Obtaining a Spectrum Easily Using a Single-Beam Spectrophotometer Miranda E. Mattson a n d William A. ~ a t t s o n ' Randolph-Macon Woman's College, Lynchburg, VA 24503 Low-cost, single-beam spectrophotometers a r e not ordinarily used to obtain spectra. Besides the spectral distribution (e.g., signal infor the analyte, adtensity versus wavelength 0.)) ditional spectral distributions (ASD's) exist for the source enkra, the detector response, and the light interactions (e.g., absorbance, refraction, scattering) with various other components in the instrument (e.g., sample cell, optics, solvent, reagents). Basic instruments do not correct far source and detectar spectral distributions. Consequently, recalibration is necessary every time the wavelength is changed. Repeated recalibration is tedious and slow. Thus, students i n a number of high school and introdudorv colleee laboratories cannot readilv obtain spedra forresearch (e.g., science fair proiects) or structured laboratow experiences. Such ipectra a r e needed (1) for a-quaiitative characterization of a synthesized substance and (2) for use in selectinia h for monitoring kinetics of a reaction or for doing quantitative analysis. Some instruments ( 1 ) a r e designed to correct adequately for ASD's:
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ae
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1. Double-beam spectrophotameters-as
h is scanned, sample and reference beam (i.e., the blank) light intensities are continuously ratined. If both beams are affected the same by the ASD's, the ratios are unaffected, resulting in a corrected spectrum. ASD differences (e.g., different euvettes) are assumed to be minimal. 2. Single-beam computerized speetrophotorneters and diode array speetrophotornetersin turn, uncorrected sample and blank spectra are obtained and stored in a computer. The corrected spectrum is obtained by ratioing the sample and blank spectra. If both uncorrected spectra are affected the same by the ASPS, the ratios are unaffected, resulting in a corrected spectrum. ASD differences (e.g., different cuvettes; signal drift in any of the
Wavelength (nm) Presented in part at the 44th Southeasternl26th Middle Atlantic Combined Reaional Meetina. American lh\ *-, Chemical Society, Ctystal C& Arlington, ~i'(12/6/92). 'Author to whom correspondence should be ad- Figure 1. Spectronic 21 spectra for a blue food color sample: (a) uncorrected blank and uncorrected sample, and (b) accepted and corrected. dressed.
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Number 6 June 1995
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spectral distributions (e.g., source intensity) caused by voltage or temperature fluctuations) are assumed to be minimal over the small time needed to obtain the two uncorrected spectra. This p a p e r describes a convenient, r a p i d method for ohtainina spectra using a process t h a t is somewhat anaiogous to that Lmpioyed in sinale-beam computerized and diode array inst&ments.
Method ~ u r n r n a r ~ ~ Using a spreadsheet for data recording, calculations, and plotting. Obtain an uncorrected spectrum of the blank. Insert the blank. Set the instrument to the X giving the greatest % T Set the 100% T control, and record % T versus ?. values for the various spectral A's. Obtain an uncorrected spectrum of the sample. Using the same Xs,repeat the steps above, substituting the sample for the blank. Obtain a corrected spectrum of the sample. For each ?. used, calculate corrected % T values by dividing each uncorrected sample reading by the corresponding uncorrected blank reading and multiplying by 100. Plot corrected % T versus 2.
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Surprisingly, the uncorrected blank spectrum, once obtained, can be reused with uncorrected sample spectra obtained on other days to produce satisfactory corrected sample spectra in about 20 min. Referring to the example in Figure 1for a blue food color? sample, Figure 1 (a)displays the uncorrected blank and uncorrected sample spectra, while Figure 1(b)presents both a n accepted spectrum, obtained using a double-beam i n ~ t r u m e n t , ~ and the corrected sample spectrum. 0
Reproducibility of the Uncorrected Blank and Spectra To further reduce t h e time needed to obtain spectra, t h e same uncorrected blank spectrum, t h a t i s quite reproducible for any particular Spectronic 21, can be used over and over, even months later. Figures 2a, b, and c Present
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(a) corrected sample spectra obtained by ratioing uncorrected spectra for the blue food color solution for three different days to the same uncorrected blank in Figure 1 (a), obtained up to a month earlier, (b) three sets of uncorrected blank spectra Le., two sets obtained an hour apart and three months apart? respectively, using two different Spectronic 21 instruments, and one set obtained on cansecutive days using a Spectronic 20~1,and
A ' very detalled procedure will be furnished on request. Unless otherwise indicated, data was obtained usin a Spectronic 21 %ec'a*Cake aqueous blue food coloring solution (200 microliterslliter 1VIV)I (Durkee French Foods. lnc.. Wavne. ~,~, NJ 074701. All accepted spectra were obtained with a Perkin-Elmer Lambda 38 UV-VIS Spectrophotometer. It should be noted that this instrument (i.e., #2) was not used much during the three-month period. Regulated Power Supply Model with a standard "blue"phototube (340-600k j . ~
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Journal of Chemical Education
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Figure 2. Spectra illustrating the stability of the uncorrected blank spectra: (a) three days of corrected blue food color Spectronic 21 spectra, obtained using the same uncorrected blank spectrum, (b)three sets of uncorrected blank spectra, and (c)three corrected blank spectra
ment-the uncorrected blank spectrum does not need
Spectronic 21's-an uncorrected blank spectrum needs to he established for each instrument.
Potential Troublesome Areas of the Spectrum Referring to Figure 2b, note t h a t for t h e Spectronic20 t h e detector sensitivity falls off significantly above 600 nm-the signal is too small for a reasonably different % T to be read for a n absorbing sample; a s given in note 7, the amplifier gain is, therefore, increased. Note t h a t i t i s 00ssible to change tht, d ( ~ c t o lto . 3 red sensitive phototube. However, this 1s not a rmsunahlc, hwh-soecd. ootlon. Fieure 3 shows 3 Soertronir I 0 2fspe'ctruk'for a blue food color sariplea absorbing in this region. 400 MO 600 700 Referring to Figures l b and 2c, note t h a t for Wavelength (nm) the Spectronic 21 there is a n area of lower preFigure 3. Accepted and corrected Spectronic 20 spectra for a blue food color sample. cision between 450 and 550 nm. Figure 2 b suggests that the signal is approaching too small a level i n this region for a reasonably different % T to be read for a n absorbing sample. Indeed, spectra of a very dilute red food color sample, having a strong absorption peak in this region, gave unacceptable results. However, when the concentration of t h e red food color was increased9 (i.e., a darker but still transparent solution having a corrected % T of about 20% maximum), a quality spectrum was obtained, shown i n Figure 4 with t h e accepted doublebeam spectrum and with a Spectronic 20 spectrum. 7-
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Wavelength (nm) Figure 4. Accepted and corrected Spectronic 20 and 21 SDectra for a red food color samble.
r, the rurrrrted hlank spcrtrn ohtamed by ralioing the t w o uncurrected blank spectra frumeach ofthe thrrc instrument.;
Note: (1) the reproducibility in Figure 2a for corrected sample spectra and in Figures 2b and c for uncorrected and corrected blank spectra, respectively, obtained on the same instrup~
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'For a Spenronc 20 !he spectra m ts for ~singthe norma Dlbe pnolotdoe are 600-340 nm. Spna s aoove 600 nm WOL 0 or0 narlly be loo m a lo a1 ow for a reasonao y a Herent Oo Tto oe read for an absorbing sample, resulting in a poor spectrum. To successfullyinclude the 700-600 nm region in the spectrum,the 100% Tcontrol on !he mslrJment is resel ai600 nm-tne ,.gang !he grealest "O Toeween 700-600 nm All examp e Spectron c 20 correcleo spectra, nclua ng the correcleo olan6 n F g u e 2 (c).were oola neo LS ng tnls ap roach. gMcCormick aqueous blue food coloring solution (400 microliterdliter 1VN))lMcComick& Co., Inc., P.O. Box 208, Hunt Valley, MD 21030-0206): gMcCormickR aaueous red food colorina solution 1400 microlit-
Suggestions for Avoiding/Resolving Problems with the Method 1. If the spectrum obtained is not smooth, consider adjusting the concentration of the analytewith less expensive spectraphotometers, the best precisions are obtained if the ahsorhances 700 for the most important parts of the spectrum lie between about 0.1 and 1.5 (i.e., about 80% T to 3% T) (2).Typically, the color of the sample should be somewhat dark hut still transparent. 2. To allow for reasonably different % T readings for a n absorbing sample, % T readings for the blank must be high enough a t all 2 s . To confirm quicklv . . that a articular instrument is probably functioning properly, ratio two blank spectra obtained on different days-a precise line at 100 % T should result, a s illustrated in Figure 2 (c).
Acknowledgment 'The authors gratefully acknowledge the contributions of the NSF J M C Rrstwrch Experieners for Undererdduates P r o g r a m , D i r e c t e d by Downey (NSF g r a n t s CHE9000748 and CHE9300261); the other faculty and staff a t James Madison University with special thanks to Donna Amenta, Tom Gallaher, J i m Leary, and F r a n k Palocsay; and Faith Anderberg, Kristi Kneas, Susannah Lomant, Jennifer Richie, Melissa Terry, Laura Young, and the first J. Chem. Educ. reviewer.
an
Literature Cited
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