1622
Anal. Chem. 1983, 55, 1622-1624
Table 111. Results of a Comparison Study for Present, Phenate, and Selective Electrode Methods for Determination of Ammonia in River Samples concn of NH,? MM selective sample present phenate electrode Kizu River 2.28 4.0 3.6 (Dec 13, 19$82) (2.2;)c Uzi River 14.5 16.5 16 (Dec 13, 1982) (1!.3)' Katzura River 227 232 250 (Dec 20, 1982) (227J' Yodo River 84.2 88.5 96 (Dec 20, 1982) (84.0)' a Average of duplicate determinations. Determinations were performed at pH 13. Determinations were performed at pH 9. determination of ammonia by this method. The interference of amines is shown in Table 11. Primary amine such as methylamine and ethylamine among the amines interfered positively a t p H 13, since they permeate through microporous P T F E membrane and react with the OPA reagent similarly to ammonia. As they are more basic than ammonia, their permeability is expected to decrease with decreasing p H more rapidly than that of ammonia. The results a t p H 9 in Table I1 indicate that this expectation is the case. At p H 9, primary amines did not interfere at the concentration level 10 times smaller than that of ammonia. However, the fluorescence intensity of ammonia derivative was reduced to three-fifths of that a t pH 13. From the above results, it is said that no primary amine exists in a sample solution when the value obtained at pH 9 coincides with that obtained at pH 13. On the other hand, if the value obtained at pH 9 is lower than that at pH 13, the contribution of primary amines must be considered for the determination of ammonia in the sample solutions. Secondary and tertiary amines which interfere with the determination by an ammonia selective electrode (6) did not interfere at the concentration level 10 times higher than that of ammonia, since they do not react with OPA reagent, even if they permeate through microporous P T F E membrane. Application. To demonstrate real sample application, recovery studies of ammonia on a spiked river sample were carried out. The percentage recovery ranged from 95 to 105% over the concentration range of lo4 to M.
Several river samples were analyzed by the present method. The results are compared with those obtained by phenate and ammonia selective electrode methods in Table 111. Below M ammonia, the calibration curve for the selective electrode method displayed some nonlinearity. Because of the considerable time required for the electrode potential to stabilize at these low levels, the accuracy of data obtained from this method is poor. The phenate method requires preliminary distillation to separate ammonia from interfering substances. I t took 45 min to collect above 95% of the ammonia by the distillation step. The total analytical time was approximately 70 min. Compared with these methods, the present method is very fast and the time required for analysis was 6 min. The concentration of ammonia in the Katzura River was exceptionally high. This may be because the Katzura River receives the discharge of effluent from the domestic waste treatment plants. For all samples, the values obtained at pH 13 were in agreement with those at pH 9 within experimental error. Therefore, primary amines do not exist a t a level of lo4 M or more in these rivers. According to the present method, the concentrations of M ammonia in rainfall a t Sakai were found to be 3.0 X and 2.3 X M. These values are in the same order of magnitude as those reported by Vijan and Wood (7)(2.9 x M), and Hendry and Brezonik (14) (0.9 X M) a t Gainsville, FL. Registry No. Ammonia, 7664-41-7; poly(tetrafluoroethylene), 9002-84-0; o-phthaldialdehyde, 643-79-8; water, 7732-18-5.
LITERATURE CITED (1) Patton C. J.; Crouch S. R. Anal. Chem. 1977, 4 9 , 464-469. (2) Ngo T. T.; Phan P. H. A,; Yam F. C. Anal. Chem. 1982, 5 4 , 49-51. (3) "Standard Methods for the Examination of Water and Wastewater", 15th ed.; American Public Health Association: Washington, DC, 1980; pp 351-366. (4) Midgiey D.; Torrance K. Analyst (London) 1972, 9 7 , 628-633. (5) Beckett M. J.; Wllson A. C. Water Res. 1874, 8 , 333-340. (6) Lopez M. E. Rechnitz G. A. Anal. Chem. 1982, 5 4 , 2085-2089. (7) Vijan P. N.; Wood R. G. Anal. Chem. 1981, 5 3 , 1447-1450. (8) Vijan P. N.; Wood R. G. Anal. Lett. 1982, 75 (B8), 699-707. (9) Roth M. Anal. Chem. 1971, 4 3 , 880-883. (10) Danleison N. D.; Conroy C. M. Manta 1982, 29, 401-404. (11) Aoki T.; Munemori M. Anal. Chem. 1983, 55, 209-212. (12) Lindroth P.; Mopper K. Anal. Chem,; 1979, 5 1 , 1867-1674. (13) Sill6n, L. G., Martell, A. E., Eds. Stability Constants of Metal-Ion Complexes"; Burlington House: London, 1984. (14) Hendry, D. C.; Brezonik, L. P. Envlron. Scl. Techno/. 1980, 14, 843-849.
RECEIVED for review Janurary 27, 1983. Accepted April 19, 1983.
Linearity Testlng of Ultravlolet Detectors in Liquid Chromatography C. D. Pfelffer," J. R. Larson, and J. F. Ryder Analyilcal Laboratories, Dow Chemlcal U S A . , Mldland, Mlchlgan 48640
The ultraviolet spectrophotometer has become the single most useful detector for high-performance liquid chromatography (LC) because of its sensitivity, versatility, and broad dynamic linear range (1). However, in a detector of this type, there must be a compromise involving the slit width between sensitivity and broad linear dynamic range (2). Increasing the slit width permits a larger amount of energy to pass through the flow cell leading to a reduction in noise. Therefore, the slit width may be increased to improve de-
tectability by improving the signal-to-noise ratio up to the point when stray light increases significantly, and a linear response is no longer obtained. Conversely, the maximum linear dynamic range necessary for highly accurate assay-type analyses is achieved by using a narrow slit width. A theoretical discussion of the relationship between sensitivity and linearity, published by Stewart (3), reached a similar conclusion. Due to the theoretical complexity in the relationship between sensitivity and linearity in ultraviolet detectors, it would
0003-2700/83/0355-1622$01.50/00 1983 Amerlcan Chemical Society
ANALYTICAL CHEMISTRY, VOL. 55, NO. 9, AUGUST 1983
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appear that a simple. experimental procedure for testing detector linearity would be beneficial. As a first step in this direction, the American Society for Testing and Materials (ASTM) recently included a static linearity test in a Standard Practice for testing fixed wavelength photometric detectors used in LC (4). In this test, ASTM suggests choosing a compound having a broad spectral band in t h e region of the chosen wavelength. While such a compound would be suitable for testing the linearity of the detector electronics, it is not adequate for testing the optical system. This is becausie in a typical multiple component mixture, it is unlikely that a single wavelength can be selected where all components have a broad spectral band. A more realistic linearity test would include p1 compound having an ultraviolet spectrum changing a t the wavelength of interest as would be the case in the analysis of a multicomponent mixture. This paper describes such a test and identifies two commonly available compounds suitable for testing the linearity of detectors at the commonly used wavelengths of 214, 2154, and 280 nm. EXPERIMENTAL SECTION Reagents. Benzaldehyde and benzoic acid of “Baker Analyzed” reagent grade were obtained from the J. T. Baker Go., Phillipsburg, NJ. Methanol of “distilled in glass” qualiity was obtained from Burdick and Jackson L,aboratories, Inc., Muskegon, MI. Equipment,. Ultraviolet spectra were obtained with a Cary 17 recording spectrophotometer. Standarld ultraviolet detectors for liquid chromatography from various manufacturers were evaluated. The specific brand names are not included with these data so that the linearity test may be judged without also comparing equipment from various manufacturers. Procedure. To test for linearity at 214 and 254 nm, a 10 mg/mL stock solution of benzaldehyde in methanol was diluted to give a series of stand4wdsranging from 2.5 to 25 pg/mL. The sample cell was then fillled with methanol using a 1-mL glass luer tip syringe and recorder output at 1.0 AUFS adjusted to zero with the balance or zero control knob of the debector. The sample cell was flushed with the 2.5 pg/mL standard (I mL) and the detector output recorded. The sample cell was then successively flushed with standard solutionrr until full scale response was obtained. A similar procedure was used to test for static linearity at 280 nm with standards of benzoic acid ranging from 25 to 400 pg/mL in methanol. RESULTS A N D DISCUSSION Nonlinearity in LC: analyses may be caused by either chromatographic effects or detector problems. Such chromatographic factors as flow variations, pump pulsation, temperature, isotherm nonlinearity, refractive index changes, or spectral band shifts due to lack of mobile phase homogeneity may all affect the linearity of an LC analysis. Thus, to evaluate the linearity of a chromatographic method, it is necessary to test the linearity of the entire chromatographic system. However, to rapidly evaluate detector linearity, it is desirable to eliminate the chromatographic variations, and this was the reason for the static test dleveloped by ASTM. I t is always desirable to monitor an LC separation a t an absorbance maximum of the cornpound of interest for maximum signal-to-noise ratio and optimum linear dynamic range. However, in a typical multicomponent mixture, it is usually not possible to select a single wavelength where all components have either an absorption maximum or broad spectral band. Therefore, a more rigorous detector linearity evaluation would include a test at a wavelength where the ultraviolet spectrum of the test compound is changing significantly. After examination of the ultraviolet spectra of numerous commonly available organic compounds, it was concluded that benzaldehyde and benzoic acid would be suitable test compounds for this purpose. The ultraviolet spectra of these compounds
w.u
200nm
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Flgure 1. Ultraviolet spectra for benzaldehyde and benzoic acid: solvent, methanol; reference, methanol; cell, 1.O cm.
dissolved in methanol are shown in Figure 1. The spectrum of benzaldehyde is rapidly changing at 214 and 254 nm while the spectrum of benzoic acid is rapidly changing at 280 nm. Thus, benzaldehyde may be used to test detector linearity at 214 and 254 nm, while benzoic acid may be used to test for linearity at 280 nm. Figure 2 shows typical test results for linear and nonlinear variable wavelength detectors at the three chosen wavelengths. The single wavelength detectors using a low-pressure mercury lamp (254 nm) have become very popular because of their excellent sensitivity and wide linear dynamic range. In order to make these single wavelength detectors more useful, many companies supply conversion kits so that the detector may be used at other wavelengths. The conversion kit typically contains a phosphor emitting light at a higher wavelength than the incident radiation from the mercury lamp. The most commonly used secondary wavelength is 280 nm, and these detectors are generally severely nonlinear because of the wide band pass used with the phosphor. The extent of this problem is shown in Figure 3, where one of the single wavelength detectors, which was linear at 254 nm (1.024 AUFS), became very nonlinear at 280 nm when the conversion kit was installed. Further evidence for the critical relationship between bandwidth and linearity may be demonstrated with an LC detector containing an adjustable slit width. Benzoic acid was chosen for this experiment and tested a t 254 nm since its spectrum is relatively flat a t this wavelength. The linearity was tested by using bandwidths of 2 and 16 nm at 1.0 AUFS to produce the results shown in Figure 4. These data show the severe nonlinearity obtained by using the 16-nm slit width
1624 * ANALYTICAL CHEMISTRY, VOL. 55,NO. 9,AUGUST 1983 LiNEAF DETECTOR AT 2.56 aufs
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in spite of the fact that the measurement was made a t a favorable wavelength on the UV spectrum. Tests a t intermediate slit widths showed nonlinear results when the slit width exceeded 4 nm. In summary, this contribution describes a rapid and simple test for evaluating the linearity of ultraviolet detectors used in LC. Test results show important differences between detectors from various mandactwers. Some detectors are linear up to 2.56 AUFS while other detectors are very nonlinear well below 1.0 AUFS. This test is designed to be very rigorous to evaluate detectors for multicomponent analyses. Detectors that are found to be nonlinear under these conditions may still produce a linear response for a compound with a broad spectral band. This procedure will allow chromatographers
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AUFS. to test their detectors, and the data contained in this paper should make more people aware of the linearity problems encountered in LC. Registry No. Methanol, 67-56-1; benzaldehyde, 100-52-7; benzoic acid, 65-85-0. LITERATURE CITED (1) Snyder, L. R.; Kirkiand, J. J. "Introduction to Modern Liquid chromatography", 2nd Ed.; W h y : New York, 1979; pp 130-140. (2) Johnson, E. L.: Stevenson, R. "Basic Liquid Chromatography", Varian Associates, Inc.: Paio Alto, CA, 1978; pp 280-294. (3) Stewart, J. E. J . Chromafogr. 1979, 7 7 4 , 283-290. (4) "Standard Practice for Testing Fixed-Wavelength Photometric Detectors Used in Liquid Chromatography"; ANSI/ASTM E 685-79; American Society for Testing and Materials: Philadelphia, PA, 1979.
RECEIVED for review August 2,1.982. Resubmitted February 14, 1983. Accepted March 28, 1983.
