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ANALYTICAL CHEMISTRY, VOL. 50, NO. 4, APRIL 1978
Autoclave Digestion Procedure for the Determination of Total Iron Content of Waters Sir: As colorimetric procedures for ferrous iron have been automated, solubilization of iron has been the rate determining step. Wilson (1) evaluated iron digestion procedures for small amounts of particulate matter in large volumes. “Standard Methods” (2) recommends concentrating the sample to one third of its original volume in hydrochloric acid-hydroxylamine media. The latter procedure is time consuming, rather dangerous, and the resultant acid fumes produce extremely corrosive conditions in the fume hoods. Digestion by autoclaving samples under similar acid-reducing conditions markedly reduces these drawbacks. The use of an autoclave has been described by Armstrong (3),but the technique is so uncommon that a detailed evaluation with respect to recovery was undertaken. A number of accepted colorimetric procedures for soluble or reactive iron are described in the iiterature, but the use of the chromophore 2,4,6-tri(2-pyridyl)-1,3,5-triazine (TPTZ) is particularly attractive as such systems are highly sensitive and very stable. This reagent was introduced by Diehl and Smith (4), and has been adapted by a number of authors to both manual (5, 6) and automated (7, 8) systems. T h e violet ferrous-TPTZ complex has a molar absorptivity (4) of approximately 2.23 X lo4 at 595 n m and thus the interference due to the natural color of samples can usually be eliminated by in-line dilution. The intensity of the colored complex does not change appreciably over the pH range 3.4 to 5.8 permitting stabilization of the system by incorporating a sodium acetate-acetic acid buffer. Accordingly, the manual T P T Z procedure described by Dougan and Wilson (6)was automated for the range 0 to 2.0 mg/L Fe. EXPERIMENTAL Apparatus. The autoclave was equipped with an automatic slow exhaust and capable of being operated at 121 OC. Culture tubes (25 X 150 mm) with Teflon lined screw caps were the sample containers, and racks to hold these tubes were utilized for the autoclave step. The digestant was added via an Oxford Pipettor. The TPTZ colorimetry utilized a single channel Technicon AAII AutoAnalyzer system and a single pen recorder. The drum of the Technicon sampler (large industrial model) was modified to accept the tall culture tubes. Reagents. The Digestion Acid contained 640 mL of concentrated hydrochloric acid plus 30 g of hydroxylammonium chloride per liter. Hydrochloric acid was the active or solubilizing agent but the hydroxylammonium chloride was included to prevent loss of iron by reduction to the divalent state, and to reduce any oxidizing agents which might be present in the sample. An Acid Wash was required for the AutoAnalyzer system to maintain an acceptable baseline. The concentration of hydrochloric acid (4% by volume) in this wash equaled that in the digested sample. Reagents associated with the TPTZ colorimetric phase were also required. Procedure. After thoroughly shaking the sample, a 30.0-mL aliquot was transferred to a culture tube via a wide mouth pipet, and 2.0 mL of the digestant acid was added, with an Oxford Pipettor. The Teflon-lined cap was screwed on tight, the vial placed in a rack, and the rack set on an enamel tray to protect the autoclave in case of breakage or leaking. The culture tubes (as many as 250) were autoclaved at 121 “C for 60 min. After the pressure in the autoclave had been discharged, the door was left ajar for 15 min to protect the operator against an exploding tube. To date approximately 15000 tubes have been autoclaved without breakage. Moreover, iron losses are minimal; after 200 tubes had been autoclaved, the trace of condensate in the enamel trays contained 0.12 mg/L Fe and had a pH value of 6.8. The samples were allowed to cool to room temperature before pro-
ceeding with the colorimetric phase. The use of the same vials in the colorimetric step avoided errors associated with transferring the samples.
RESULTS AND DISCUSSION In selecting the experimental conditions for solubilizing the iron, the autoclave temperature was not varied; it remained 121 “C throughout. T o select the volume of digestant, 30.0-mL aliquots of ten routine samples were autoclaved for 40 min in the presence of 1.0 mL and 2.0 mL of digestant; the same samples were also digested according to “Standard Methods” (2); using the latter data as reference, the average recovery of iron was 93% for 1 mL of digestant and 100% for 2 mL of digestant. T o select the required period of autoclaving, 20 routine samples were autoclaved for 20,40, and 60 min (2.0 mL of digestant per 30.0 m L of sample); again the same samples were digested according to “Standard Methods” ( 2 ) , and the latter data were used as reference. As the digestion period increased from 20 t o 60 min, the average recovery increased only from 99 to 101%. Although 40 min appeared sufficient, the 60-min digestion period was selected to provide a safety margin. T o confirm the overall procedure, solutions of ferrous sulfate, ferric chloride, potassium ferrocyanide, and potassium ferricyanide were analyzed. T h e measured and theoretical iron concentrations differed by less than 1% . A linear calibration was obtained for the autoclave-TPTZ procedure for total iron. T h e precision of t h e method was estimated by analyzing routine water samples in duplicate on different days, and determining the mean standard deviation of the differences for two concentration ranges. For the concentration range 0 to 0.4 mg/L Fe, the mean standard deviation was 0.017 mg/L Fe ( n = 48), and for the range 0.4 to 1.0 mg/L Fe, the mean standard deviation was 0.037 mg/L Fe ( n = 28). These levels of between-run precision also reflect the sampling difficulties associated with particulate matter, and are considered satisfactory for routine analyses. The interference study showed that iron was recovered with 2% of the theoretical value in the presence of common ions; ten cations and six anions were tested a t concentration levels in excess of those normally encountered in water samples. Neither the disodium dihydrogen salt of adenosine-5’-triphosphoric acid nor pyrophosphate anions affected the recovery of iron at the 5 mg/L P level. Sulfide, linear alkylate sulfonate, humic acid, and tannic acid were also tolerated at the 5 mg/L concentration levels. T o complete the evaluation of the autoclave-TPTZ procedure, the iron contents of 610 routine water samples from Central and Southern Ontario were determined by the subject method and by the acid digestion-phenanthroline procedure recommended by “Standard Methods” (2). The samples were divided into three groups; (a) domestic water supplies and landfill leachates, (b) river, lake, and snow samples collected in the winter months, and (c) river and lake samples submitted during the spring runoff. With the exception of the leachates (about IO%), group (a) were the “cleanest” samples. T h e linear regression analyses for the three sets of data (Table I) indicate that the two procedures give comparable results; the slopes for the comparisons were within 1% of 1.00. The mean differences and standard deviations for the three groups were (a) 0.003 mg/L Fe ( u = 0.024), (b) 0.000mg/L Fe ( u = 0.037), and (c) 0.003 mg/L Fe (a = 0.073). T h e lowest standard deviation was obtained for group (a) which normally contain little or no particulate matter while the largest standard deviation was associated with spring runoff samples which
0003-2700/78/0350-0658$01.00/0 0 1978 American Chemical Society
ANALYTICAL
Table I. Comparison of Autoclave-TPTZ and Standard Methods Procedures by Linear Regression Analysis
Statistical Parameter No. of Samples
Correlation Y o n X regressiona Slope Intercept (mg/L Fe 1
Std dev (mg/L Fe ) X on Y regression Slope Intercept (mg/L Fe 1 Std dev (mg/L
Type of water sample Domestic Rivers and Rivers and water suplakes, lakes, plies and Winter Spring leachates months runoff 251 0.9973
194 0.9948
165 0.9893
1.0097 - 0.0044
0.9982 0.0025
1.0024 0.0038
0.0 24
0.0369
0.0726
1.0043 - 0.0032
0.9879 0.0057
0.9811
0.024
0.0366
0.0720
0.0163
Fe 1
X refers t o the data obtained using the procedure from "Standard Methods" ( 2 ) .
contain so much silt t h a t it is extremely difficult to select a representative sample. Winter samples contain small amounts of particulate matter and the standard deviation was intermediate (0.037 mg/L Fe). If the autoclave digestion technique was inadequate, the measured iron content would tend to be
CHEMISTRY,
VOL. 50, No. 4, APRIL 1978
659
lower than that determined by the "Standard Method" procedure (2). Such a bias (5 to 10% lower) was found when creek and snow samples from two locations were analyzed. Although this weakness can be tolerated in view of its infrequent occurrence and the size of error involved, analysts should evaluate the autoclave digestion procedure for their particular waters before adopting the technique. LITERATURE CITED (1) A. L. Wilson, Analyst. (London), 89, 402 (1964). (2) American Public Heah Association, "Standard Methods for the Examimtion of Wafer and Wastewater", 14th ed., APHA, Washington, D.C., 1976, p 208. (3) F. A. J. Armstrong, J . Mar. Biol. Assoc. U.K.. 36, 509 (1957). (4) H.Diehi and G. F. Smith, "The Iron Reagents: Bathophenanthroline, Bathophenanthroline-Disulfonic Acid, 2,4,6-Triphyridyl-S-triazine, Phenyl-2-pyridyl ketoxime". 2nd ed.,G. Frederick Smith Chemical Company, Columbus, Ohio, 1965. (5) P. F. Collins, H. Diehl, and G. F. Smith, Anal. Chem., 31, 1862 (1959). (6) W. K. Dougan and A. L. Wilson, Water Treat. Exam., 22, 100 (1973). (7) R. D. Britt, Jr., Anal. Chem., 34, 1728 (1962). (8) A. Henriksen, Vaftenhygien, 22, 2 (1966).
Joan C r o w t h e r Ontario Ministry of the Environment Laboratory Services Branch Water Quality Section Box 213 Rexdale, Ontario Canada M9W 5L1
RECEIVED for review October 17, 1977. Accepted January 3, 1978.
On-Line Coupling of a Micro Liquid Chromatograph and Mass Spectrometer through a Jet Separator Sir: High performance liquid chromatography (HPLC) has become almost comparable to gas chromatography (GC) in separation capability and analysis time. The former, however, still has some disadvantages in its versatile detection system. Therefore, on-line coupling of HPLC to the mass spectrometer (MS) has been studied as one of the most promising approaches to develop a universal detection system: using a moving wire ( I ) , a silicon membrane separator ( 2 ) ,direct introduction of a portion of the column effluent into the chemical ionization source through a fine glass capillary (3-5) or atmospheric pressure ionization (6). The first commercial LC/MS interface was produced by Finnigan Ltd., where the moving wire was replaced with a continuous belt to enable efficient sample loading. Recently, Ishii has developed a micro LC (7, 8 ) , which requires a flow rate of only a few microliters per minute and has separation ability almost comparable to the conventional LC. In our study, an ordinary one-stage jet separator for GC/MS was used as an interface for coupling of the micro LC with MS. T h e total LC effluent solution was continuously introduced into the heated separator through a stainless steel capillary tubing and evaporated. The sample-enriched vapor is transferred to the chemical ionization source in MS where t h e solvent acts as the reagent gas. With this method, it is possible t o maintain a constant pressure of the ionization chamber even under various LC flow rates by adjusting the evacuation rate a t the jet separator, and the solute concen0003-2700/78/0350-0659$01,00/0
tration is enriched since the solute molecule is usually larger than the solvent molecule. EXPERIMENTAL Apparatus. A schematic diagram of the micro LC/MS system is shown in Figure 1. The micro LC is a FAMILIC-100 from Japan Spectroscopic Co., Ltd. (JASCO, Tokyo, Japan) equipped with a gas-tight micro syringe (250 pL in volume). Flow rates of 2, 4, 8, and 16 pL/min can be selected. The UV spectrophotometer is a UVIDEC-100 from JASCO and the flow cell (8 pL in volume) attached to the spectrophotometer was replaced by a quartz capillary cell (ca. 0.5 pL in volume). One end of the cell was directly connected to the outlet of a micro LC column of polytetrafluoroethylene (PTFE) tubing. The other end was combined with a PTFE tubing (0.1-mm i.d.) to a stainless steel capillary (30 cm in length, ca. 4 pL in volume). The capillary was directly connected to the separator through a silicon septum. The total eluent from LC was introduced into the separator, which is evacuated by a rotary pump at the maximum pumping speed of 80 L/min. The separator orifices are 0.1 and 0.3 mm, respectively, with a distance of 0.4 mm used at a temperature of 150 "C to enable the rapid vaporization of the column effluent. The mass spectrometer used is a JMS-Q1OA quadrupole mass spectrometer from Japan Electron Optics Laboratory Co., Ltd., equipped with an EI/CI ion source and an oil diffusion pump (1000 L / s pumping speed). Column. A micro column used for separation is a PTFE tubing (0.5-mm id., 7 cm in length) packed with Silica ODS SC-01 from JASCO. MS Detection. MS detection was carried out using the CI source under the following conditions: ionizing voltage of 250 eV, C 1978 American Chemical Society
