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Anal. Chem. 1984, 56,629-633
Automated Determination of Inorganic Anions in Water by Ion Chromatography Julia A. Mosko Calgon Corporation, Calgon Analytical Laboratories, P.O. Box 1346, Pittsburgh, Pennsylvania 15230
This paper describes the development and application of automated chemically suppressed ion chromatography for determlnatlon of lnorganlc anlons In a variety of Indusirlai water matrices. Chromatographlc conditions, sample pretreatment, and results of interference studles are descrlbed. Various problems and pltfalls In the appllcatlon of the technique are discussed as well as method development required to obviate unexpected interferences. Accuracy and precislon data for the method are provided.
Ion chromatography (IC) has brought about a revolution in the approach to determination of anions in water by providing an alternative to classical methods of analysis. In many laboratories, the technique of IC (1, 2) has been utilized to improve accuracy and precision of measurements previously determined by nonselective interference-prone colorimetric and turbidimetric methods (3-5). Introduction of the packed hollow fiber suppressor and the so-called high-performance analytical columns (6) has been leading to the obsolescence of the conventional IC technology used in this work (conventional analytical and suppressor columns). However, many ion chromatographs presently in operation still employ the conventional technology. Although longer analysis times are required with the conventional columns, about the same chromatography is achieved. Laboratories geared to analysis of hundreds of water Samples per day w e automated wet chemical methods extensively as production tools. However, not all samples meet the basic criteria (clear and colorless) for analysis by automated wet chemical methods. The automation of IC (7) provided an attractive supplemental methodology to mventional automated methods. The purpose of this work was to develop the application of automated IC to the determination of chloride, orthophosphate, nitrate, and sulfate in industrial water samples which were not amenable to present automated methods. Also, the automated IC system would serve as a backup unit when needed despite its lower productivity due to longer analysis times. Therefore, the automated IC method was required to approach and exceed, if possible, the sensitivities and dynamic ranges of the conventional automated methods. Additionally, the IC method would provide an automated supplemental methodology for measurement of fluoride, nitrite, and bromide which were currently determined by manual methods. Factors to be addressed included development of an approach for accurate measurement of nitrite despite the inherent problem associated with chemical reaction in the suppressor column (8)and evaluation of the effects of automatic sampling on sample integrity. Two major problems were encountered during development of the method-(A) the variability of nitrate retention time with concentration and (B) the sudden change in slope of the orthophosphate calibration curve. The change in nitrate retention time with concentration was found to be greater than
originally reported by Jenke (9). The change in orthophosphate response with concentration surfaced during the developmental work. Although the possibility of such a response as observed with orthophosphate does exist for IC determination of polyprotic species, the literature has not reported this observation. Developing an automated method that would successfully deal with these phenomena without sacrificing accuracy presented a major challenge in this study. Two types of analytical columns (standard anion and “fast-run” series) were available during the time the automated method was being evaluated. The standard anion column provided separation capabilities which permitted measurement of the seven anions of interest. The ”fast-run”columns were evaluated as a production tool since the analysis time was about half that required with the standard anion column. However, the “fast-run” columns could not be used for samples containing nitrite or bromide due to resolution problems. Exhaustive interference studies were performed for both the standard anion and “fast-run” columns. Interferences due to poor resolution, peak overlap, and retention time shifts were investigated. Final validation of the automated IC method involved generating comparative data and performing recovery studies on industrial water samples which had been previously analyzed for the majority of anions of interest by traditional automated or manual wet chemical methods. Precision data were developed on real samples which were representative of typical industrial water matrices.
EXPERIMENTAL SECTION Reagents and Standard Solutions. Reagent grade chemicals were used throughout this work and low conductivity water (1-1.5 pmhos/cm) prepared by polishing demineralized water with a strong mixed bed resin exchanger was used to prepare all standards and eluents. Standard eluent (0.003 M NaHCO3/0.W24 M Na2C03)was used for the chromatography. Periodic conditioning and regeneration of the separator columns and suppressor column employed the use of 0.1 M Na2C03and 1 N H2SO4solutions, respectively. An appropriate volume of a 1 1 solution of sodium bicarbonate/sodium carbonate (50 g/L solutions) was added to standards and samples to produce a matrix background similar to the eluent. This minimized interference from the “water dip” that is common to IC analyses. Instrumentation. The chromatography was conducted with a Dionex Auto Ion System 12 Analyzer (Dionex Corp., Sunnyvale, CA). The system consists of a Model 12 ion chromatograph (Dionex Corp.) coupled with a Gilson autosampler, a Columbia Scientific Industries Supergrator 3-A integrator, and a Gilford Instrument Laboratories dual pen strip chart recorder. A concentrator/precolumn (4 X 50 mm, Dionex 30825) and separator column (4 x 250 mm, Dionex 30827) containing low capacity, .strong base anion exchange resin were used for the standard seven anion determination. Two anion concentrator/precolumns (4 x 50 mm, Dionex 30825) connected in series (“fast run” series) were employed for the five anion analyses. A high-capacity suppressor column (9 X 100 mm, Dionex 30828) containing a strong acid cation exchange resin in the hydrogen form was used for all chromatography. The ion chromatograph was equipped with a 2 5 0 - ~ Lsample loop. A conductivity full-scale sensitivity setting of 30 pmhos was routinely used. Eluent pump flow rate of 35-40% (161-184 mL/h) maintained system pressure between 500 and 700 psi. Both the sample and
0003-2700/84/0358-0629$01.50/0 0 1984 American Chemical Society
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ANALYTICAL CHEMISTRY, VOL. 56, NO. 4, APRIL 1984
830
Table I. Typical Retention Times and Peak Heights of the Calibration Standard
-. ~
20
approx approx peak retention height, pV time, min
concn, mg/L
anion
5.0 5.0 20.0 25.0 25.0 5.0 50.0
fluoride (F) chloride (Cl) nitrite (NO,) orthophosphate (PO,) bromide (Br) nitrate (NO,) sulfate (SO,)
2.1 3.5 4.5 7.0 9.0
5000
15.5
50700
Table 11. Detection Limits and Calibrated Range for Sequential Seven Anion Determinations
anion fluride (F) chloride (Cl) nitrite (NO,) orthophosphate
0.1
(PO,)
bromide (Br) nitrate (NO,) sulfate (SO,) a
0.1 0.1
0.2
0.2-50.0
0.5-50.0 0.2-30.0 0.5-100.0
,"l
54000 30000 50000 24 000 24 300
11.0
detection calibrated limit, range, mg/L mg/L 0.02 0.1-15.0 0.02 0.1-20.0 0.25 0.5-30.0
c
90 b
limit of linearity, mg/L >20a > 2oa 50 50-60
0
'C
20
Maximum concentration level tested.
regeneration pumps were operated at 50% flow rate (230 mL/h). Procedure. The samples were analyzed by IC using the operating conditions previously described. Ten milliliters of sample were filtered through a 0.22-rm membrane and the filtrate was collected in a 15-mL capacity polystyrene vial containing 100 r L of 1 1 sodium bicarbonate/sodium carbonate solution. Calibration was established initially and after every six sample analyses by using the combination anion solution indicated in Table I. The calibration standard was prepared fresh daily from separate lo00 mg/L stock solutions and was fortified in the same manner as the samples with bicarbonate/carbonate solution. Analysis time per run (seven anion determination) was about 24 min. Table I1 contains information on the calibrated range of the automated IC method.
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RESULTS AND DISCUSSION Standard Seven Anion Determination. Sensitivity and linearity for each of the seven anions were established by analysis of a series of combination anion standards (series A, fluoride, orthophosphate, and nitrate; series B, chloride, bromide, and sulfate; series C, nitrite) of increasing concentrations. Both peak height and peak area measurements were plotted vs. concentration. Comparable results were obtained for both modes of quantitation and it was decided that peak height (easily confirmed by manual calculation) would be employed for the automated quantitation. Variation in sensitivity of orthophosphate was noted with relation to concentration range as indicated in Figure 1. Linear responses were obtained for concentration ranges 0-25 mg/L PO4 and 30-80 mg/L PO,; however, at a concentration between 25 and 30 mg/L PO4 the sensitivity changed. To establish a broad linear range, the data were evaluated by using the method of linear regression. Up to a concentration of 55 mg/L PO4, the actual phosphate values did not vary significantly from the linear regression plot. Attempting to quantitate phosphate a t concentrations above the 55 mg/L level would result in significant positive error. The degree of error would increase as the phosphate concentration increased. Because of the linearity problem the dynamic range of the method was limited to 0-50 mg/L PO4. Calibration of the system was established by using a concentration of
CC
50
MG/L ORTHOPHOSPHATE
71
53
00
90
[Poi)
Figure 1. Linearity of orthophosphate obtained by using an automated IC method for sequential determination of seven inorganic anions in
water. Table 111. Change in Retention Time with Concentration of Nitrate
nitrate, mg/L
retention time, min
retention time normalized to 5.0 mg/L NO,, min
0.2
12.4 11.2
t1.2 0
10.9
-0.3
10.9 9.2
-0.3
5.0
>200a 150 120
30
20.0 25.0 45.0
-2.0
phosphate standard (25 mg/L PO,) which was mid-range for the method. Also, the phosphate concentration used for calibration fell within the range where the change in sensitivity occurred. Precision and accuracy data confirmed accurate calibration over the 0-55 mg/L range, using a 25 mg/L PO4 standard. A decrease in retention time as concentration increased was noted for nitrate. The average retention times observed during development of precision data are listed in Table 111. This phenomenon (peak migration) was a source of concern in the automated method because the integrator identifies peaks on the basis of retention time. The program entered into the integrator permitted only a 10% maximum deviation in retention time between the sample peak and a standard calibration peak. If the sample peak retention time deviated more than 10% from that of the calibration standard, the sample peak would be identified by the integrator as an extraneous peak or possibly would be incorrectly identified as some other anion having a similar retention time. A minimum reporting limit of 0.2 mg/L NO3 was a preestablished requirement for the automated IC method. The concentration of nitrate used to establish calibration had to possess a retention time with a 10% window that encompassed the retention time of a 0.2 mg/L NO3peak. Calibration with a 5.0 mg/L NO3 standard provided a reasonable compromise whereby the widest dynamic range in which identification of the required minimum reporting limit (0.2 mg/L NO3) was attainable. With this concentration of nitrate used for calibration, the range of the method was 0.2-30 mg/L NO3. Concentrations of nitrate >30 mg/L NO3resulted in retention times outside the 10% window and were identified by the integrator as extraneous peaks or sometimes incorrectly identified as bromide (anion eluting immediately before nitrate). Reproducibility of nitrite with relation to suppressor column capacity was investigated by continuous analysis of a 20 mg/L NO2 solution over a 6-h period. During this period (18 analyses performed), a 24% increase in sensitivity was observed; however, it was noted that satisfactory results (within 10% deviation) were obtained if calibration would be reestablished after every six sample analyses. Partial exhaustion (after 2 h of operation) of the suppressor column was required to
ANALYTICAL CHEMISTRY, VOL. 56, NO. 4, APRIL 1984
Table IV. Precisiona for Sequential Determination of Seven Anions in Low Conductivity Water mean nominal concn concn, (analyte), mg/L mg/L 0.087 0.1 ( F ) 1.99 2.0 ( F ) 6.59 7.0 ( F ) 13.3 14.0 (F) 0.136 0.1 (Cl) 1.73 2.0 (Cl) 7.09 7.0 (Cl) 15.9 14.0 (Cl) 0.392 0.5 (NO,) 4.56 5.0 (NO,) 10.2 10.0 (NO,) 23.5 20.0 (NO,) 0.167 0.2 (PO,) 5.0 (PO,) 4.46 27.3 25.0 (PO,) 47.6 45.0 (PO,) 0.519 0.5 (Br) 5.30 5.0 (Br) 22.3 20.0 (Br) 44.0 40.0 (Br) 0.227 0.2 (NO,) 4.74 5.0 (NO,) 16.5 20.0 (NO,) 23.4 25.0 (NO,) 0.484 0.5 (SO,) 5.0 (SO,) 4.67 20.0 (SO,) 19.9 88.9 90.0 (SO,) a
std dev, re1 std mg/L dev, % 0.009 0.149 0.392 0.548 0.022 0.045 0.282 1.06 0.067 0.292 0.739 2.62 0.011
0.129 0.563 1.16 0.020 0.070 0.305 0.719 0.010 0.056 1.04 0.444 0.022 0.052 0.112 0.929
10.3 7.5 6.0 4.1 16.2 2.6 1.1
6.7 17.1 6.4 1.3
11.2 6.6 2.9 2.1 2.4 3.9 1.3 1.4 1.6 4.4 1.2 6.3 1.9 4.6 1.1 0.6 1.0
bias, % -13.0 -0.5 -5.9 -5.0 t 36.0 -13.5 t 1.3 t 13.6 -21.6 -8.8 t 2.0 + 17.5 -16.5 -10.8 t 9.2 t 5.8 t 3.8 t 6.0 t 11.5 t 10.0
t 13.5
-5.2 -17.5 -6.4 -3.2 -6.6 -0.5 -1.2
Based on 1 2 replicate analyses.
Table V. Summary of Findings of the Interference Study concn (mg/L)/ analyte
concn (mg/L) of anion at which interference begins to occur
lO/Cl 10/NO, 2O/PO, 10/Br 10/NO, 5O/SO,
50 F > 2 0 c1 >200 NO, 150 PO, 80 Br 500 NO,
degree of interference
+ 10% +15% t 10%
negligible -100%0
negligible
a Nitrate peak present as shoulder on bromide peak and not detected by integrator.
achieve sufficient sensitivity for measurement of the desired 0.5 mg/L NO2minimum reporting level. Since use of a strong eluent to partially exhaust the suppressor column would also decrease operating time, it was decided that any sample requiring nitrite quantitation would be analyzed after the second calibration of the system (calibration performed after every six samples). No nitrite data were reported for samples analyzed by using a freshly regenerated suppressor column. The range of the method for fluoride, chloride, nitrite, and sulfate was set at that concentration for which the peak on the chromatogram as read from the strip chart recorder (lo00 mV full-scale response) would remain on-scale or slightly beyond (chloride range was slightly extended for minimization of sample dilutions). Orthophosphate range was based on the highest concentration that could accurately be measured considering the change in slope of the phosphate calibration curve. The nitrate range was limited by the fact that retention time changed with concentration and identification was based on retention time. A range of 0.5-50 mg/L bromide was
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ANALYTICAL CHEMISTRY, VOL. 56, NO. 4, APRIL 1984
Table VII. Precisiona for Sequential Determination of Seven Anions in Industrial Waters type of sample boiler water
anion F
nominal concn, mean concn, mg/L mg/L
10.0
cooling water 2.0
nitrite treated
1.0 10.0 10.0
wastewater
2.0 100.0
2.81 61.6 8.58 0.203 1.40 3.26 494 4.26 16.1 1.44 0.499 1.40 4.30 19.2 1.04 21.9 36 2 13.7 11.7 8.18 88.9 1.89 183 87.6 15.3 149 22.8 250
std dev, mg/L
re1 std dev, %
0.159 3.18 0.749 0.165b 0.034 0.117 3.84 0.302 1.39 0.064 0.036 0.205 0.085 0.126 0.100 1.02 18.1 0.386 0.202 0.183 0.693 0.146 12.5 4.59 0.302 2.27 0.663 1.43
5.7 5.2 8.7 81.5b 2.4 3.6 0.8 7.1 8.6 4.5 7.2 14.6 2.0 0.7 9.6 4.7 5.0 2.8 1.7 2.2 0.8 7.7 6.8 5.2 2.0 1.5 2.9 0.6
bias, %
-14.2
-28.0
t 4.0 i 37.0
+ 17.0 -5.5 -12.4
Four of the orthophosphate peaks were not measured by the integrator and were a Based o n 1 2 replicate analyses. included as zero in the calculation of standard deviation and relative standard deviation. selected to minimize possible interferences in nitrate determinations. At higher concentrations bromide and nitrate peaks merge. The precision for sequential anion determinations developed on standard solutions prepared with low conductivity water is listed in Table IV. In general, the precision was considered to be quite good. Most relative standard deviations were within 10%. The majority of anions exhibited an increase in standard deviation with increase in concentration of analyte. Potential interferences related to coelution problems were evaluated by analyzing combination anion standards bracketed as indicated in Table V. In all cases, the concentration of the interfering anion a t which poor resolution or coelution occurred was higher than the calibrated range of the test (Table 11)for that anion. The study indicated that any sample which was sufficiently diluted to a concentration level within the calibrated range for each anion of interest would result in minimum interferences due to poor resolution. Depending on sample matrix, analysis of as many as three separate diluted aliquots was required to quantitate all anions of interest. Comparative and recovery data were developed for typical industrial process water samples which had been previously analyzed by traditional methods. The sample selection included a cross section of waters with varying concentration levels of anions. Each sample was analyzed and fortified with known concentrations of at least three different anions selected by significance or historical data based on typical matrix for sample type. In general, the largest number of problems with recovery of added anion were encountered with orthophosphate at the lower concentration range of the test where precision data had indicated a negative bias. Table VI contains some examples of comparative data. Most results agreed within experimental limits for the specific concentration range and test method used. Traditional methods of analysis used for comparison purposes were as follows: fluoride, ion selective electrode method; chloride, automated mercuric thiocyanate colorimetric method nitrite,
permanganate titration method; orthophosphate, automated ascorbic acid reduction colorimetric method; nitrate, automated copper-cadmium reduction colorimetric method. Sulfate was measured with a barium-methylthymol blue colorimetric automated method. None of the samples had been analyzed for bromide prior to this study. Listed in Table VI1 are precision data obtained on samples of varying water types. Matrix appeared to have minimal effect on precision since the calculated relative standard deviations for anions in the sample matrices were about the same (510%) as those calculated for standards prepared in low conductivity water (Table IV). The major exception was orthophosphate precision for a boiler water sample containing orthophosphate a t a concentration near the minimum reporting limit of the test. For the precision study, if one of the anions of interest was not present in the sample, a known concentration of that anion was added (added anion concentration indicated in Table VI1 as nominal concentration). Five Anion Determination. Sensitivity, linearity, precision, interference, comparative, and recovery studies were repeated by using analytical conditions described for the “fast runn sequential determination of five anions. Increases in sensitivity (0.01 mg/L detection limit) with decreases in linear range (10 mg/L limit) were observed for the two earliest eluting anions, fluoride and chloride. The previously described problems encountered with orthophosphate and nitrate determinations were also present during evaluation of the five anion method. Overall, precision was considered satisfactory. However, apparently as a result of reduction in scan time, precision for the five anion determination was not as good as the precision found using the conditions employed for the seven anion determination. Although not quantitated by the five anion method, nitrite and bromide were included in the interference studies and were found to cause significant interferences when present at concentrations as low as 100 mg/L NOz or 40 mg/L Br. A recommendation was made that all automated se-
633
Anal. Chem. 1904, 56,633-638
quential anion determinations be performed by using the method developed for the seven anion determination since the seven anion method provided wider dynamic range and fewer potential interferences. Effects of Automated Sampling. The possibilities for sample contamination from the polystyrene vials used in the autosampler and carry-over from the probe and inlet line to the injection loop of the chromatograph were investigated. Several polystyrene vials were filled with the eluent used for chromatography and then analyzed after several hours of contact. The results obtained were below the minimum reporting limits for the seven anions of interest and indicated that no pretreatment of the vials was necessary. Evaluation of potential carry-over was investigated by analyzing six series of vials (three vials per series) with contents alternating between eluent and calibration standard. Immersion of the sampler probe in a rinse cell (continuously flushed with low conductivity water at a dropwise rate) during advancement of the autosampler carousel was sufficient to
cleanse the outside surface of the probe. Carry-over due to previous sample was eliminated by flushing the inlet line and sample injection loop with sample for a period of 1 min prior to injecting the sample into the eluent stream.
LITERATURE CITED Small, tiamish; Stevens, Timothy S.; Eauman, Wllllam C. Anal. Chem. 1975, 4 7 , 1801-1809. Pohl, C. A.; Johnson, E . L. J . Chromatogr. Sci. 1980, 18, 442-452. Rawa, J. A. I n “Ion Chromatographic Analysis of Environmental Pollutants”; Mullk, J. D., Sawlcki, Eugene, Eds.; Ann Arbor Science: Ann Arbor, MI, 1979; Vol. 11, pp 245-269. Wetzel, Roy; Smith, Frank C., Jr.; Cathers, Elizabeth I n d . Res. Dev. 1981, 23 (I), 152-157. Stevens, Timothy S.; Turkelson, Vlrgli T.; Albe, Wllllam R. Anal. Chem. 1977, 49, 1176-1178. Stevens, Timothy S. I n d . Res. Dev. 1983, 25 (9), 96-99. Lipskl, A. J.; Vairo, C. J . Can. Res. 1980, 13,45-48. Koch, Willlam F. Anal. Chem. 1979, 51, 1571-1573. Jenke, Dennis Anal. Chem. 1981, 53, 1535-1536.
RECEIVED for review July 20,1983.
Resubmitted January 9,
1984. Accepted January 12, 1984.
Determination of Monosaccharides as Aldononitrile, 0-Methyloxime, Alditol, and Cyclitol Acetate Derivatives by Gas Chromatography Gordon 0. Guerrant* and C. Wayne Moss Division of Bacterial Diseases, Center for Infectious Diseases, Centers for Disease Control, Atlanta, Georgia 30333
Mlxtures of neutral, alcohol, and amine monosaccharides were separated by gas chromatography after conversion to volatile derlvatlves. Neutral and amine sugars were derlvatired as aldononltrlle acetates whlle we simultaneously derlvatlzed alcohol sugars as alditol or cyclltol acetates. I n addition, neutral and amine sugars were derlvatlzed as 0 methoxlme acetates, with alcohol sugars as alditol or cyclitol acetates. Derlvatlratlon was facllkated by use of the catalyst 4-(dlmethylamlno)pyrIdlne. Stable derivatives were readily formed by both of the proposed methods, and a mixture of 28 sugars was analyzed that Included sugars contalnlng five through nlne carbon atoms. The usefulness of these methods was demonstrated by analysis of carbohydrates In whole bacterial cells.
Carbohydrates are important components of biologic materials and are present in organisms as polysaccharides attached to glycoproteins. The monosaccharide moieties of polysaccharides can be liberated by hydrolysis and subsequently analyzed to determine the individual neutral, alcohol, and amine sugars present. The identification of these constituent sugars can provide useful biologic information for recognition and differentiation of cells (1). Gas-liquid chromatography (GLC) has been used to determine small amounts of neutral sugars after conversion to volatile alditol acetate derivatives (2,3). However, the sample preparation procedure is timeconsuming for removal of excess borate used to reduce neutral sugars before acylation. Recently, reduced neutral and amino sugars were acylated without removal of borate by use of a
catalyst 1-methylimidazole (4,5). Also, by this method neutral and alcohol sugars cannot be differentiated since neutral sugars are reduced to alcohols before derivatization. Separations have been significantly improved by use of the fused-silica capillary column; recently 20 neutral and amino sugars have been determined in a single chromatographic analysis (6). The fused-silica column has also been used for the separation of trimethylsilyl (Me,Si) derivatives (7) and trifluoroacetyl (TFA) derivatives (8) of monosaccharides. However, multiple peaks from isomeric forms of sugars are produced with both Me3Si and TFA derivatives which complicate interpretation of chromatograms; also Me3Siderivatives are often unstable in storage (9). Aldononitrile acetate derivatives have been used (9-11) instead of alditol acetate derivatives because of easier preparation, greater stability, and good chiomatographicseparation and for good mass spectra (12). Although the neutral pentose and hexose sugars readily formed aldononitrile acetate derivatives, the derivatizations of glucosamine (GlcN), galactosamine (GalN), and mannosamine (ManN) were not reproducible (10). Therefore, the hexosamine sugars were derivatized (10) as the 0-methyloxime acetates which were stable and could be readily chromatographed. Glycoproteins were analyzed (IO)by first separating the neutral and amine sugars of hydrolyzed glycoproteins using ion exchange chromatography. These respective sugar fractions were derivatized separately as aldononitrile acetates and 0-methyloxime acetates and then the two derivatives were combined for a single chromatographic analysis. Aldononitrile acetate derivatives of neutral sugars and glucosamine have been prepared by using 1-methylimidazole
This article not subject to U S . Copyright. Publlshed 1984 by the American Chemical Society
