21
Anal. Chem. 1984, 56,21-24
maximum of the latter two compounds. Shielding the detector from laboratory air currents is quite important. Noise levels are more than ten times greater when the apparatus is operated in the open air than when it is enclosed in a box. However, no special vibration isolation is necessary. We have successfully operated the system on a wooden table and on a honeycomb optical table. The flexibility of a wooden table is undesirable, but any rigid metal base is apparently adequate. Photothermal deflection is not sensitive to scattered light, unlike conventional densitometry or fluorescence. The detector has not been optimized for laser spot size. Photothermal deflection and dual beam thermal lens systems operate best if both beams have approximately the same diameter. In these experiments we have reduced the argon ion laser beam size to match the helium-neon beam. This approach requires only one lens but produces a detector which samples only a small fraction of the cross section of the chromatographic spot. In future experiments we will explore the effects of expansion of both beams to more closely match spot diameters. The use of cylindrical lenses or prisms as beam expanders suggests the possibility of making a detector with slit-like cross section which approximates the ideal shape of a densitometer for thin-layer chromatography. Photothermal deflection has been shown to be a simple and sensitive method for quantitation of thin-layer chromatograms. The detector is easy to align and stable for long periods. Only modest laser powers are needed to reach picogram sensitivity. Further reduction in laser power is possible with some refinement of the system. Thus, the device should be operable with inexpensive lasers, including devices which operate in the ultraviolet. Experiments toward these goals are under way in our laboratories.
Registry No. 1,2-Naphthoquinone,524-42-5; phenanthrenequinone, 84-11-7; a-ionone, 127-41-3. LITERATURE CITED Fried, B.; Sherma, J. “Thin-Layer Chromatography: Techniques and Applications”; Marcel Dekker: New York, 1982; pp 15-16. Fenimore, D. C.; Davis, C. M. Anal;, Chem. 1081, 53, 252A-266A. Ziatkis, A., Kaiser, R. E., Eds. High Performance Thin Layer Chromatography”; Eisevier: New York, 1977. Poiiak, V. “Advances in Chromatography”; Giddings, J. C., Grushka. E., Cazes, J., Brown, P. R., Eds.; Marcel Dekker: New York, 1979; Voi. 17, pp 1-52. Coddens, M. E.; Butler, H. T.; Schuette, S. A,; Poole, C. F. LC 1083. 1, 282-289. Rosencwaig, A.; Hall, S. S. Anal. Chem. 1075, 4 7 , 548-549. Castleden, S. L.; Elliott, C. M.; Kirkbright, G. F.; Spiliane, 0. E. M. Anal. Chem. 1070, 51, 2152-2153. Burggraf, L. W.; Leyden, D. E. Anal. Chem. 1081, 53, 759-764. Flshman, V. A,; Bard, A. J. Anal. Chem. 1081, 53, 102-105. Lloyd. L. B.; Yeates, R. C.; Eyring, E. M. Anal. Chem. 1082, 5 4 , 549-552. Harris, J. M.; Dovichi, N. J. Anal. Chem. 1080, 52, 695A-706A. Kliger, D. S. Acc. Chem. Res. 1080, 13, 129-134. Haushaiter, J. P.; Morris, M. D. Appl. Spectrosc. 1980, 34,445-447. Fujiwara, K.; Lei, W.; Uchiki, H.; Shimokoshi, F.; Fuwa. K.; Kobayashi, T. Anal. Chem. 1982, 54, 2026-2029. Mori, K.; Imasaka, T.; Ishibashi, N. Anal. Chem. 1082, 5 4 , 2034-2038. Mlyaishi, K.; Imasaka, T.; Ishibashi, N. Anal. Chem. 1082, 5 4 ; 2039-2044. Jackson, W. B.; Amer. N. M.; Boccara, A. C.; Fournier, D. Appl. . O.p t . 1081, 20, 1333-1344. Aamodt, L. C.; Murphy, J. C. J. Appl. Phys. 1083, 54, 581-591. Murphy, J. C.; Aamodt, L. C. J. Appl. Phys. 1080, 51, 4580-4586. Low, M. J. D.; Lacroix, M.; Morterra, C. Appl. Spectrosc. 1982, 36, 582-584. - - - .. Buffett, C. E.; Morris, M. D. Anal. Chem. 1082, 54, 1824-1825. Rosencwaig, A. ”Photoacoustics and Photoacoustic Spectroscopy”; Wiley: New York, 1980.
__
RECEIVED for review June 6,1983. Accepted October 3,1983. This work was supported by Research Grant GM28484 from the National Institutes of Health.
Comparison of Suppressed and Nonsuppressed Ion Chromatography for Determination of Chloride in Boric Acid H. H. Streckert* and B. D. Epstein G A Technologies, Inc., P.O. Box 85608, S a n Diego, California 92138
A comparison was made of suppressed and nonsuppressed Ion chromatography ( I C ) to determine chlorlde Ion in borlc acld solutions. On first Inspection, It appears that good separatlon between borate and chiorlde Is obtalned wlth suppressed IC. However, quantkatlon of Chloride is compilcated by an unldentlfied matrix effect. This determination can be accompllshed wlth nonsuppressed IC. Chloride Is concentrated on a small precoiumn prior to separation on a sllicabased anion exchange resin. Chiorlde can be determlned at concentrations as low as 2 ppb In borlc acld solutions.
Ion chromatography (IC) is attracting considerable interest as an analytical technique for the determination of ions in solution (1-3). Small et al. (4) developed the method of using conductometric detection to monitor ion-exchange separations. Ions are separated on a column containing a low-capacity ion-exchange resin. The effluent from the separator column is subsequently passed through a suppressor column which converts the highly conducting eluent to one of much lower 0003-2700/84/0356-0021$01.50/0
conductance. With the high background conductivity thus reduced, the ions of interest are detected conductometrically. Fritz et al. (5,6) described a method of IC which does not require the use of a suppressor column. A low-capacity resin is employed in conjunction with a low conductivity eluent which can be passed directly through the conductivity detector. Both suppressed and nonsuppressed techhiques have been employed for anion analyses in water samples (1-6). Quantitative determination of chloride ion impurity in boric acid solutions is an important requirement in the nuclear reactor industry. Boric acid is added to the primary coolant in pressurized water reactors to aid in the control of reactivity. Chloride ion can lead to stress corrosion of reactor components. Typical procurement specifications allow a maximum of 0.4 pg of chloride/g of boric acid (ppm) (7). Due to the limited solubility of boric acid in water (-50 g/L), a maximum solution concentration of -20 pg/L (ppb) chloride will be obtained by dissolving boric acid at ambient temperature. Chloride can be determined at the parts-per-billion level in aqueous media by a spectrophotometric technique (8). Solutions of ferric ammonium sulfate and mercuric thio1983 American Chemical Society
22
ANALYTICAL CHEMISTRY, VOL. 56, NO. 1, JANUARY 1984
cyanate are added to the sample. The chloride ion reacts with the mercuric thiocyanate to liberate thiocyanate ion. Thiocyanate complexes with ferric ion to form red ferric thiocyanate which has been shown to obey Beer’s law. This method has several disadvantages. One drawback arises from high blank readings produced by the ferric ammonium sulfate and mercuric thiocyanate reagents. A high blank coupled with a low incremental absorbance due to the ferric thiocyanate complex a t low chloride levels limits the usefulness of this technique. Two methods of IC, suppressed and nonsuppressed, for the determination of chloride in boric acid are compared in this paper. It is demonstrated that suppressed IC can give erroneous results for the impurity chloride Concentration. Nonsuppressed IC yields adequate sensitivity and specificity for the analysis. Boric acid solutions have been analyzed successfully for low ppb concentrations of chloride with nonsuppressed IC. EXPERIMENTAL SECTION Apparatus. A Dionex Model 2010i ion chromatograph, modified as described below, was used throughout the study. Chromatrograms were recorded either on a Hewlett-Packard Model 3390A recording integrator or a Linear Instruments Corp. Model 255 strip chart recorder. In all cases the detector response parameter used was peak height. Suppressed IC. A 3 X 50 mm anion guard column was used in series with a 3 X 150 mm anion separator column (Dionex, HPIC-ASP) and a fiber suppressor (Dionex). The eluent consisted M Na2C03and 2 X M NaOH. The of a solution of 3 X flow rate was 2.3 mL/min. The fiber suppressor was continuously regenerated with 0.025 N HzS04. A Dionex Model CDM-1 conductivity detector was employed. 1nje.ctionvolumes ranged from 50 to 150 pL. Nonsuppressed IC. A 4.6 X 250 mm standard anion column (Wescan) was used with a 3.9 X 10” M potassium acid phthalate (pH 4.1) eluent. The flow rate was 2.3 mL/min. A concentrator column consisting of a 4.6 X 30 mm Ion-Guard Anion Cartridge in an Ion-Guard Holder (Wescan) replaced the sample loop in the injection valve and was loaded “on-line” in order to avoid sample contamination. Samples were loaded either manually with a plastic syringe or with a Cole-Parmer Model 7013 peristaltic pump at a rate of -1 mL/min. A Wescan Model 213A conductivity detector was employed. A calibration c w e was obtained by injecting freshly prepared standards containing 4, 8, 16, 32, and 64 ppb chloride, respectively. Reagents. Chemicals which were used as received were NaOH (Baker), NaZCO3,and potassium acid phthalate (Mallinckrodt). NaCl (Baker) was dried at 120 “C for 3 h prior to use. Chloride standard solutions were prepared from a lo00 mg/L stock solution. Boric acid samples were obtained from Southern California Edison, San Onofre Nuclear Generating Station. Water used throughout the study was distilled water which was subsequently passed through a Barnstead Ultrapure mixed bed resin. It was analyzed by IC to contain S0.5 ppb chloride. All eluents were made from this water after boiling. RESULTS AND DISCUSSION Suppressed Ion Chromatography. Most of the work was performed with a pellicular anion-exchange resin containing partially sulfonated polystyrene/divinylbenzene beads and M an eluent consistiiig of a 3 x M NaZCO3and 2 X NaOH. Initially, we sought to determine whether chloride can be detected with adequate sensitivity in the presence of a large excess of boric acid. A 150-pL sample loop proved sufficient to obtain a good chloride response. A typical chromatogram of the impurity chloride in boric acid is shown in Figure 1. The negative peak, A, commonly referred to as a “water dip”, results from the dilution of the eluent by the water sample. The large peak B, having an elution time of 1.3 min from the point of injection, is due to borate. After nearly base line resolution the chloride elutes at -3.8 min, peak C. Peak D is due to sulfate and is observed
-
I
I
I
I
L
0
TIME, MINUTES
Figure 1. Chromatogram of a 3 % boric acid solution obtalned under suppressed I C conditions (see Experimental Section). Peak A is the “water dip”. Peaks B, C, and D are due to borate, chloride, and sulfate, respectively.
Table I. Chloride Analysis in Boric Acid detector response for chloride sample suppressed nonsuppressed (% boric acid)‘]! IC IC 0.5 1.0 2.0 4.0
62 120 40 47
-b
10 18 45
a Four grams of boric acid was dissolved in 100 mL of water. Successive dilutions of this sample were made N o peak above the noise to obtain the other samples, level was observed for this samole.
a t -13 min. For this comparison only the chloride was considered. At first inspection it appears that good separation between borate and chloride is obtained under the chromatographic conditions employed in this study. The good sensitivity and apparently adequate separation obtained in these chromatograms seemed to allow the quantitative analysis of chloride. However, the chloride response depends not only on the chloride concentration but also on the boric acid concentration as evidenced by the following experiment. Samples containing 40, 20,10, and 5 g of boric acid per liter were analyzed. Eliminating adventitious sources of chloride contamination, the chloride response should be in proportion to the boric acid content. The results given in Table I show that a matrix effect is occurring. As the boric acid concentration increases the chloride response increases initially but then declines. This type of behavior prevents quantitation of chloride by a calibration curve obtained in the absence of boric acid. Attempts to quantify the chloride concentration by the standard additions method were also limited by this type of matrix effect. As the chloride concentration is purposely increased by spiking the sample with a standard chloride solution, a “leveled” chloride response is obtained. Plotting chromatographic response vs. standard added results in a straight line with a slope which is too low and an apparent chloride concentration which is artificially high. This can be demonstrated with a recovery study. Spiking a sample with 0.8 pg of chloride yielded a recovery in excess of that of an unspiked sample of -1.2 pg of chloride. It should be noted that during the course of these experiments no change in retention times was observed, indicating that the capacity of
ANALYTICAL CHEMISTRY, VOL. 56, NO. 1, JANUARY 1984
23
~~
0
1
Table 11. Recovery Study for Nonsuppressed IC pg of C1total pg of net pg of % addeda C1- found C1- foundC recovery 0 0.40 0.80 1.60
0
I
I
I
1
6
10
15
20
I
T I M E MINUTES
Flgure 2. Chromatogram of a 4 % boric acid solution obtained wlth nonsuppressed IC (see Experlmental Section). Peak A Is the “water dip”. Peak B is attributed to be a solvent front disturbance. Peak C represents -8 ppb chloride. Peak D has been identified as carbonate which coelutes with sulfate at the pH employed. Note the scale change for this peak. Borate Is not retained under the condtlons used to obtain this chromatogram.
the column was not exceeded and the column did not change with time. In an attempt to minimize these matrix effects we diluted the eluent incrementally up to a factor of 2. This change resulted in better peak separation but did not improve the quantitation. Dissolving boric acid in the eluent, in the hopes of preequilibrating the sample, gave the same type of response. Adding lo-* M mannitol to the eluent, in order to form a boric acid-mannitol complex also did not eliminate the matrix effects. Nonsuppressed Ion Chromatography. Further investigation demonstrated that nonsuppressed IC could also be used to analyze for chloride in boric acid solutions. Good separation results with use of a low-capacity bonded-phase M potassium acid silica anion exchanger and a 3.9 X phthalate eluent. To obtain the required sensitivity 10 mL of sample was preconcentrated on a small column containing the same ion-exchange resin as the analytical column. Because the boric acid itself is not retained on this type of column at the pH employed it is not concentrated and does not interfere with the chloride loading. The use of a preconcentrator column was precluded in this application of suppressed IC because the pellicular resin employed concentrates both chloride and borate. The preconcentrator column used for nonsuppressed IC cannot be employed for the suppressed IC because the silica-based resin in that column is not compatible with the eluent used for suppressed IC. The concentrator column is substituted for the sample loop in the injection valve. After the sample is loaded onto the concentrator column, the ions that were retained are swept into the analytical column by directing the eluent flow through the concentrator column. It has been demonstrated previously that sample ions are quantitatively retained on the concentrator column during the loading process (9). A typical chromatogram of a boric acid sample under these conditions is shown in Figure 2. The chloride elutes at -5 min adjacent to a large peak attributed to be a solvent front disturbance. Sulfate and carbonate coelute at -22 min. Varying the boric acid concentration has no adverse effect on the chloride response as demonstrated in Table I. No significant chloride peak was detected for the most dilute solution under our chromatographic conditions. However, at the 1% boric acid level a chloride peak was observed. The chloride peak increased monotonically as the boric acid content was increased to 4%. A calibration curve was established which was linear from 4 ppb to 64 ppb in chloride with a correlation coefficient of 0.993. The detection limit in solution was determined to be about 2 ppb. This limit could be lowered by loading more sample onto the concentrator column. The absence of matrix
0.24 0.62 1.05 1.94
-
-
0.38 0.81
95 101
1.70
106
a Amount of chloride added to 50 mL of 4% boric acid sample; 50-pL aliquots of an 8 ppm chloride standard were added incrementally. Sample peak height was converted to concentration via interpolation of the calibration curve. The total chloride was then calculated from the sample volume and chloride concentration. Amount of chloride found in excess of the amount found in the unspiked sample.
Table 111. Chloride Analysis in Boric Acid
sampleb 842C-1 84 2C-1 3 842C-9 6 52C-7 3
chloride, ppm in solida spectrononsuppressed photometric IC methodd 0.20 0.18 0.14 0.09
0.27 0.16
0.16 0.13
a Concentration of chloride in 4% boric acid sample was determined by the indicated technique and then converted to micrograms of chloride per grams of solid Samples of boric acid obtained from boric acid. Southern California Edison, San Onofre Nuclear Measurements made by nonsupGenerating Station. pressed IC are estimated accurate to 10% based on The ferric ammonium sulfate/mercuric recovery data. thiocyanate method was employed. Chloride was quantified by standard additions. Table entries are estimated to be accurate to +30%.
effects and analytical utility of this technique was demonstrated by adding known amounts of chloride to a solution of boric acid. Three 50-mL samples of a 4% boric acid solution were spiked with 0.4,0.8, and 1.6 bg of chloride. The results in Table I1 show that the recoveries ranged from 95 to 106%. The relatively consistent chloride recovery data indicate that there were no major interferences in the analysis. The results of chloride analyses for boric acid solutions are presented in Table 111. The values obtained are compared with data from the ferric ammonium sulfate/mercuric thiocyanate spectrophotometric method. Relatively good agreement between the two techniques is observed; the two determinations generally differ by less than 30% and are well within the error bars associated with each technique. These samples, obtained from Southern California Edison, San Onofre Nuclear Generating Station, are also in compliance with the procurement specifications (vide supra). The absence of certified standards for chloride in boric acid makes it difficult to evaluate the absolute accuracy of the technique. However, the recovery data indicate that the accuracy of the quantitation of chloride can be in the range of 5-6% relative to the chloride present in excess of background. This finding coupled with the comparison to the spectrophotometric technique indicates that nonsuppressed IC could serve as a routine method for the determination of chloride in solid boric acid at the 0.4 ppm level. In summary, while apparently good separation of chloride from borate can be obtained by using suppressed IC, quantitation of low level chloride is complicated by an unidentified matrix effect under the chromatographic conditions employed in this study. Quantitation of impurity chloride in boric acid can be accomplished by using nonsuppressed IC and a con-
Anal. Chem. 1984, 56,24-27
24
centrator column to ensure the necessary sensitivity.
ACKNOWLEDGMENT We thank John Dixon for performing the spectrophotometric chloride analyses. We are also grateful to Southern California Edison for providing boric acid samples and verification test plans. Registry No. Boric acid, 11113-50-1.
LITERATURE CITED (1) Rocklin, R. D.; Johnson, E. L. Anal. Chem. 1983, 55, 4-7. (2) Green, L. W.; Woods, J. R. Anal. Chem. 1981, 53, 2187-2189. (3) Pohlandt, C. S A f r . J. Chem. 1982, 35, 96-100.
(4) Small, H.; Stevens, T. S.;Bauman, W. C. Anal. Chem. 1975, 47, 1801-1809. (5) Gjerde, D. T.; Frltz, J. S.;Schmuckler, G. J. Chromarogr. 1979, 186, 509-519. (6) Gjerde, D. T.; Schmuckler, G.; Fritz, J. S. J. Chromafogr. 1080, 187, 35-45. (7) Verification Test Procedure for Safety Related Borlc Acid; Southern Callfornia Edison Go., San Onofre Nuclear Generating Station, San Onofre, CA. (8) Iwasaki, I.; Utsumi, S.; Ozawa, T. Bull. Chem. SOC.Jpn. 1952, 25, 226-229. ~~. (9) Wetzel, R. A.; Anderson, C. L.; Scheicher, H.; Crook, G. D. Anal. Chem. W79, 51, 1532-1535. ~~
RECEIVED for review June 30,1983. Accepted September 19, 1983.
On-Line Extraction, Evaporation, and Injection for Liquid Chromatographic Determination of Serum Corticosteroids Kitaro Oka, Kazuo Minagawa, and Shoji Hara* Tokyo College of Pharmacy, Horinouchi, Hachioji, Tokyo 192-03, Japan Makoto Noguchi, Yasuo Matsuoka, Michinori Kono, and Shoichiro Irimajiri Kawasaki City Hospital, Kawasaki 210, Japan
To construct an on-line system for cleanup of bloiogicai fluids, closed-bed columns packed with fine diatomaceous earth granules were developed. Three cascaded columns were coated with neutral water, aqueous sodium hydroxide, and sulfuric acld since the extraction of neutral components with a hlgh degree of selectivity is possible by such stationary phase iiqulds. The recovery of steroid solutes from sera was almost quantltatlve, using aqueous diethyl ether as the carrier. The effluent from the aqueous liquid-iiquld partition columns was introduced Into a line evaporator made of glass tubing. The injection of the sample into a liquid chromatography column was performed by inserting a steel stick for eliminating the dead space in the evaporator to resolve the residuie A model of an on-line equipped extraction, evaporation, and injection system was constructed by the incorporation of a valve switching procedure. This technique was applied to a clinical assay of corticosteroids In sera and was found suitable as a highly sensitive method for monitoring steroidal drugs.
For determination of constituents in biological fluids such as serum and urine samples, preliminary fractionation and/or enrichment of target compounds is usually recommended. Elimination of contaminants from the fluids consisting of a complex matrix enhances the sensitivity of analysis of the compounds under consideration. In many instances, the cleanup of biological fluids has been carried out by manual batch operation prior to high-performance liquid chromatography (HPLC) analysis. Recently, two new procedures have been introduced: (1) liquid-liquid distribution using columns packed with diatomaceous earth support and (2) prechromatography involving either a normal or reversed phase system employing a silica gel or octadecylsilyl silica columns. Commercially available disposable short columns such as Extrelut by Merck, Darmstadt, and Sep-PAK cartridges by Waters, Milford, MA, are being widely accepted by analytical chemists. However, it is difficult to transfer the
effluent from the cleanup column directly into the main analytical system. Sample preparation by collecting the fraction and evaporation of the solvent is often necessary. Hence these prechromatographic techniques require off-line processes. At our laboratory, column switching extraction and chromatography for programmed flow preparation (PFP) have been developed (1,2). PFP has improved the preparation system and avoids the defects encountered in former procedures using batch processing in chemical laboratory experiments. Through the PFP concept, an attempt has been made to develop an on-line technique for fractionation, evaporation, and injection in microchemical analysis. First, a suitable design for the liquid-liquid distribution column system for on-line procedure was investigated. Instead of the open-bed cartrige columns commonly employed in the laboratory, the closed-bed columns slurry packed by fine diatomaceous earth granules were introduced ( 3 , 4 ) . These cleanup columns were found to have high efficiency and could be used repeatedly if the columns are flushed by water soon after the injection of certain amounts of biological fluids. Secondly, a new type of evaporator made by glass tube was developed to afford quantitative recovery of small amounts of solutes. There was hardly loss of any sample when the residue in the evaporator was injected into the analytical column (3). As a result, construction of the on-line system for switching extraction, evaporation, and injection into the HPLC column (LEEI system) was achieved. Several applications of this system revealed high sensitivity and efficiency for HPLC analysis of biological samples. In this paper, the application of this technique to the clinical analysis of serum corticosteroids is reported.
EXPERIMENTAL SECTION LEEI System. A model for a switching fractionation and chromatography system is illustrated in Figure 1. This sytem is integrated by four functional operations: (a) presaturation of water for the mobile phase solvent; (b) sample application and extraction of the constituents; (c) evaporation of the effluent; (d)
0003-2700/84/0356-0024$01.50/00 1983 American Chemical Society
