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Spectrophotometry: Mechanics and
5) Repeat the ahove process, in increments of 50 nm, until a wavelength of 800 nm is reached. Record the data obtained. 6 ) By looking over the data, determine the wavelength regions where a peak of absorbance (minimum %TI occurred. In these regions, scan (repeat steps 3-5) the solution in increments of 10
Measurement Susan M. Diehl-Jones Brother Rice High School 7101 Lahser Road Birmingham. MI 48010
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7) Again, where a peak of absorbance occurs (minimum%T),scan
Purpose This experiment is designed to h e an introduction tospectrophotometry and t o general spectral analysis techniques. Students will hecome acquainted with the basic components of a soertro~hotometerand their functions. will use a soectrophotomeier in an open-ended experiment, and wilfuse Beer's Law in several different ways. In addition, they may compare the detectahility (tolerance) of the spectrophotometer with visual detection limits a s an optional activity. T h e concepts involved in these experiments are not new. T h e arrangement and method of presentation is, however, thought t o he unique a t the secondary level. Equipment Spectrnphotometer Pipets (1-ml through 25-ml, in 5-ml graduations) Volumetric flasks (100-ml) 100-200 ml of stock solution for each unknown notes)
(see
special
Procedure Because of the open-ended nature of these experiments, students d o not necessarily need a detailed, step-by-step procedure. They may only be given a general procedure and a n "unknown" solution. Both t h e general procedure and one ahhreviated step-hy-step procedure are given in the following sections. Spectrophotometric Determination of the Concentration of an Unknown Solution General Procedure Students first determine the wavelength of maximum ahsorhance of a colored "unknown" solution by scanning. They prepare a series of solutions of this "unknown" of varying concentrations (0.01-2.0 M ) . These solutions are from this point referred to a s standards. Then, a t the optimum wavelength (identified earlier), students determine the ahsorhance of each of the standards. Suhsequently, the calibration curve is prepared by graphing t h e results of ahsorhance versus concentration. Using this graph, t h e students apply Beer's Law t o identify t h e concentration of t h e original "unknown" solution. Abbreviated Procedure: The Absorption Spectrum 1) Prepare the spectrophotometer for use, as directed. 2) Fill one cell with distilled water and the other cell with your colored "unknown" solution. 3) Adjust the wavelength selector knob to 350 nm; determine the percentage transmittance of your unknown solution at this wavelength. 4) Record the wavelength and corresponding percentage transmittance obtained.
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chemishy through the use of the laboratory are provided in this feature. Experimemswill be fully detailed and will be field tested before they are " published. Contributions should be sent to the feature editor.
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the solution again; first in increments of 5 nm, and then in increments of 2 nm, to determine the exact wavelength of minimum transmittance. It should be noted that the regions scanned in these smaller increments should only be less than 50 nm. This entire procedure is called scanning a spectrum. 8) Calculate the absorbance from the percentage transmittance data and graph the results (absorbance versus wavelength).This graph is known as an absorption spectrum. Abbreviated Procedure: Calibration CurvelBeer's Law 1) Using volumetric flasks, pipets, and distilled water, prepare a series of solutions of your "unknown" from a stock solution identified by your instructor. (Instructor's Note: Usually 1 M stock solut&s are used.) At least four solutions of varying concentrationsshould be prepared: for example, 0.1 M, 0.25 M, 0.50 M, and 1.0 M solutions might he considered a series of standards. Be sure to record solution preparation methods in your notebook. 2) Prepare the spectrophotometer for use, as directed. 3) Adjust the wavelength selector knob to the maximum absorption peak determined in the Absorption Spectrum Procedure, which will be called the optimum wavelength. If two strong absorption peaks occur, choose the wavelengths nearest 500 nm, that is, choose the wavelength nearest the center of the visible spectrum, rather than at one end. 4) At this optimum wavelength, determine the percentage transmittance for each prepared solution as well as for your unknown. Record the data. (The reference solution should be distilled water.) 5) Calculate the absorbance from your percentage transmittance data. Graph the results, absorbance versus concentration for all the standards prepared. This graph is known as a calibration curve or a Beer's Law plot. 6) From the linear relationship graphed, read the concentration of your unknown. Record this concentration and report it to your instructor. Limits of Detectability (Optional) General Procedure Students oreoare several solutions (10-20) of varvine concentratink df their "unknown" ranging from very d c u t e t o very concentrated and determine the ahsorbances a t t h e optimum wavelength. T o determine the limits of detection, the students continue t o vary the concentration of t h e solutions until a concentration is reached which exceeds 85% transmittance (lowest limit) or transmits less than 20% (upper limit). These two concentrations, then, are identified a s t h e limits of detectahility for t h e unknown substance. Abbreviated Procedure: The Limits of Detectability 1) Prepare 1 M, 0.10 M, and 0.01 M solutions of the chemical compound you studied previously. Record preparation methods in your notebook. 2) Prepare the spectrophotometer for use as directed. Adjust the wavelength to the optimum wavelength determined in the AbWrption Spectrum procedure. 3) At this optimum wavelength, determine and record the percentage transmittance for each prepared solution. The reference solution should be distilled water. 4) If needed, prepare more dilute solutions and repeat the measurement procedure (step 3) until a solution concentration is Volume 61
Number 3
March 1984
255
Llmlts of DetectabilHy (Student Data) for Co(NO&. 6H,O wavelength = A = 512 nm dilution preparation from 1 Msolution
molarlty
slit = 0.05 mm 0V C
% T
abS0rba"CB
(calc)
mlor dk. red
-
400
3W
5W
6W
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m0
--
red red n. red It. red n. red n. red
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pink pink n. pink nale oink
BOO
WAVE L E N O T l l h I
Graph of absabance versus wavelengm far dnermining Uw c@mmwavelengh for CO(NOQ)~. 6H,O.
reached, that, when tested, transmits 80435%of the light. This solution represents the lower limit of detection concentration. 5) If needed, prepare more concentration solutions and repeat the measurement procedure (step 3) until a solution concentration is reached, that, when tested, transmits only 15-20% of the light. This solution represents the upper limit of detection concentration. Be sure to record all data, including the solution preparation method. 6) In order t o make a comparison between the instrumental detection limits and the visual detection limits, note the color and shade of each solution tested. 7 ) Calculate the nlmlrhsnce from the percentage transmittanre data fur each solution tested, and put in tahular furm with the corresponding concentrations (in molarity). 8) (optional) Plot the data (absorbance versus concentration) t o determine whether Beer's Law is obeyed over the entire range of concentrations tested. S p e c i a l Notes 1) This experiment offen the students the opportunity t o explore spectrophotometry while gaining the experience, practice, and confidence necessary for independent research. Although students are not usually given a detailed, refined procedure, they are given the general instructions presented here, along with a list of background articles (1-9). Therefore, this experiment would he especially useful in an AP or second-year chemistry class. Other references appropriate for the instructor andlor student are listed below (10-20). 2) The preparation for this experiment is extensive the first time, since all accepted values must be determined experimentally. On a modern spectrophotometer (like the Beekman DB-G), where manual and automatic scanning can he done interchangeably, the absorption spectrum of several colored solutions can he ohtained. For each of thesesalutions, the concentration is adjusted so that no percentage transmittance (300-800 nm) falls below lo%, and so that the major peaks of absorption (400-700 nm) are clearly indicated, preferably with a t least a minimum transmittance of 25%. These solutions, then, become the student "unknowns."Forstudent use,they should he labeled with their chemical name hut without a concentration. I t is usually convenient, a t this time, t o prepare 1M)-200 ml of 1M stock solution for each compound used. In this way, solutions are available for many of the standards and weighing can be kept t o a minimum. All solutions should he stored in amber bottles. Alternatively, students may prepare their own stack solutions. The followine.. comnounds seem t o work best as student "unknowns": nickrhll, rhoride hydrate, nickelrll) nitrate hydrate, col,alt(ll~chloride hydrarp, uol,alt(ll~nitrate hydrate. copper(I1) nitrate hydrate, copper(I1) sulfate hydrate, chromium(II1) nitrate hydrate, iron(II1) chloride hydrate, iron(II1) nitrate hydrate, potassium permangauate and potassium chromate. The yellow solutions (iron chloride, iron nitrate, and ~ o t a s s i u mchromate) are more difficult t o work with because the rolur remain. intense w e n at i w e r cunccntrations. Neither mangancsrtlll suliarr nur polasa:um dichnmste are rrrommended. Sample student d a u and agraph r,latwrhance versus wavelength for determining the optimum wavelength are given in the table and figure for Co(N03)26H~O.
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256
Journal
of Chemical Education
C = clear(Mt vialbly detectable) ' = dstectable llmits (spectrophotomefrlc)
0 =opaque V = visibly detntabb
3) This instructor scanned the solutions listed on s Beckman DH-G. The data ohtained, with the rorreaponding absorbanres, wm avaihlAe for ampariaon as a ret of "rhec,rer~cally-orcepcedSced v.dues. Therelore, errors were quickly idrntifiable. The most common student errors are: (1)dilution errors in either calculation or volumetric equipment usage and (2) the choice of a minor absorption peak (on one end of the visible spectrum) as the optimum wavelength. Students find i t helpful t o develop a computer program to calculate absorbance and print out the data calculation. The only disadvantage of the experiment is the extensive solution preparation required. The fact that it is an introductory experiment does mean that students may do more retrials than is usual for an experiment. However, in spite of this, all students seem t o find the experiment interesting and a hit of a challenge, and are able to complete it well within eight hours of laboratory time.' Editor's Note:To ohtain further informafion on student handout materials, lalxmwn. prrxedurcs, and DH-C rUarceptedvalues") data, contact the author directly. ~~~~
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Literature Cited 11) One or more equipment manuals for the speetrophofometerin use. For e m p i e , in usingS~ecuonie20:BauachandLomb,AnslvtidSystemsDiuision."Swho~ho~
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t o m i t e i & i ~ ~ t i ~ " .Menual" l (nodate). 12) Beckman Instruments, "An lntduction toSp3xwhotometry,"Bulletin 295-D.INine, California, Beckmen Instruments, (Nodate). (3) Davis, Jeff C.. Chemlslry, SO (November, 1977). The antire aeries dartidea 1%9)i. available ae Chemistry remint 128, "lntmduetion to Spdmeeopy" fmm the American C h e m t d S&& I41 Davis. Jeff C., Chemistry, 48.W (December. 1975). (5) Davis, JeffC., Chomisfry,48,15 (July-Auguaf 1975). (B) Dsvis. JeffC., Chemkfry, 48.19 (May, 1975). 17) Dsvis. JdfC., Chemistry. 47.6 (October, 1974). (81 Dsvis. JeffC.. Chemistry. 48.11 (January.1975). (9) Davis, JeflC., Chemistry,49.18 (November, 1976). (101 Ewing. Calm W.,"lnsuumental MethodsofChemiealAnalyrir~Ithed.,M&raw-HW, New York. 1975. Not neasssrilyforstudentuse.Thur book,leferences(18)or(191, or acurrent instrumental text could be iwd done. (11) Kaiser,H., "TheLimitofDeteetionaf aCompleteAnslytiealPdm,"ITm~lator: Menzies. A. C.), Hafner Publishing, New Ynrk, 1969. (121 Macslady. Donald L., Chemistry. 48.27 (July-August 1915). (13) Meloan, Clifton E., and K k r , Robert W.. "PrablemrsndExprimentsinlinhlllochllloctal Analysi%"CharlesE. Merrill Publishing. Coiumbuli, OH, 196J.Eithel thk or reference (141 would be sufficientforbackground. (14) ReiUey, ChdesN.. andSsanier.DonaldT.."Erp.imcnlsforlnstrumantal Methods," McCraw-Hili.NewYark, 1961.
115) Sandell,E. B. (Editor),"Colorimetric Determination dTracer of Mstsls," 3rd d., "Inter-SciencePublishers Chemical Analysis,"voLII1,John Wilw sndSona,Ncw York, 1959. Notneasssrily for student uss 115) sienko, ~ i e h a eJ., l plane. Robert A,. and ~ a r m Stan~w~.."ExperimentalCbema istry: 5th ed.. McCrsw-Hill,New York, 1976, 1171S k w g , Douglas A,. and West, Donald M.,"Fundament& of A n a l y t i d Chemistry," 2nd sd., Holt, Rioehart,and Winaton. New York, 1969.Notnecegsarily forstudent we
(18) Skwg,DouglasA..and West, Donald M., "Principlesofh~entalAnalyaia," Holt. Rinehart, and Winaton, Nsw York. 1971. Not mcessariiyfor student use. 119) Willard, H. H.,Merritf, L. L., and Dean, J. A,. "lnatrumentsl Methods of Aoaly8k." 5th ed.. D. Van Nostrand. New York. 1974. Not ne-arily foratudent we. (20) Wineforher. J. D.(Editor). 'Trace Analysis: Spfftrmpic Methods for the Elements," "inter-SeieneePublishers:Chemieal A n w : Volume XWY, John Wllw and Sons, Now York, 1976. Not necessarily for student use. (21) Diehl-Jones,Susan, J. CHEM. EDUC.,60,986(19831.
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In the program described in the November, 1983 issue of the JOURNAL OF CKM~CAL EWCATION, a three-week period was alloned and was sufficient (21).
