Anal. Chem. 1990, 62, 2405-2408
furthermore, the B / F separation in immunoassay will be achieved by polarization or time-resolved fluorometry. For this purpose, a new fluorometric reagent is necessary for labeling protein in the near-infrared region. Registry No. Insulin, 9004-10-8.
1 .o
LITERATURE CITED
. -'0
0.5
I
0
2405
50
100
150
Concentration/(pU/mi) Figure 1. Analytical curve for insulin. I, and I are fluorescence intensities at 0 and 15 min after initiation of the enzyme reaction, respectively.
In heterogeneous enzyme immunoassay, an additional incubation time is required for the enzyme reaction. However, more rapid competitive binding assay is used in radioimmunoassay: the sample is readily measured after the immunological reaction and the succeeding phase separation of bound and free (B/F) antigens. Competitive binding fluorescence immunoassay based on semiconductor laser spectrometry may provide us with a more practical means for fluorometric determination of protein: low background fluorescence in the near-infrared region is essential in ultratrace analysis, and
(1) Lidofsky, S. D.; Imasaka, T.; &re, R. N. Anal. Chem. 1979, 5 7 , 1602. (2) Lidofsky, S. D.;Hinsberg, W. D., 111.; Zare, R. N. R o c . Natl. Acad. Sci. U . S . A . 1981, 78, 1901. ( 3 ) Hinsberg, W. D., 111.; Milby, K. H.; Zare, R. N. Anal. Chem. 1981, 5 3 , 1509. (4) Imasaka, T.; Ishibashi, N. Anal. Chem. 1990, 62, 363A. (5) Sauda, K.; Imasaka, T.; Ishibashi, N. Anal. Chem. 1986, 5 8 , 2649. (6) Imasaka, T.; Tsukamoto, A,; Ishibashi, N. Anal. Chem. 1989, 67, 2285. (7) Imasaka, T.; Okazaki, T.; Ishibashi, N. Anal. Chim. Acta 1988, 208, 325. (8) Handbook of Chemical Substances (Kagaku Binran), Fundamental I I ; The Chemical Society of Japan, Ed.; Maruzen: Tokyo, 1975; p. 1490. (9) Imasaka, T.; Yoshitake, A.; Ishibashi, N. Anal. Chem. 1984, 5 6 , 1077. To whom correspondence should be addressed
Totaro Imasaka Hiroyuki Nakagawa Takashi Okazaki Nobuhiko Ishibashi* Faculty of Engineering Kyushu University Hakozaki, Fukuoka 812, Japan
RECEIVED for review May 7, 1990. Accepted July 19, 1990. This research is supported by Grants-in-Aid for Scientific Research from the Ministry of Education of Japan and Naito Foundation.
TECHNICAL NOTES Reversed Injector Loading Technique for Simultaneous Determinations by Flow Injection Analysis Jose Luis PBrez Pavbn,* Carmelo Garcia Pinto, Bernard0 Moreno Cordero, and Jesiis Hernandez MBndez Department of Analytical Chemistry, Bromatology a n d Food Sciences, University of Salamanca, Salamanca, Spain Usually flow injection methods are based on the measurement of a single signal depending on the analyte concentration. However, this methodology also permits multidetection and multidetermination, the difference between these two terms having been established by Luque de Castro et al. (I). The same authors have reviewed the proposed configurations allowing multidetection and multidetermination by flow injection analysis (FIA) (2). The more usual ways to carry out multidetermination are sequential injection and sample splitting ( 3 , 4 ) . The use of a two-valve injector (5, 6) or an eight-port valve (7) allow simultaneous determinations in FIA. In this paper a six-port valve is used for the first time to carry out multidetermination by a single injection.
PRINCIPLE The term "reversed injector loading technique" is used to indicate that in the inject mode the flow through the sample loop is opposite to the flow in the loading mode (Figure 1). If a chemical reactor (i.e. a reducing column) is included in
the loop, when the valve in turned to the inject mode, two zones of sample are inserted into the carrier stream, one of them having undergone a differentiating chemical process, thus originating two signals in the detector.
EXPERIMENTAL SECTION Reagents. Stock solutions of uranium and thorium (2.0 X M) prepared by dissolving appropriate amounts of uranyl nitrate hexahydrate (Merck) and thorium nitrate pentahydrate (Merck) in water. Stock solutions (lo-* M) of Fe(II1) and Fe(I1) were prepared by dissolving appropriate amounts of their chlorides in 0.1 M HC1. Stock solutions of nitrate and nitrite (10" M) were also prepared from sodium nitrate (Panreac) and sodium nitrite (Panreac) in aqueous 1%NH4C1 (Panreac). Carrier solutions: 3.6 M HCl for the spectrophotometric determination of Th and U; 0.1 M HC1 in 0.3 M NaCl for the spectrophotometric simultaneous determination of Fe(I1) and Fe(II1);aqueous 1% NHICl for the amperometric determination of nitrate and nitrite. Reagent solutions: 2.0 X 10"' M Arsenazo I11 in 3.6 M HC1 (in the presence of 1%Triton X-100) for the spectrophotometric
0003-2700/90/0362-2405$02.50/00 1990 American Chemical Society
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*
ANALYTICAL CHEMISTRY, VOL. 62. NO. 21. NOVEMBER 1, 1990
R . .
LOAD
(01A
B2
4 20s 1
R INJECT
C-
-~
Flgure 1. Injection system: C. carrier; R. reducing minicolumn; B,, nonreduced sample loop; El,. reduced sample loop. Figure 3. Fiagrams for the Simultaneous determination of Th and U: I. 4.0 x 10-8 M ~ h2.4.0 : x io-EM v: 3, 2.0 x i o - e ~ m + 2.0 x 10.' M U.
Figure 2. Manifolds: a, spectrophotometric determinations: b. amperometric determination: C, carrier: R, reagent: P, peristaltic pump; I. injection system: L. reaction coil; D, detector; W. waste. determination of T h and U; 0.25% 1,lO-phenantroline monohydrate (Panreac) in 0.2 M citrate buffer (pH = 5.2). Apparatus. The flow system (Figure 2) comprised a peristaltic pump (Gilson Minipuls 2 HP-4) and a Rheodyne 5020 six-port injection valve. All connections were 0.5 mm i.d. Teflon tubing. For the spectrophotometric determinations (Figure 2a) a Perkin-Elmer Coleman 55 with a 1-cm flow-through cell (18 pL, Hellma 178 12-QS)was used. For the ampemmetric determination (Figure 2h) a Metrohm E-656 electrochemical detector furnished with glassy carbon, silver/silver chloride, and gold as working, reference, and counter electrodes, respectively, a Metrohm E-611 potentiostat, and a E-586 Labograph recorder were used. The reducing minicolumn was a 5 cm length of 2 mm i.d. glass tubing; it was packed with 0.1-0.3 mm lead powder for Th/U and Fe(II)/Fe(lII) systems or with =0.5 mm copperized cadmium (NOj/NO;). Small glass wool beds at each end prevent the escape of the material. Procedures. SpectrophotometricDeterminntions. The uurier solution and the reagent solution are pumped into their respective lines at equal flow rates of 1.7 mL mid' (total flow rate Q, = 3.4 mL min-'). The sample is injected into the carrier stream from 3-m knotted tube; B,, 2-m coiled tuhe) which the sample loop (BE, includes the reducing minicolumn. The length of the reaction coil L is 1 m. Absorbance is monitored at 665 nm for Th/U and 512 nm for Fe(II)/Fe(III). Amperometric Determination. The carrier solution is pumped a t a flow rate of 1.7 mL min-'. The sample is injected from the sample loop and the signal provided by the nitrite was measured at +1.000 V. The coil between the injection valve and the electrochemical cell was a 1-m coiled Teflon tube. R E S U L T S A N D DISCUSSION This new injection mode has been tested in three systems: (A) the simultaneous spectrophotometric determination of T h
and U with Arsenazo 111, (B)the simultaneous spectrophotometric determination of Fe(I1) and Fe(II1) with 1,lOphenantroline, and (C) the amperometric simultaneous determination of nitrate and nitrite A. Simultaneous Spectrophotometric Determination of Th and U w i t h Arsenazo 111. The injection mode described above is adapted for the simultaneous determination of thorium and uranium. Most of chemical and FIA variables were kept the same as in the previous work (6,8,9) and the values are given in the Experimental Section. A typical recorder output (fiagram) for solutions of thorium and uranium and for a mixture of both ions is shown in Figure 3. When an uranium solution is injected into the system, the plateau signal (section B,, nonreduced sample) is much lower than the peak signal due to the low molar absortivity of the U(VI)-Arsenazo 111 complex; the peak signal (section B,, reduced sample) corresponds to the U(IV-Arsenazo 111complex and is similar to the signal obtained when a thorium solution is injected into the system. We optimize the length of the two sections in order to get enough separation between the plateau signal and the peak signal. The influence on the dispersion of increasing the B1 loop length is less significant for knotted tubes; thus a 3-m knotted loop was chosen for the determination because it provides adequate fiagrams to measure both plateau and peak signals. The signals were found to he proportional to the cation content for concentrations up to M, the data fitting the following equations: uranium plateau
h = ((1.91 f 0.05) X 103)[U] + (0.0016 f 0.0004) r = 0.9971 uranium peak
h = ((4.12 f 0.03)
X
104)[U]
+ (0.005 f 0.003)
r = 0.9997 thorium plateau
h = ((5.57 f 0.03)
X
104)[Th] - (0.0002 f 0.0017)
r = 0.9999 The calculated detection limits (2 X noise) were 9.59
X
lo*
M for uranium and 7.18 X 1O"M for thorium. The relative standard deviations (10 identical samples, 2.0
X
U and T h were 0.91% and 0.51%, respectively.
lo4 M)for
ANALYTICAL CHEMISTRY, VOL. 62, NO. 21, NOVEMBER 1, 1990
t
After the accuracy of the method was tested with synthetic mixtures and with a standard ore (pitchblende S-12) it is being currently used in our laboratory for the simultaneous determination of thorium and uranium in ores provided by ENUSA (National Uranium Enterprise). B. Simultaneous Spectrophotometric Determination of Fe(I1) and Fe(II1) with 1,lO-Phenantroline. Several procedures have been proposed for the determination of Fe(I1) and Fe(II1) by flow injection techniques (10-12) based on the spectrophotometric measurement of the iron(I1)-1,lOphenantroline complex. We used a procedure including the injector proposed in this paper and using lead as redudor with 0.1 M HCl + 0.3 M NaCl as carrier as well as reduction medium. NaCl was added to the carrier solution and to the samples in order to avoid the formation of lead chloride in the column. The signals were found to be proportional to the cation concentration, the data fitting the following equations: Fe(I1) plateau
h = ((4.32 f 0.01) X 103)[Fe(II)] + (0.001 f 0.001)
r = 0.9999 Fe(II1) peak
h = ((4.36 f 0.01) X 103)[Fe(III)] + (0.004 f 0.001)
r = 0.9999 The calculated detection limit (2 X noise) for both Fe(I1) and Fe(II1) was 9.2 X M and the relative standard deviations (10 identical samples, 2.0 X M) for Fe(I1) and Fe(II1) were 0.73% and 0.91%, respectively.
C. Simultaneous Amperometric Determination of Nitrate and Nitrite. The reversed injection mode was also adapted to the simultaneous amperometric determination of nitrate and nitrite with a glassy carbon electrode. The electrode was electrochemicallypretreated at +1.750 V for 5 min as recommended by Fogg et al. (13). The reduction minicolumn was filled with copperized cadmium. The carrier solution and reaction medium were a 1 % (w/v) NH4Cl as recommended by Trojanowicz et al. (14). Typical recordings for blank, nitrate, nitrite, and a mixture are shown in Figure 4. The signal provided by the blank solution (when using carrier as a sample) could be attributed to some impurities in the carrier and in the minicolumn that can be oxidized at +1.000 V. In fact a change in the baseline is observed when the valve is turned to the inject mode, which implies the carrier passing through the reducing minicolumn before reaching the detector. In addition, owing to the strong influence that changes in the medium may exert on the electrode response, a decrease in the nitrite signal is observed for the subsample that passes through the minicolumn. However, no special difficulties arise from this fact and the measurement is made in each sample zone from the corresponding baseline. The signals were found to be proportional to the ion concentration up to 8.0 X M, the data fitting the following equations: nitrite plateau h(nA) = ((4.294 f 0.009) X 1 O 7 ) [ N O ~-] (14 f 3) r = 0.9999 nitrite peak h(nA) = ((2.98 f 0.01) X 1O7)[NO2-]+ (2 f 3)
r = 0.9999 nitrate peak h(nA) = ((2.759 f 0.003) X 107)[N03-]- (1 f 1)
r = 0.9999
2407
I I d -
I
Figure 4. Fiagrams for the simultaneous amperometric determination of nitrate and nitrite: 1, blank; 2, 5.0 X M nitrite; 3, 5.0 X M nitrate; 4, 2.5 X M nitrite 2.5 X M nitrate.
+
The calculated detection limits (3 x s / m ; s, standard deviation of the blank signal; m, slope of the calibration graph) were 2.5 X M for nitrite peak and 2.8 X M for nitrate peak. The reproducibility of the signal was studied for three different concentrations: 5.0 X M, 2.5 X M, and 5.0 X lo4 M. After 20 consecutive injections 9% and 3 % loss M, of signal are observed for 2.5 x 10” M and for 5.0 X respectively. No appreciable loss of signal is observed for 5.0 x lo4 M (relative standard deviation 1.0%).
CONCLUSIONS In this work a reversed injector loading technique is proposed as a new way for FIA simultaneous determinations with a six-port injection valve. Neither sequential injections nor sample splitting is required. The simplicity and the tested applicability of this injection mode offer interesting perspectives for those simultaneous determinations and speciationswhich involve a differentiating chemical process. An interesting possibility is the creation of more than two zones in the sample loop by including several differentiating chemical reactors. Work along these lines is presently under development.
Registry No. Th, 7440-29-1;U, 7440-61-1;Fe, 7439-89-6;NO,, 147976543; NO;, 14797-65-0. LITERATURE CITED Luque de Castro, M. D.; Valdrcel Cases, M. Trends Anal. Chem. 1986, 5 , 71-74. Luque de Castro, M. D.: Valdrcel Cases, M. Analyst 1984, 109, 413-419. Valdrcel Cases, M.; Luque de Castro, M. D. Flow Injection Anaksis. Principks and Applications. Ells Hoorwood: Chichester, 1987. FIAstar Flow Injection Analysis Bibliography. 1974-1964, 1985, 1986, 1987-1988. Dasgupta, P. K.; Hwang, H. Anal. Chem. 1985, 5 7 , 1009-1012. Plrez Pavbn. J. L.; Moreno Cordero, B.: Herngndez M6ndez. J.; IsMro Agudo, R. M. Ana!. Chem. 1989, 61, 1789-1791. Alonso, J.; Bartroli, J.; del Valle, M.; Escalada, M.; Barber, R. Anal. Chim. Acta 1987, 199, 191-196. Plrez Pavbn, J. L.; Moreno Cordero, B.; Rcd&uez Garch, E.; Hernindez Mlndez, J. Anal. Chim. Acta 1990, 230,217-220. Plrez PavBn, J. L.; Moreno Cordero, B. Anal. Chim. Acta, in press.
Anal. Chem. 1990, 62,2408-2412
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(10) Faizullah, A. T.; Townshend, A. Anal. Chim. Acta 1985, 767, 225-23 1. (11) Mortatti, J.; Krug, F. J.; Pessenda, L. C. R.; Zagatto, E. A. G.; J0rgensen, S. S. Analyst 1982, 107, 659-663. (12) Alonso, J.: Bartroli, J.; del Valle, M.; Barber, R . Anal. Chim. Acta 1989, 219. 345-350.
(13) Chamsi, A. Y.; Fogg, A. G. Ana/yst 1888, 113, 1723-1727. (14) Hulanicki, A.; Matuszewski, W.; Trojanowicz, M. Anal. Chim. Acta 1987, 194, 119-127.
RECEIVED for review April 16,1990. Accepted June 26,1990.
Bottle-Callbration Static Head Space Method for the Determination of Methane Dissolved in Seawater Kenneth M. Johnson,*J Jeffrey E. Hughes, Percy L. Donaghay, and John McN. Sieburth Graduate School of Oceanography, University of Rhode Island, Narragansett, Rhode Island 02882 Interactions between physical structure and microbial populations play a critical role in controlling the bacterial production and consumption of gases in estuarine and oceanic environments, especially in poorly ventilated water bodies. In the permanently stratified anoxic lower basin of the Pettaquamscutt River in southern RI, fine-scale profiles (1-cm vertical resolution) of the physical structure (salinity, temperature, density, light intensity), particle structure (chlorophyll a fluorescence, light transmission, microbial abundance), and chemical structure (oxygen, methane, hydrogen sulfide, and carbon dioxide) showed an approximately 2 m thick oxic-anoxic transition zone (OATZ) of intense microbial activity usually situated between the 2.5- and 5.5-m depths ( I ) . To determine the association between gases and the chemical, physical, and particle structure would require gas profiles of similar resolution. Current methods for the collection and analysis of gases, however, allow only a small fraction of the 200 or so required samples to be processed in a reasonable time. The primary techniques for gas analysis are dynamic and static head space techniques (2). In dynamic analysis the analytes are stripped from solution with a purging gas (nitrogen, helium, etc.), trapped, and then desorbed or refocused prior to detection. Dynamic analyses for dissolved COz in seawater followed by coulometric detection (3, 4 ) showed quantitative recoveries of C 0 2 with high precision but at a rate too slow for this work (7-10 min sample-'). In static analysis a sealed sample container (usually a serum bottle) is incompletely filled with solution so that the dissolved gases in the liquid phase can partition into the small head space (gas phase) until the partial pressure of the gas is equal in the two phases. The equilibrium gas concentration in the head space can be increased by reducing the pressure in the head space, heating, adding electrolytes, or by keeping the ratio of head space volume (gas phase) to sample volume (liquid phase) small (5-8). The latter technique is simplest in terms of manipulation and calculation but requires tedious gravimetric phase volume determinations for each analysis. In this paper we describe a simple technique for simultaneously calibrating serum bottles for both gas and liquid phases to better than f O . l mL. Once calibrated, the bottles may be reused without recalibration and volume changes due to temperature and salinity can be calculated. These bottles can serve for sample collection, preservation, gas concentration, incubation, and possibly as a reaction vessel in other analytical sequences. With this calibration technique and the static head space method, we have collected up to 100 samples during a
* To whom correspondence should be addressed.
'Present address: Department of Applied Science,Oceanographic and Atmospheric Sciences Division, Brookhaven National Laboratory, Upton, NY 11973.
profiling run, equilibrated them, and analyzed them for CH, by gas chromatography (GC) in a single day.
EXPERIMENTAL SECTION Gas- and liquid-phase volumes of 50-mL serum bottles (Wheaton, Millville, NJ, No. 223745) were calibrated with distilled water. The empty bottles and a gas impermeable butyl rubber septum stopper (Belco Glass, Vineland, NJ, No. 2048-11800) were weighed to four decimal places to get a total tare weight (WJ. After weighing, the bottles were immersed in a distilled water constant-temperature bath (Model RTE-8, Neslab, Portsmouth, NH) at a calibration temperature of 25 "C. When the bottles came to temperature, a Gilson 5-mL white polypropylene disposable pipet tip (Gilson Medical Electronics, Middleton, WI, No. P-5OOO), hereafter called the calibrator, was forced into the filled serum bottle until the tip contacted the bottle bottom, as shown in Figure 1. The calibrator sealed the bottle, which was then inverted and shaken gently from side to side until water no longer drained from the calibrator. After the calibrator was carefully removed from the upright bottle, leaving both a liquid and gas phase, the bottle was stoppered, dried, and reweighed ( Wz). The liquid-phase volume in milliliters at the calibration temperature (V1,,,) was calculated from the difference (W, - W,) (9) corrected for the buoyancy of air (10). The bottle was reimmersed to displace the gas phase, and the bottle's stopper, pierced with a 20-gauge syringe needle, was reinserted so that the water displaced by the stopper flowed through the needle until the stopper was completely seated. After the needle wai removed, the stoppered bottle was dried and reweighed (W3). The gas-phase volume in milliliters at the calibration temperature (V,,J was calculated from the corrected (IO) weight difference in grams ( W , - W2).The total volume of + the bottle at the calibration temperature (EVet) equals V,,,,. This procedure was repeated three times for each bottle with a different calibrator tip each time. Field samples were collected from a stable donut-shaped floating platform (11)moored in the deepest basin of the Pettaquamscutt River (12) by siphoning through a 1 cm i.d. black polyethylene hose (13) to prevent alterations by pumping. The siphon tubing was attached to an electronic profiler (Sea Bird Electronics, Belvue, WA), and the siphon was maintained by discharging into a sump tank extending 1 m below the water surface in the center of the platform. Gas samples were obtained as in Figure 1. The serum stopper used to seal the bottle was pierced with a 20-gauge hypodermic needle to ensure that the gas phase remained at ambient pressure when the stopper was seated. After seating, it was secured with a 20-mm aluminum seal (Belco Glass, No. 2048-00150 or equivalent) and hand crimper (Wheaton No. 224303). A simple plastic tool shown in Figure 2 was fabricated to aid in the insertion and withdrawal of the calibrator from the serum bottles. By use of the tool, the average time for insertion, withdrawal, and crimping was about 25 s, but insertion and removal can be made without the tool in about the same time if bottles with narrow mouths that cause the calibrator to seal too tightly are discarded. To retard microbial activity, the sample bottles were placed on ice until the liquid and gas phases were equilibrated in a constant-temperature shaking bath at 100 revolutions min-' for at least 12 h.
0003-2700/90/0362-2408$02.50/00 1990 American Chemical Society
