer titration. However, interferences are known including oxidizing agents, unsaturated compounds, and thio compounds ( I ) . Thermal conductivity detection gas chromatography (GC) is probably the second most used method, often resulting in a water peak that elutes rapidly, e.g., 1-2 min, and with good sensitivity, e.g., 1 ppm (2). However, with GC the other components of a sample can take much longer to elute than water and can even decompose on-column and interfere with the analysis. We were faced with the need to determine water in commercial formulations of dibromonitrilopropionamide (DBNPA), an antimicrobial product of The Dow Chemical Co. DBNPA is an oxidizing agent and reacts with iodide to yield iodine and thus interferes with the Karl Fischer method. DBNPA is thermally labile and decomposed on-column in a GC. The products of the decomposition (believed to include HBr) corroded and eventually severed the filaments of the GC detector. We thus considered high-performance liquid chromatography (HPLC). Blasius et ai. determined water by using a cyclic polyether column with a refractive index (RI) detector but water and other nonionic components eluted without retention ( 3 ) . Fehrman et al. determined water by gel permeation chromatography using a RI detector. Toluene was used as the eluent which significantly improved separation of water from other low molecular weight interfering components ( 4 ) . However, the DBNPA formulation was not miscible in toluene, and water itself has a limited solubility in toluene. Bjorkquist et al. reacted phenyl isocyanate with water to form N,N’-diphenylurea (NN’-DPU), with a total h, and then analyzed the NN’-DPU reaction time of about by reverse-phase HPLC ( 5 ) . We wanted a simpler and faster procedure than this. Roof et al. used an anion-exchange
EXPERIMENTAL SECTION The chromatographic system included an LDC Constametric I11 pump, a Rheodyne 7120 injection valve, a Chromatronix CM1-A conductivity meter with MCC-75 flow cell, and a Sargent SRG-1 recorder. The chromatographic columns used included a Whatman Partisil 10-ODS, 10-ODs-3,10-SAX, and 10-SCX as well as a Du Pont Zorbax SIL, all 4.6 X 250 mm, and a Hamilton PRP-1 column, 4.6 X 150 mm. The glass column was an LDC Cheminert LC-9-MA-13,9-mm inside diameter (i.d.) packed with Bio-Rad AG-1x2 anion-exchangeresin, 200-400 mesh, or Bio-Rad Aminex 50WX4, cation exchange resin, 20-30 pm, by slurry packing with eluent. Solvents used included Fischer HPLC grade methanol and acetonitrile. The acids used included Baker Analyzed concentrated sulfuric acid (96%) and hydrochloric acid (37%). The p-toluenesulfonic acid was obtained from Eastman Chemicals. The inorganic salts used were Baker Analyzed grade sodium chloride and lithium bromide. Water was determined in samples of carbon tetrachloride, toluene, and in eluent by using a Metrohm Model 652-KF coulimetric Karl Fischer analyzer and all water results reported herein are in units of micrograms of water per milliliter of sample (pprn) or in grams of water per 100 mL of sample (%). The following chromatographic conditions were used to generate Figures 1-7. Figure 1: eluent, 0.0024 M NaCl in methanol at 2 mL/min; column, 9 X 54 mm, Aminex 50WX4, NaC ion form, 20-30 pm; injection, 10 pL loop; detection, 7.5 ps cm-I per 10 mV; recorder, 10 mV span, normal polarity. Figure 2: eluent, 0.0024 M NaCl in methanol at 1 mL/min; column, Partisil-10-ODs-3, Zorbax-SIL, or Partisil-10-SCX; injection, 10 pL loop; detection, 7 . 5 ps cm-’ per 10 mV; recorder, 10 mV span, reversed polarity. Figure 3: eluent, 0.0015 M p-toluenesulfonic acid (PTSA) in methanol, or 0.0012 M HC1 in methanol, or 0.0012 M H,S04 in methanol, at 1.5 mL/min; column, 9 X 21 mm, Aminex 50WX4, HCion form, 2C-30 pm; injection, 30 pL loop; detection, 30 ps cm-’ per 10 mV; recorder, 10 mV span, reversed polarity.
l Present affiliation: Dow Patent Department, 1776 Bldg.. Midland, MI.
a
0003-2700/87/0359-1716$01.50/0 1987 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 59, NO. 13, JULY 1, 1987
Y
H2 0
e
1717
3
2 3 3
0
I
2 I-
L
U 0
2
U 0 2 Minutes
U 0
2
I
RESULTS AND DISCUSSION Eluents Containing Salt. It was concluded that the addition of an electrolyte to a nonaqueous eluent would increase the conductivity of the water peak as it passed through the detector from the column. Thus, we began our work with eluents containing NaCl. Instead of positive peaks, negative peaks (dips) were seen as shown in Figure 1. Two dips were seen in the chromatogram for each injection. The one a t about 0.8 min was explained as the void volume upset resulting from the lack of NaCl in the injected sample. The one at about 2.1 min was explained as the water “peak”, and its size was proportional to the amount of water in the injected standard. Doubling the amount of NaCl in the eluent doubled the water “peak” height and also doubled the background conductivity of the eluent from about 250 p s cm-’ to about 500 ys cm-’. As expected, the base line noise increased with background conductivity, and the limit of detection observed with either eluent was about 0.1% water (in a standard) with a 500-pL injection. The use of LiBr in the eluent instead of NaCl resulted in similar system performance. When the eluent contained no added salt, only a small response was seen for the same injections shown in Figure 1. Reversing the recorder polarity resulted in upscale “peaks” and this became standard operating procedure. Other Columns. The conventional microparticulate HPLC columns listed in the Experimental Section were also
J
0
2
4
0
2
4
6
6
0 . 2 4 6
Flgure 1. HPLC determination of water with NaCl in the eluent.
Figure 4 eluent, 0.0012 M HzS04 in methanol at 1.5 mL/min; column, 9 X 21 mm Aminex 50WX4, H+ ion form, 20-30 pm; injection, 1 p L ; detection, 60 ps cm-l per 10 mV; recorder, 8 mV span, reversed polarity. Figures 5 and 6: eluent, 0.0012 M HzSO4 in methanol at 1.5 mL/min; column, 9 X 21 mm Aminex 50WX4, H+ ion form, 20-30 pm; injection, 50 p L loop; detection 7.5 p s cm-* per 10 mV; recorder, 10 mV span, reversed polarity. Figure 7 : eluent, 0.0012 M HzS04in methanol at 1.5 mL/min; column, 9 X 18 mm Aminex 50WX4, H+ ion form, 20-30 pm; injection, 100 pL loop; detection,Wescan model ICM conductivity detector, range 1, 10 mV output; recorder, 2 mV span, reversed polarity.
I
-
Minutes
Flgure 2. HPLC determination of water using microparticulate normalphase, reverse-phase, and ion+xchange columns with NaCl in the eluent.
tested and with the exception of the Hamilton PRP-1 column, all yielded a retained water peak whose height was proportional to the amount of water injected in a standard. However, the water peak in each case was little retained (k’less than 1)and tailed. For example, Figure 2 shows the performance for the Du Pont Zorbax SIL column, the Whatman Partisil ODs-3 column, and the Whatman Partisil SCX column. With the Hamilton PRP-1 column, the water peak coeluted with the void volume upset. The best performing microparticulate column, the Partisil 10-25-SCX, was judged to perform poorer than the one filled with Aminex 50WX4. The retention mechanism with the use of the columns packed with ion-exchange resin is probably liquidfliquid partition given the known affinity of ion-exchange resins for water (8). It is more difficult to explain the retention mechanism with the use of the columns packed with silica based microparticulate normal- and reverse-phase packings but it may well be a size exclusion chromatography mechanism. Eluent Containing Acid. In dilute aqueous solution a strong acid has about 5 times the specific conductivity of an equal concentration of the salt of the acid formed by neutralization with a strong base. The use of HC1, H2S04,or p-toluenesulfonic acid (PTSA) in an eluent of methanol on a 9 X 21 mm column of H+ ion form Aminex 50WX4 resulted in significantly improved performance. With the NaCl containing eluent of Figure 1, a 50-pL injection of 1% H,O in methanol resulted in a water peak 0.11 p s cm-’ tall. Use of any of the above acids (also of a concentration sufficient to give an eluent background conductivity of about 250 p s cm-’) resulted in a water peak about 18 ps cm-’ tall (see Figure 3), an increase in sensitivity of about 160-fold! Explanation of the Negative Water Peak. It was originally expected that the water peak would be positive with the use of conductivity detection and an eluent of methanol containing an electrolyte, i.e., an increase in conductivity with
1718
ANALYTICAL CHEMISTRY, VOL. 59, NO. 13, JULY 1, 1987
ae , I 0
-
0
2
4
6
Minutes
Figure 4. HPLC determination of water in a formulation of DBNPA.
u
u
u
0
0 2 4 Minutes
0
2
4
2
4
6
Flgure 3. HPLC determination of water with varlous acids in the eluent. Table I. Measured Conductivity of Methanol-Water Mixtures Containing 0.0012 M Sulfuric Acid eluent 1 2 3 4
5 6
7 8 9 10 11
PPm HzO
conductivity,’ ps cm-’
200 1200 2 200 4 200 8 200 16 000 31 000 61 000 113000 203 000 340 000
252 228 212 191
170 156 146 140 144 167 238
Measured with the Chromatronix CM1-A detector and MCC-75 cell at a flow rate of 1.5 mL/min. the addition of water. However, when the conductivity of methanol/water mixtures containing a constant concentration of H2S04(0.0012 M)was measured, the data in Table I resulted. The data in Table I indicate that only above about 6% water does the conductivity increase with increasing water content. The data in Table I parallel the work of Jones et al. who found a minimum in the conductivity of hydrochloric acid in mixtures of methanol and water at about 10% water (9). Jones et al. also found a minimum in the conductivity of many salts in mixtures of methanol and water, e.g., at about 35% water for KI and at about 50% water for LiN03 (IO). Thus, the negative peaks obse~edin this work and the greater sensitivity of the determination of water with the use of an acid added to the eluent relative to the use of a salt added to the eluent are both consistent with the data of Jones et al. The data in Table I suggest that there is a possibility that a calibration curve could show the same response for two different sample water concentrations. However, dilution of the injected sample in the eluent through the system prevented this, i.e., an injection of 100% water resulted in less than 6% water at the detector as long as the injection size
u 0
2
4
Minutes
Figure
HPLC determination of water in
done-I1 soil fumigant.
was less than about 25 pL. The data in Table I also show that the calibration curves of this procedure are not linear. However, the calibration curves are essentially linear to about 1000 ppm of water at the detector. Effect of Water in the Eluent. A “vacancy chromatography” (11) water peak appeared in the chromatogram at the retention time for water when the eluent contained water and a relatively dry sample was injected. As expected, the magnitude of the vacancy peak was proportional to the concentration of water in the eluent. Best results were obtained when the eluent contained little water, Le., about 100 ppm water or less. Other Examples. Figure 4 shows a chromatogram for the injection of a 20% DBNPA formulation in a water/glycol based formulation. This formulation was injected 10 times and a statistical evaluation of the data indicated an average water content of 20.1% with a standard deviation of 0.22%. During the development of the HPLC procedure for the determination of water in DBNPA formulations a headspace gas chromatographic (HSGC) procedure was successfully developed that eliminated the need to inject the formulation directly into the GC. A comparative study of the HSGC and HPLC results for nine DBNPA formulation samples showed a difference of from 0% to 15% relative with an average bias of 7% relative. This was considered to be a good agreement
ANALYTICAL CHEMISTRY, VOL. 59, NO. 13, JULY 1, 1987
-
0
u 0 2 4 Minutes
Flgure 6. HPLC determination of water in 2-mercaptoethanol.
between the methods. The HPLC procedure was preferred as being less complicated and faster. Figure 5 shows a chromatogram for an injection of Telone-II soil fumigant (isomers of dichloropropene, a product of The Dow Chemical Co.). As is generally the case, water was the last peak to elute in the chromatogram and the level of water estimated in the sample was 67 ppm. A GC analysis of the same sample estimated a water level of 71 ppm. In the GC analysis, the water elutes at about 2 min followed by dichloropropene peaks for the next 45 min (12). Thus, the HPLC procedure was faster overall. Water cannot be determined in Telone-I1 soil fumigant by the Karl Fischer method since iodine adds across the double bonds of the dichloropropenes. Figure 6 shows a chromatogram for an injection of 2mercaptoethanol. Water cannot be determined in many thio compounds by the Karl Fischer method (1). Interferences. As a rule, water elutes as the last peak in the chromatogram well resolved from the other components of a sample. However, dimethyl sulfoxide (Me80) eluted just before water and seriously interfered. It is clear that the procedure could be used to determine Me,SO and perhaps other compounds in favorable matrixes. Samples containing hydroxide ion would interfere when the eluent contains an acid. Samples containing ketones or aldehydes interfere due to their reaction with methanol to form ketals and acetals with the resultant liberation of water just as when a Karl Fischer reagent contains methanol ( I ) . A possible solution to this problem, as with the Karl Fischer method, is to replace the methanol with another nonreactive solvent. Acetonitrile i n the Eluent. Water eluted later (k’= 58) and with poor efficiency ( N = 144) when using an eluent of 0.0012 M H2S04in acetonitrile at 1.5 mL/min and a 9 X 16 mm Aminex 50WX4 column. However, unlike the use of methanol in the eluent the water peak was positive. A 50-wL injection of a 1% water standard resulted in a water peak that was 2.4 ~s cm-’ tall on a background conductivity of 7.2 ws
1719
2 4 Minutes
6
Flgure 7. HPLC determination of water using a low noise detector.
cm-’. A comparison of the peak area (in units of fis cm-’ mL) for the water peaks in Figure 3 and the water peak obtained by using the acetonitrile-based eluent indicated that both had about the same area (10 vs. 9, respectively). Since the background conductivity obtained when using the acetonitrile based’ eluent was 36 times lower, it was concluded that a system using an acetonitrile-based eluent with a more efficient and less retentive column might result in rapid analyses, improved detection limits, and reduced interferences. Of the columns listed in the Experimental Section the only one that even came close to meeting this potential was one filled with AG-1x2, but poor peak efficiency ( N = 16) severely limited practical usefulness and sensitivity. The poor efficiency obtained with the acetonitrile-based eluent is explained by slow mass transfer in the stationary phase due to the limited swelling of the ion-exchange resins in acetonitrile. Further work, perhaps with smaller diameter resin or with partially sulfonated or aminated ion-exchange resins that may swell more in acetonitrile, is planned. In addition, size exclusion chromatography columns of appropriate pore size could prove to be useful with this technique since they have already been used with refractive index detection (4). Refractive Index Detection. An Altex Model 156 RI detector was evaluated on the basis of a signal to noise comparison. It was estimated that the detection limit with this RI detector was about 10-50 times poorer than with the conductivity detector. Detection Limits. The relatively high background conductivity (and its associated noise level) of this method, upon which the water peak or dip is seen, is a major limitation of detection limits. This situation is analogous to “single column ion chromatography” (13)where the commercially available conductivity detectors are especially designed to work at relatively high background conductivity with minimum noise. One such detector is the Wescan Model ICM conductivity detector, which places the column and conductivity cell in a temperature-controlled enclosure. Figure 7 shows the performance of a system using the Wescan Model ICM detector. The data in Figure 7 indicate that at a signal to noise ratio
1720
Anal. Chem. 1987, 59, 1720-1725
of 2.5 to 1 the detection limit for water would be about 2.5 PPm.
ACKNOWLEDGMENT Richard Solomon of our laboratory developed the headspace GC method and used it for the comparative determinations of water in formulations of DBNPA. Danial Martin of our laboratory performed the comparative GC determination of water in Telone-I1 soil fumigant. Registry No. DBNPA, 10222-01-2; H20,7732-18-5;Telone.11, 542-75-6; 2-mercaptoethanol, 60-24-2. LITERATURE CITED (1) Mitchell, J., Jr.; Smith, D. M. Aquametry, Paif 111: Wiley-Iciterscience: New York, 1980. (2) Mitchell, J., Jr.; Smith, D. M. Aquametry. P a f I ; Wiley-Interscience: New York, 1977. (3) Blasius, E.; Janzen, K. P.;Adrian, W.; Klein, W.; Klotz, H.; Luxenburger, H.: Mernke, E.; Nguyen, V. B.; Nguyen-Tien. T.: Rausch, R.; Stok-
erner, V.; Toussaint, A. Talanfa 1980, 27. 127. (4) Fehrman, V.; Schnabel, W.; Fresenius’ 2.Anal. Chem. 1974, 269(2)
116. (5) Bjorkquist, B.; Toivoner, H. J . Chromatogr. 1979, 778, 271. (6) Roof, L. B. U.S. Patent 3935097, 1976. ( 7 ) Smith, D. M.; Mitchell, J., Jr.; Aquametry, fart I I ; Why-Interscience: New York, 1984. (8) Helfferich, F. Ion Exchange; McGraw-Hill: London, 1962; p 104. (9) Jones, H. C.; Carroll, C. G. Am. Chem. J . 1904, 32, 521-583. (10) Jones, H. C.; Lindsay, C. F. Am. Chem. J. 1902, 2 8 , 329-370. ( 1 1) Snyder, L. R.; Kirkland, J. J. Introduction To Modern Liquid Chromatography, 2nd ed.; Wlley: New York, 1979; p 812. (12) Analytical Method ML-AM-81-45. available from the Methods Editor,
The Dow Chemical Company, Michigan Divlsion, Analytical Laboratories, 574 Building, Mldland, Michigan 48667. (13) Gjerde, D. T.; Schrnuckler, G.; Fritz, J. J . Chromatogr. 1980, 787, 230.
RECEIVED for review July 21, 1986. Resubmitted February 17, 1987. Accepted March 16, 1987. A United States Patent Application bas been filed covering the subject of this contribution.
Estrogen Conjugates in Late-Pregnancy Fluids: Extraction and Group Separation by a Graphitized Carbon Black Cartridge and Quantification by High-Performance Liquid Chromatography Franca Andreolini,’ Claudio Borra,l Federica Caccamo, Antonio Di Corcia,* and Roberto Samperi
Dipartimento di Chimica, Universitci “La Sapienza” di Roma, Piazza Aldo Moro, 00185 Roma, Italy
A stepwise elution system was elaborated for the fractionatbn in classes of conlugatlon of estrogens in pregnancy body fluid samples by exploiting the presence of charged actlve centers on the surface of graphitized carbon black (Carbopack). After biological samples were percolated through an experlmental Carbopack cartridge, flve classes of estrogens were eluted In the followlng order: unconjugated, A-ring glucuronides, D-ring glucoronides, monosulfates, double conjugates. After solvent elirnlnation, each class of conjugation was subfractionated and quantifled by Ion-pair high-performance liquid chromatography. The major advantages of this procedure over those based upon ion exchangers are that minimum sample manipulation is required, It permits analysis of double conjugates, and the time of analysis is drastically reduced. Recoveries of all compounds of interest ranged between 90% and 98%. The levels of estrogens and thelr conjugates in late pregnancy urine, serum, and amniotic fluid samples were measured.
It is known that only a fraction of the steroid hormones are present in biological materials as unconjugated forms, the major part of them being conjugated with sulfuric and glucuronic acids. Although steroid conjugates do not possess a direct biological activity, they can act as precursor hormone reservoirs able to be converted to either uncorijugated precursor steroids or physiologically active steroids (1. 2 ) . Moreover, the level of each conjugate in biological materials is controlled by complex metabolic and transport mechanisms, which may be altered by pathological disturbances. For these Present address: Department of Chemistry, Indiana University,
Bloornington, IN 47406.
0003-2700/87/0359-1720$01.50/0
reasons, a more complete understanding of the relationship of steroids with diseases may be achieved from the knowledge of the levels of these conjugates in normal and abnormal status. The recent trend in clinical chemistry is that of devising analytical systems for measuring one selected intact steroid conjugate (3-5) or all the conjugated forms of a single steroid (6-9) or the conjugation profile of one class of steroids. Group separation of urinary steroid conjugates has been performed by anion exchangers (10-12). After fractionation, steroids are deconjugated, derivatized, and analyzed by capillary gas chromatography. With this procedure and the use of the chromatograph coupled to a mass spectrometer, impressive results have been obtained as to the determination in pregnancy urine of the conjugate profile not only of the three main estrogens but also of their metabolites (13). Although various modifications have been tried in order to simplify and increase the speed of all steps, this methodology is time-consuming, prone to errors due to excessive manipulation of the sample, and thus unsuitable for routine analysis. In addition, the procedure cited above does not permit determination of double conjugates and it has not been employed for body fluids other than urine. The elution and separation of synthetic mixtures of intact estrogen conjugates have been successfully performed by ion-pair “high-performance” liquid chromatography (IP-HPLC) (14, 15). By the use of this technique, we succeeded recently in determining the conjugation profile of estriol in various body fluids of pregnancy (9). The purification of the biological specimens was accomplished by solid-phase extraction with Vulcan (Carbopack B), a well-known example of graphitized carbon black (GCB),which has been extensively used for isolating analytes of clinical interest from biological materials (16-18). Interestingly, during the experiments we made in order to find the most effective eluant phase for simultaneous de1987 American Chemical Society
