Evaluation of Derivative Ultraviolet Spectrometry for Determining Saccharin in Cola and Other Matrices An Instrumental Methods Experiment Rkhard J. Stolzberg University of Alaska, Fairbanks, AK 99775 Advances in micro~rocessor-controlled or -assisted instrumentation and theavailability of microcomputers in the laboratory have increased the options available to the chemist. We can operate instruments more creatively and reliably than has been possible previouslv, and we interpret data more rapidly and accurately, using more sophisticated approaches. As a result, chemometrics is now becoming a part of the undergraduate curriculum a t a number of schools ( I ) . The new capabilities of speed, reliability, and flexibility must be balanced against increased conceptual complexity and the chance for blind error based on helief that computers alwavs give correct answers. p a r t o i tKe challenge of teaching an instrumental methods course is to make students aware of capabilities and limita.tions of advanced methods of data acquisition, manipulation, and interpretation. This includes recornition that choices must bemade among approaches and that trade-offs exist. Improvements in signal to noise ratio (SIN) may rem i r e lenithv " data aconisition or loss of fidelitv in the sienal (2). Improvement in resolution may he accompanied by decreased SIN. Finally, computer-aided techniques are susceptible to errors in method, and detection of those errors mav be difficult. Derivative spectrometry (3)can he used to introduce students to enhancement of spectral data. T h e intensitv of narrow absorbance hands ;ncreases compared to hioad hands. Thus, sharp features of low intensity may he ohserved even if they lie within a broad, intense band. T h e method has been used analytically for avariety of compound types, and capabilities and limitations have been described (4-6). Although the method looks foolproof, theoretical studies show that the choice of a n inappropriate derivative level or method of measuring response can result in bias or unnecessarilv laree random error (7).The correct methodology depends o n relative analyte a n d interfering hand intensities and widths, and the success of derivative spectrometry will vary among analyte and sample types. This experiment allows students to make choices and to address limitations that are often present in analysis. The statement of the problem is that only distilled water matrix standards and a blank are available, and the "best" method must he applicable to a wide variety of matrices. Three samples of saccharin-a nickel la tine solution. a dilute cola drink, and a more concentrated cola matrix-are analyzed and the data are interpreted using five methods. Precision and accuracy are evaluated, and the best method is chosen. Significant differences among methods are observed.
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Exprlmental Aqueous standards of 0, 3, 6, and 9 ppm saccharin (Sigma) are prepared from a 1000-ppmstandard. The Watts nickel plating solution containingapproximately80 ppm saccharin is prepared according to the literature (8)and is diluted 10-fold for analysis. The cola unknowns are prepared from saccharin-free cola. The pH of the eola is adjusted to 5 to 6 with NaOH, and the cola is diluted 50-fold
(normalcola) or 10-fold (super cola).Saccharin is then added to give 5 to 6 ppm, which corresponds to levels found in diet cola that has been diluted by a factor of 50. Both cola samples are labeled as "diluted 50.0Xm. A Perkin-Elmer 555 UV-VIS spectrometer is used with 1-cm ouartz cuvettes and with water as the reference. A slit width of 2 nm. scan rate 60 nmlmin, and response time 0.5 s rlirst derivative)or 1 s (secund derivative) givegood resulw. It isalso possible loderivati7. digitbed absorbanr~spertraoff-li,>e (9). Students are instructed to determine the appropriate wavelength range (260 to 210 nm) for analysis and to obtain a single absorbance curve for all standards and unknowns, duplicate first and second derivative spectra of standards and blanks, and six replicate first and second derivativespectra of the three unknowns. Data interpretation is made bv measurine two first derivative resoonses-a baseline method 11Si and a oeako-oeak method 11~);and three second drr~vnr~ve method-n bnselme method (2R) and t w o peak topeak methods (21'F. 2PN) The terminology la that used hy O'Haver and Green (7). Students construct the five calibration curves and calculate concentrations of the unknowns. They evaluate the standard deviation of the technique using two methods: from regression data and the number of unknown replicate measurements performed (10) and from repeatability of the unknown response. Re~Its Absorbance and first and second derivative spectra of saccharin in distilled water are shown in Figure 1. Compara-
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WAVELENGTH ( n m 1 Figure 1. Absorbance (A), first derivative (A'), and second derivative (A") Spectra of 5.92 ppm saccharin in distilled water. lnsbumemal conditions as described in text, except response t i r e = 0.5 s for A".
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Table 1.
Mean Concentration of Saccharin In Three Matricesa
Method 1B 1P 2PF 28 2PN true value
Watts
Cola
Super Cola
80.03 79.8 79.9 81.0 80.2 80.0
355 266 270 269 267 265
705 284 239 230 219 288
ble spectra for saccharin in cola matrices are shown in Figure 2 and Figure 3. In all three sets of data, derivatization suppresses the broad absorbance feature and enhances the narrow shoulders near 226 nm and 234 nm. Noise increases with derivatization and the magnitude of the noise is greater for the highly absorbing cola samples. The spectra in Watts solution are nearly identical to those in distilled water. Student results show that the accuracy of derivative spectrometric determination of saccharin depends on the sample matrix, derivative order, and the method of interpreting the data. A set of data is shown in Table 1. The Watts solution, which has a small matrix absorbance, can be analwed accurately using any of the five methods investigated. The saccharin determination in cola will generally be biased when the 1B method is used, due to the difficulty in accurately locating the curving baseline. This could explain the biases reported by Fix and Pollack (8). The other four methods typically give results accurate to 1 to 3% (relative) with cola. Accurate analysis of super cola can be made only if the 1P method is used with distilled water standards. Table 2 shows sienificant errors 1>5%) occurrine in over half of the anal$5 for all other dethods of interpretation. The moderatesized nositive and neeative biases for second derivative spectra of'super cola suggest personal error rather than error in method. The high noise in the spectra causes bias if signal
Table 2.
Percent Error In Saccharin Determlnatlons In Super Cola studem MG
Method
MM
SM
JS
KM
1B 1P 2PF 28 ZPN
73 3 8 2 14
30 -3 4 4 16
...
147 -1 -16 -20 -23
4 6 6
25
178 -1 -14 -18 -21
MA
BM
CL
14 -2 8 1 -4
0 -18 2 -12 -11
5 4 42 23 4
size is measured from the top (or bottom) of the noisy pen trace, rather than from the middle. There are significant differences in the precision of the technique among samples and among methods of evaluation (Table 3). Random error increases with sample absorbance and with derivative order. The relative standard deviation should be calculated from the response of replicated unknowns herause the standard deviation calculated from the reeression does not include the extra mectrometer noise pr&t with the highly absorbing colas and is therefore an underestimate of the true value. The reproducibility of the saccharin signal in distilled water standards and in Watts solution is 52%. In contrast. the recision of replicate ~ e a k measurements is 2 to 4% RSD i n cola and 3 ti8% RSD in super cola. The intensity of the background absorbance in the colas introduces significant noise into the trace. The more sloping background in the soda derivative spectra makes the subjective location of baseline less reproducible. Both factors degrade precision of the analysis with respect to distilled water standards and Watts unknowns. Derivatization increases the analytical usefulness of absorption spectralike that of saccharin. For the samples studied, the first derivative accentuates the shoulders sufficientIv for ouantitative evaluation. and the ~ e a k - t o - ~ e atechk nique gives accurate and reasonably precise results using simple standards. In general, most effective use of derivatization requires flexibjlity inacquiring and interpretingdata. For example, the most accurate results will be observed for
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WAVELENGTH (nm Figure 2. Absorbance (A), first derivative (A'), and second derivative (A") specba of 5.92 ppm saccharin in coia. Conditions as in Figure 1.
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Figure 3. Absorbance (A), first derivative (A'), and second derivative (A") Spectra of 5.92 ppm saccharin in super cola. Conditions as in Figure 1.
Table 3.
Average % RSD for Saccharin Concentration Cola
Watts Linear Regre~sion Method
(N = 12).
Replication
Linear Regression
(N= 11)
(N = 13)
0.2 ppm saccharin when the second derivative is used be-
cause the curving baseline of the first derivative becomes a major source of bias when the signal size decreases (9). Literature Clted (1) Howery, D.C.;Hirsch, R. F. J. Chrm.Edue. 1983.60.656.
(2) Binkley. D.: Dmy,R. J. ChamEduc 1979,56,148.
Super
Cola
Replication
Linear Regression
Replication
(N= 10)
(N= 2)
(N = 7)
(3) 0'Hauer.T.C.Anol. Chem. 1979,51,91A. (4 Talsky,U.:Mayring,L.; Krevzer,
[email protected]. (Intermf.Ed.) 1978.17.758, (5) O . H ~ V ~ ~c.: , TB. ~ ~ ~ ~ . T them. . A ~ 1981.53.1876. ~ I . (6) Crimths, T. R.: ~ i n gK.; . ~ubbard.H.; schwing-wei~~, M.: M ~ ~J. A"GI. u Chim.Aelo 1982,143, 163. (7) 0'Haver.T. C.: Gleen. C. L. AnoLChem. 1976.48.312. 181 Fir, G. L.:Pollaek, J.D.And.Cham. 1980.32. 1589. (9)Marshall, 8.. unpublished report (1985). (101 Skaog, D. A: West, D. M. "Fundamentah of Analytbal Chemistry", 4th ed.: Holt, Rinehart, and Winstan: New York. 1982: p 71.
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