Revised 2009 Dithizone Extraction + UV-Vis SYNOPSIS Lead is selectively removed from an aqueous phase into an organic phaseby a chelating agent. The molecular electronic transition of the chelating reagent is perturbed by the presence of lead causing a UV-Vis absorption band which can be monitored. The selectivity of the process will be monitored by difference spectroscopy in the presence of Zn2 . !tudents also learn how to deconvolute a spectra to account for bac"ground absorbance. READINGS #ead pages $%&-$%' in the (ritical #eviews. #eview the section on soluble lead chemistry on pages 2)$-2)'. *ttached is an article describing some of the chemistry of dithi+one. The attached article is not as complete as the (ritical #eviews discussion. Testable Skills ,. Ability to er!or" a #econ$ol%tion on a set o! ex eri"ental #ata 2. &reate a baseline s%btracte# stan#ar# c%r$e 3. Ex lain 'hy UV-Vis absor tion ban#s can be consi#ere# to be (a%ssian in sha e in the !re)%ency #o"ain* 4. Ex lain the che"istry o! the #ithizone "etho#* The Dithizone +etho# This method was the first truly sensitive measure of lead. *doption of its use lead to the discovery of wide spread child lead poisoning in -altimore in the ,'$%s. The method relies upon the development of a UV-Vis absorption band for a comple. of lead dithi+one that is resol$e# from the absorption bands of the unreacted dithi+one. 1 5 5 5 − A 5 5 5 .u n r e a c te d + A 5 5 5 . /espite the fact that the lead dithi+one pea" is reasonably resolved from the unreacted dithi+one the absorbance measured is the sum of both reacted and unreacted dithi+one0 A m e a s u r e d .6 8 0 )b [ F r e e d ith iz o n e ] 122 The second calibration curve should be at the wavelength at which you will measure the comple. fr e e d ith iz o n e = ε ( fr e e d ith iz o z n e . +etho# . cm8.6 8 0 fr e e d ith iz o n e The correct absorbance of the lead dithi+one pea" is then0 A 5 5 5 .ample 56&%-67% nm8.5 5 5 )b [ F r e e d ith iz o n e ] 1$2 !olve the two equations for the absorbance contribution from free dithi+one at 999nm. fr e e d ith iz o n e = ε A 5 5 5 .5 5 5 = A 5 5 5 .-ecause the lead band is resolved from the comple.le a d − d ith iz o n e 1. 4999 nm8.2 To get an accurate calibration curve you must subtract the absorbance contribution of the unreacted dithi+one. it can be measured in the presence of the unreacted dithi+one.6 8 0 A 5 5 5 .u n r e a c te d . *gain: determine the molar absorptivity of the free dithi+one at this wavelength from the slope of the calibration curve.* The first is to create two calibration curves for the pure dithi+one. A 5 5 5 .5 5 5 fr e e d ith iz o z n e .5 5 5 )b [ F r e e d i t h i z o n e ] ) (ε A ) fr e e d ith iz o z n e . 3ne calibration curve should be at a wavelength that is unaffected by the presence of the lead dithi+one 4for e. /etermine the molar absorptivity for free dithi+one at this wavelength from the slope of the calibration curve and the "nown thic"ness of the cuvette 4typically . fr e e d ith iz o n e ( = (ε ) = b F r e e d ith iz o n e [ ] 680 fr e e d ith iz o z n e . There are three ways to do this. A 680 = ε ( fr e e d ith iz o z n e . 1&2 +etho# -* The second method is to estimate the contribution of the unreacted dithi+one by imposing a baseline on the spectra 2 .le a d − d ith iz o n e = A m e a s u r e d . (ε A 680 fr e e d ith iz o z n e . The corrected absorbance for the 999 nm pea" is then0 A 5 5 5 . pea"s when plotted an .bac"ground< at 999 nm is estimated from the baseline value at 999 nm.The absorbance of the . a. le a d − d i th i z o n e = A m e a s u r e d . =n the third method an assumption is made that the absorbance pea"s are >aussian in shape with respect to energy.5 5 5 − A 5 5 5 .b a s e lin e +etho# .is representing energy are >aussian0 192 3 . 4 . This is illustrated with some data that is not for dithi+one or lead dithi+one.The energy levels: hc E = hν = λ 162 are randomly split by temperature so that the absorption when plotted as a function of energy is randomly centered around a pea". 5 . The summed >aussian is subtracted from the measured absorbance: the difference squared: and plotted 4dashed line8. ?otice that for this particular plot the sum of squared difference is large: indicating that the estimated >aussian fit is poor. a.?otice that the pea"s swap position on the . 6 . =n a previous lab we introduced the normali+ed >aussian0 1 x−µ − 1 2 σ y = exp σ 2π 1)2 =n order to match the pea" heights in this lab we use0 y = A exp − 1 x−µ 2 σ 172 =n the ne.is: but more importantly become distinctly more .>aussian< in shape: and: as such: can be modeled as >aussians.t graph the data has been modeled as individual >uassians 4dotted lines8 summed. 6 0.t plot the sum of squared differences has gone very nearly to +ero: indicating that a good fit has been obtained0 1.8 Absorbance 0.2 0 10 15 20 cm -1 25 30 35 =n the ne. ) .4 0.4 0. &n t e fig"!e above fo! exa%p#e.6 0.2 0 10 15 20 cm-1 25 30 35 Once a “good” fit is obtained t e cont!ib"tion of eac band to t e signa# can be ta$en f!o% t e individ"a# pea$ eig ts.1. t e abso!bance at 22 c%'1 is t e s"% t(o cont!ib"tions eac of ( ic can be dete!%ined.2 1 0.8 Absorbance 0.2 1 0. . . 4%. !et the range 4G28 to &%%-)%% nm.7g in .%%% mLF %. .. . *dd .%% mL volumetrics !eparatory flas" *ssorted -ea"ers Bicropipettes: 2 mL pipette SO0UTIONS The dithi+one must be made fresh on the day of use as it is unstable.%%% ppm stoc" Ab..%%.INST/U+ENT (0ASS1A/E @A 7&92* /iode *rray. /%n blank 3"ethylene chlori#e4 4G78.2 B anhydrous sodium carbonate from 2.%%% ppm Ab stoc" solution.H dithi+one in (@2(l2 solution over &%%nm to )%% nm. *dd 9 ml of stoc" dithi+oneD(@2(l2 solution to a separatory flas".9-2% seconds.%%% mL.2 B sodium bicarbonate: diluted to . !can 2 mL of %.periment with new additions of dithi+one in (@2(l2 three times.%% Jl of . The green dithi+one solution should turn KcarmineK red or pin".% mg dithi+oneDL Bethylene chloride: (@2(l2 2% g of E(? diluted to . !et the y scale to some preset value and print.%%% ppm Zn stoc" solution &AUTION22222 =f you use gloves to protect your hands from the organic reagents the gloves should be free of powder as the powder will cause a false positive.port data for further wor" in I. P/O&EDU/E *.cel.2 B anhydrous sodium bicarbonate from . *lso: e. !i. L in the buffer -uffer0 229 mL of %. 8 . This lab was a staple 4standard method8 for &% years: but any lac" of cleanliness will cause this method to fail. 15Y66 $. #epeat this e. 2.2 g in . Cavelength scanning.%%% mL8 results in p@ .%% JL of concentrated ?@&3@ 4What is the purpose of NH4OH?8 and .2 B sodium carbonate: 29 mL of %. !ha"e . . (How would you prove this to yourself?) Cash bottle of acetone ..6. +eas%re the exact n%"ber o! secon#s* Do not esti"ate* 3ne student should be sha"er and should use the same sha"ing procedure every time. 9 .#emove the organic layer of the solution from bottom of separatory funnel and monitor itLs absorbance from &%%nm to )%% nm. &. !uperimpose the plots 10 of the dithi+one and the lead and note the region where the lead chelate absorbs as compared to the free dithi+one. . cel format. I.5.port and save data to 11an e. . % ml of cyanide mi. *dd & mL of dithi+one solution and .-. minute..ture. I. Yo% 'ill ha$e to "ake these sol%tions % * . to & ppm. Alace . 12 (alibration curve* Gor this method you should be able to get a calibration curve linear from %. !ha"e well for .% mL of lead standard in separatory funnel.port the data. . +eas%re this ti"e exactly 'ith a secon# han# 'atch* *llow the phases to separate.. Aull (@2(l2 solution from bottom of separatory funnel scan between &%% and )%% nm. #inse the cuvette and funnel between samples.13 with Be3@ and then acetone 2. . #epeat for the Zn standards. 3. : band2: band$: band&: sum: sqr diff... =n cells I&: I9: and I6 enter your band. &. Alot your computed absorbance bands in your previous plot.. pea" frequency: absorbance and standard deviation.%. $.%:%%%D*2 and copy down to the end of data in column *. type the labels0 band. .$ NIQ9R4ISA4-%. /econvolution is necessary in this e.$ type the I. . =n cell =. =f you measure the pea" absorbance and plot it vs the added lead you will often get poor calibration curves because the pea" absorbance contains absorbance from the un-reacted dithi+one. 3pen your data in e.% rows: so that the labels for the columns are in row =n column /: rows & through ) type the following labels0 pea" frequency: pea" *: s. 9. =nsert a new column between * and -.cel formula0 Nsum4I. Type in cell I. /o this for each of the bands you thin" you have. . =n cells I.9R44Q-.$-IQ&8DIQ68T288 (opy down to the end of the data: and into columns G: >: and @.%: and @.cel to deconvolute the spectral data. This represents the reciprocal of the wavelength 4..periment because there will be a large amount of unreacted dithi+one present which will cause the baselines to vary substantially.$8 and copy down. Mou should have the wavelength in column *: and the absorbance in column -.Dnm and some other pea" at different locations...Dnm8 which is a measure of energy.Dnm vs *. (opy this formula into cells G.% type the formula Nma. 2. -ac"ground (orrection for the /econvolution Use I.cel. through O. =n cell O7 type the label . . (ompute the signal contributed by band .%%2 . =n cell b2 type the formula0 N. . Gind your pea" frequencies: and pea" absorbances by plotting . 4Mou may need to insert columns: if you thin" you have more than & bands8... Mou should find a maPor pea" frequency of interest near %.$.%.14(alibration (urve by (. 7. Istimate the standard deviation of the pea"s from the base width of the pea"s.@.%: >. =n cell I..sum sqr diff<. 6.4data string for column I8 to find the modeled pea" absorbance. '.. =nsert . ). 2. D* =n cell @.perimental data.cept that it must be adPusted for the presence of the nitric acid.% through @. =f your sample absorbance is large 4U28 dilute the sample.cel 15 formula N 4(.traction of lead into the organic phase by dithi+one requires the dithi+one to have lost some protons. .%.$ type the I.%%. .solver< in your tools tab. 9. /EPO/T . 2.9. Chen diluting the sample: dilute into the buffer. Use the value obtained by deconvoluting the lead dithi+one pea" for your calibration curve.%.tract should be adPusted to p@ .$.. &.'V Chat constitutes a blan" in this procedureV Chat are the sources of error embodied in the standard deviation of the blan"V @ow would you determine the source of error attributed solely to . . #emember: when the value of the sum of the square of differences goes to +ero then you have a perfect match between your modeled set of pea"s and the e.7.subPect to constaints< that the values you are changing are not allowed to go to negative numbers0 Q/Q&0QhQ)U%. .&. additions. The p@ of your 9 mL of sample e. =n addition to materials: methods: and results: report0 Chat are the figures of merit for your method and how do they compare with any of the other methods you have triedV Chat is the chemistry of the separationV Chat is the purpose of adding citrate and cyanideV Chy does the p@ have to be adPusted to .%.$8T2 and copy down =n cell O' type the formula to sum the entire column @..'. . =n order that solver does not wander off into finding solutions that are not plausible set . $.6.$-=.solver< set the target cell to O'. #epeat with each of the spectra obtained with lead En$iron"ental Sa" le =f you have digested some environmental sample for analysis procedure is the same: e. (hec" the p@ of the diluted sample and adPust to p@ .' 4use: a p@ electrode8. The e.%. =n . . The modeled pea" absorbance of each of the bands can be found in cells I. !olver will start changing values in cells Q/Q&0Q@Q) 4your estimated values of pea" location: pea" height and pea" standard deviation for your four estimated bands8 one by one to try and drive the value in cell O' to +ero.). 3pen up . 7.ed with leadV . * chromophore is the portion of the molecule that absorbs light. ). the UV-Vis spectrometerV 16 Chat was the estimated time to analy+e one single standardV *re there any problems with disposal of ha+ardous materialsV =dentify the chromophore in dithi+one.6. Chat electronic perturbation gives rise to the change in color when comple. 9. 11. 12. 10. absorption pea" from the unreacted dithi+one pea"sV Chy can you treat absorption bands as >aussian curvesV Chat is the selectivity of the method for lead as compared to ZnV Questions to consider if you run an environmental sample . @ow does deconvoluting the 1) data to remove bac"ground or blan" absorbance compare to doing a baseline subtractionV Chich do you preferV @ow well resolved is the lead dithi+one comple. if at a##. 14. @ow does the L3/ compare with the cutoff between what is considered to be contaminated and uncontaminated soilsV *o( does sa%p#e %at!ix affect +o"! !es"#ts.. 18 =f you are using a soil or dust solution: convert the ppm of your L3/ to ppm in the soil using the dilutions and measured quantities of soil used in the e.$.traction procedure. .