EXPERTMENT 2

W.YISIBLE DETERMINATION OF AN TJNKNOWNCONCENTRATION OF KMnOr SOLUTION

TMORY/ BACKGROIJND

All absorbancc spectrophotometers coatain a light source, a sample comparheot, aud a detector.Many speckophotometers a[5s sontzin one or more monochromators, a device used to separate lightinto its component wavelengtls. The Spectronic 20 operates in the visible range only and uses a priimas its monochromator. Speckophotometer that rleasure in the UV ana visibte iegion are of fwogeneral tlpes: scenning aud diode-array. A diode array qpeckophotometer imparts light of allwavelengths ts thg 5amPle at onc time. In contrasl 3 5sanning UV-Vis spectophoiometer coatains amonocbromator, usually consis';rg of. holographic gratings, which allows light of iadividualwavelengths to be sequentially imparted io the sample.

-

Spectroscopy involves the observations of absorption or emission of electomap.etic radiationresulting from kansitions of atoms or molecules from one etrergy level to auother. Wdn a molecule atits "ground state" (the state of lowest energy) absorbs

"o"rgitf some g4re, the molecule is said to

rmdergo a "bansition" to a higber energy state. The higher energy state is refered to as an ..excitedstate"- A molecule can only absorb energJ i1 16s input energy exacfly matches a molecular bansitionfrom one energy level to aaother. Examples of some traasitions that can occur in a molecule includebond vibratiou or rotation around a bond due to interaction with infi=ared energy (IR spectoscopy), orchanges in tle elcctonic skucture of a molecule due to iateractions witfr v;siUrc oi ultaviolet radiation(Visible and UV Speckoscopy). The transitioas betweeu ground and excited safs5 r rirhin moleculesare a firoction of the types of stuctures that are found within a molecule aud the eavironments thatthese structures are located u.ifhin a giveu moldcule- So if we know something about tle absorption ofenergy in a given molecule, then we can predict the types of structures or fiinctioual groups that wemight have within a molecule.

Many orgaaic aud biological'molecules have Fansitisus fhat occur betwem energ-y levels of electronicstates of atoms or molecules. Therefore, our focus will be ou the visible and ul#viot"t ."gio"s J tl"electromaggetic spectum iu this lab. The visible and ultaviotet region of isterest is fouia between170 and 800 nm; though ttre most useful region for experimental use is between 250 and 700 nm.

Principles of IfV Spectroscopy

UV-Vis qpectoscopy is based on the selective absorption of electomapetic radiation in the 1g0-7g0lm wavelength range. tfV-Vis radiation hac sumcient eoergy to cause furritio* in bouding electons(as opposed to atomic inner shell or valence electons) and thus, is correlated best with the behavior ofbo''ds and fuactional groups in the analyte.

Absorption in the W-Vis range is mainJy a study of molecules and their electronic transitions.FJechons particrpating directly in bond formation eJ 1s rrnshared, outer electons that are ]ocalizedabout electouegative atoms (such as oxygen, the halogens, sulfirr and nitoge,a) can be promoted to ahigher energy molecular orbital. Each of these molecular o6ital represents i aifferent .r.rgy level asshown below.

10

Antlbondlng

Antibonding

bonding

Bondlng

Bondlng

Most IJV-Vis spectra involve n---tc* and r-r" Eansitions. r'_>E* transitions are generally moreintense fhan a--+76*-

Transitions to a zc* orbital requires the preseuce ofan unsaturated firnctional group (chromophord tosupply the z* orbitals. Radiation in the 200-700 Em raoge brings about these transitions makiegmolecules with cbromophores convenient for analysis using a LIV-Yis spectrophotometer. Organicmolecules with conjugated double bonds, carbouyl groups, carboxyl groups, and nitro groups are thebest absorbers in the W-Vis rrnge. Each firnctioual group has a wavel*gth associated with an

absorption maximum. that can be used for qualitative ideutification in arr unknown sample-

t* a--- Electronic iransitions

1elrulz.

t'

C6HpCH:CH2

CsHnC=C-CH3

oII

CH3CCII3O

oil

cH3cor{

CH3NO2

CHrN:NCHs

177 13000 lr+tc*

178 i0000 1t---r'*

186 -1000 n-+g*

4l rr--+rE*

ZZ \-+tt*

5 B---+n*

204

280

339

Molar absorptivities (e) of up to 10s are suitable for use in IIV-Vis absorbauce speckoscopymeasurements. Transitions with e < 103 are howev'er considered to be of low intensity.

TT\f.\_I:. --^1-

^^- L- ^Lh.:-^l c---:------:- ^L^^::-+ 1--.!-- ---L -1 -::::ij:::: t!----.:---i -i -r-r:- jv-.

--r-bE t) urvy--e v.vsvAr .vuo,

lanthaaides and actinides as well as inorganic complexes or charge transfer complexes-

11

v3"

Transition Mdals" Spectra from taasitiou eleuent ions arisc &om the 3d and 4d electons- Thesespecka are quite broad, often in thc visible (solutions are brighfly coloured), and are significantlyaffected by ligands aad solveots.

loI64,,

o300 400 500 t50() 700 8rx,

400 500 600 700 8m

v>r

oo

5

C'o=

20

15

r0

5

ot:300

o.r0o$8o.06,

o.o4o-oE,

O;0O

l.{#.

400 500 600 ?N 8@

Lanthanides and adinide* LIV-Vis qpectra of lanthanide ions arise 6o* 4lsrbitals and 5f orbitals forttre acrinides. These are rather well-screened from outside i.,Rr*""s and as such the spectra arecomparatively sharp, and oniy weakly influencedby ligands and solvents.

&I;

a

JSO c{8, isCI

rt,-

I

s60 (so ?flo

L,,i. l

po9

€sEo*

.$sfi

/--\\\ \

fIIII Qf+

t2

Ifnorganic complaa- laorgan'is somFlexcs made up of metal ions with surrouuding ligands, or chargetransfer complexes, caa undergo absorption processes where the eleckon jrrrnps from an orbital mostlycentred ou the ligaud to au orbital Eostly centered ou the metal ion (the opposite c2n occur, but lessfrequeutly)-

\:,>r

-a

o

50004000

3000

2000

IOO0

o

12000roooo80006000{ooo2000

o

25000

20000

15o00

toooo500()

o

FcscN"+

.+oo +50 5{)0 550 650

Fc(phen)l*

400 450 550 600 650 700

Stcrch-t]

400 450 soo 550 60(} 6so 700Wavelength. n.m

IfV speckoscoPy can be used to quatify the amouut of au absorbing material present. Absorption isproportional to the path length b through a saurple solution and the eonces.kation c of an absorbingspecies according to Beer's Law: A: ebc

The proportionality constant in Beer's Law, g is tbe molar absorytiity, and is expressed in u1its Lmolr cm-r wheu c (concentration) is expressed in molarity and b (pah ligh of lighi) 1a @.

Using Beeds Law, the absortance of a sample cau be related to its concentration- Absorbau.ces areadditive, and Beer's Law can be used to determine the concentations of a mixhrre of species, as longas they are not interacting. Bee/s Law is limited in that it applies ouly to dilute solutions. Inconcentrated solutioas (> 0.01 M) particles iateract altering the ,"alysfs

"LiUty to absort a certain

wavelength of radiation which causes uon linear deviations from Beels Law. Changes in the refractiveindex of the solutior, and chemical reactioo wiil also cause error in applyng geert taw to a sarnple.Finally, Beer's law is only valid for monochromatic radiation- Thus,-the most acc,rate result will beobtaiued by using a source able to produce inteuse radiation at a siagle wavelengtb-

UV-Yisible Sp echophoto meters

Speckophotometers are made uP of stable source of radiaot energy, a ransparent sample container, adevice for isolating specific wavelengtb, a radiation detector which coavers ta$mitted radiation to ausable sipal, and a sipal processor aad readout. LIV-Vis spectra can be used to detect for thepresence of absorbing fuuctional $oups or chromophores.

13

tIV-visible speckoscopy is a valid, 5imple and cost effective method for determiaing the coacentrationof absorbing species if applied to pure compounds, and used with the appropriate standard curve. AstaDdard curve relating absorbance to concenhation can be developed ficr any 66'r,found, and used todetero.ine the concentration of sanples containing tie srme compouud- The analysis should be done ata wavelength with naximum absorptioa, and located in relatively flat region of the specta so tlatabsorbance will be high and coustznt in a narrow r:rnge around the chosen wavelengtb- The optimalwaveleirgth should ensure good absortance of the analyte aud low absorbance by other spccies in thesolution. This waveleugth will allow valid absorption measuremcnts to be made on *"iy" samplesthat contain mixhrres of materials-

Sources: A radiation sonrce for spectroscopy must generate a beam with sufficient power, wavelengthrange and subility for detectable ard reproducible results. Many t-IV-Vis speckophotometers r6e adeuterium t:mf for the UV range and switch to a tungsten filament lamp at 350 nm for the visibierange. The eleckical excitation of deuteri,'m at low pressure results in a continuous spectum ofemitteil radiatioa from 160 nrn to the beginning of the visible (375 nm). At around :iO nm tneinskument switches its radiation source to a tuagsteu filament. The radiaat euergy emiued from aheated tungsten filament approaches that of a black body and thus, is temperature depeudent.

Sample containers: $amPle containers (cells or cuvettes) must be coastucted of a material that istranspiareat to radiation in the wavelength raage of interest. Coutainers of quarE or fused silica arenecessitry * ft: YV raugq and can be used into 700-3000 ,,m infra-red region. Glass and plastic canbe used in the visible reglotr as well.

Wavel en gth Selectors : Filters and monochromators.

Detectors: Phototube, photomultiplier tube (pMT), pbotodiode, photodiode array-

OBJECTTYES

To determine the maximum waveleugth of potassium permanganate.To plot the calibration curve of potassium permaagaaate.To determine the concentration of an unknown56lution of potas5inm pennangaaate.

APPARATUS

Beaker

Burette

Glass rod

Volumetric flask 100 mL

D'opper

CHEMICALS

Potassinm p ermanganate (KtvInO4)

Distilled water

t.2.3.

t4

PROCEDURE

During this laboratory expcrimcnt you will rnake a series of dilutions, and thea take the absortancc ofthe dilutions at L*. The permanganate iou absorbs at 534 om aud it is at this wavelength mat we willdetermine the absorbance values for the solutions. Iu this experiment, an iaitial peroxanganate stocksolution is prepared and the solutions to be measured are diluted &om a dilutiou of the stock- Ouce tbeabsorbaner values are taken, generate a Beer's law plot for trGdnOa and determine the concentration oftbe 'nlcnsvm solution.

A- Preparation of the KI\{nO4 Standard Solutions

l. Weigh accurately 0O1 g KlvInOa, to the nearest 'ng, on a wgjgbigeBper. Record ffis rc3ding.Usiug a funnel tarsfer the solid to a@

2. Dissolve the solid with a few '',T- of $stil&k+Ier. Stopper aad shake the flask Attd distilledwater to the oark, using a medicine dropper io aaa t}re iast few &ops. Stopper the flask andshake several times to homogenize the solution.

3. Pour the 'stock' solutiou iuto a beaker. Label the bealer as '100 ppm':

4. Pipet 5.00 mr of the 'stock' solution and dilute with distilled water in a 100 'nT- y6lrrmeEic

flaslc

5. Transfer iato abeaker a:rd label as it as '5 ppm'.

6. Repeat Step 4,using 10 mr , 15 mL and 20 mT stock solution and tansfer into small beakers.

7- Label the beakers as'10 ppm', '15 ppm' and'20 ppm', respectively.

Preparatior of the Unknown

1. Pipet between 5.00 to 20-00 rDl of the 'stock' KlrllaOa solution and dilute with distiiled wateriu a 100 'nI- voluaetric flask.

2. Traasfer into a beaker aad labelas it as 'I]nknowu'.

Determination of Absorption Maximnm (L.r)

Take the absorbance reading of each of your samples in successioo, starting with the least

CAATTON - Yhen using spedrophotometers, alt of your data must be taken on the sameinstnamen$ d the sqme time If you need to change instruments you must stort over and takeall of the datafor the spedrum again

Remember that your blark is always the s^*e as the solvmt, in this case, distilled water. Eachspectrophotometer wili need one cuvette for the blarh and each student will need one cuvette forthe sample. I:r order to 'clea.n' the cuvette so that a sample is at the appropriate concentration, adda srnall amount of lhe 'new' solution using a medicine &opper and rinse &e sides of the cuvettewith the solution- Rinse the cuvette three seoarate ti"'es in this wav. and then onlv fill with thesample before tqking the absorbance reading-

B.

C.

15

once this series of absorbauce readings has beeu recorded, let your lab instructor check theresults' rt may be that you need to re-make one or more of the diluted solutions. The mostcorumon error in this experiment is not measuriag the vol,mes accurately.

Rinse aud retura your cuvettes at the end gf the experiment. RINSE with distilled water only - donot use a brush or soap on thesc oryou risk scratching thcm.

D. Operation of the {fv-Vls Spectrophotometer

Instramenb Vadan/ Cary 50 W_Vis Speckophotometer

Operating Instructions

1. Select Cary'Win (fV icon

2. Click on 'Scar,

3' Go to setup, click on ttre 'GARY', then key in the required start and stop scan wavelength' (Dna)

4:.

5.

6.

7.

8.

9.

10.

11.

12.

13.

14,

q) Y-mode (rnia:0 aad max: l)(b) 'X-mode: 800-200 nm ^{v- . r.r(c) Bearn 6qds:Dual Beam - ,/ u. ^1.e''+ l?t\

Select cycle mode ifmore than I cycle is r-equired >- *I*l iU fSelect "scan control speed" -+ Fast \eCY

\ ';

17^ X(" 4, u' t

,,,J*cliik on 'Baserine' icon aud check .Baselile correction Fr:action, \6r \qt) rv

's * ,r" -'dAt the peak table option, select maximum peak /rn\*

$? \' ,#

In the Auto Store icon, set .storage on @ompt At Start), ,, X--_ * f'

To save the method, Go to the f,re and save the scan method ' nY - 6\qtr.l 1 - i"

Fill the'BLANK, cwette solution with distilled water il ' ^ \ *'

"-[$' ,v

Put the'BLANK' cuvette solution and crick '!a5sline, \fi t

4lRemove the 'BLANK' cuvette and put the .sAMpLE,

cuvette (you may use ttre 20 ppmsolution)

Click 'Start' icon to start the Deasurement-

E. Determina6on of the Unknowu Concentration1- Click oa the conceukation icon

2, Go to Sehrp, click on Cary icoq then key ia maximum wavelengtb tr*.3. Select Replicate : 3

4. click on'standard' icon, check the 'calibrate During Run' fimction

16

,l

I-t

II+

5. Set the calibration standard unit (mg/L) and the number of tle st"nd.ard srmples

6. Select the Fit Type (Linear Dircct)

7 ' Go to sample icon, select the number o1'ths sarnples and key in unknowq

8. Go to the Report icon, key in operator name and the commsrl

9. In the Auto Store icon, set 'storage on (hompt at Start)'

10. To save thc method ciick 'File -+ Save Method As -+ Ok

11. Put the 'BLANK' cuvette aad click 'Zero,

12. Remove the 'BLANK' cuvette andput the .SAMpLE' cuvette

13- Click 'Start' icon to start the conceukatiou measurements

GRA?EING USING MICROSOF'T EXCEL

Use the computer and open Excel and create a graph of absorbance vs. coocenkation (in nnits of ppmor m€ll) on the computer. The graph should have a descriptive title that includes the particular SoftDrink you aaal'yzed and the concentrations at which all of tne absorbances were obtaiaed. The axesshould bave approp;ate.laf{g (units). The li''e should fill the graph adjust the axes appropriately.Leclude the trendli:re and the R' value, through the Excel saphintfirnctions.

To Make tle Graph

If you are unfamiliar with the latest version of Excel,part oJyour rqort.

1. To Open Excel:

this will help you. You will submit this graph as

. click on START in the lower left corner and

. click on Prograos

. click on Microsoft OfEce

. click on Microsoft Office Excel 2007 toopea Excelo Notes on Graphing in Excel

2. Input da4:

' X-axis data in the left-most col ,,n,r Y-axis data in the next columl. Higbiight the data (and not the headers for the data) aud. click on the INSERT tab at the top of the screenr from the meuu select. SCATTER with markers but no lines.. This shouldproduce tle appropriate graph.

' Once the graph is made, right click on one of the marters, and select ..ft6prtling.,,

' This should produce a skaight line that may or may not go through all of the points onyour graph-

' Next, right click ou the trendline (not on a marker) and select .,Equation,, aa.d

separarely, -'t(-".

' Both the equation of the [iue, and the rf value should appear on your graph. Make. sure that the box containing the equatiou aud the R2 value does not interfere with

t7

reading the graph- The box caa be moved around using the mouse to select it and dragit.. Next in the top tab, select LAyOI-ff' Select: Chart Title and write iu "visible Absorption Speckum of (Braad aud Flavor),,. SelectAxis Tifle and add Wavelength (nm) to the X_axis. select Axis Title a,,d add Absorbance (no uuits) to the y-axis. Rjght click on Series I and delete iL' Click on Axes - to format X-axis: start at 400 not at 0 nm. you sho,ld always

maximize your graph space appropriately. Show ouly 400_700 nm.

3. ToSAVE:

' If you do NoT have Excel 2007, wheu you saye the wortbook, select SAVE AsExcsl 97-2003 and it should be compatibre on most computers.

REPORT

l' You-will record ail observations and tabulate your data. Then use Microsoft Excel to constructthe Beer's Law plot to determine the unknown concentation of KMuOr.

Attach the print suf slfeined from the LIV-Vis instrument into your lab report aloug with yourMicrosoft Excel spread sheet.

Discuss the sigaificance of the corelation coefficient for the graph you obtaiaed_

Discuss the principal of operation of the uv-vis instumeat you used and include the fuuctionof each component.

PRE-LABORATORY QTTESTIONS

1' Show how you will PiePare a 5 ppm solution fr-om a 100 ppm KMno4 stock solution using a 100-T volumekic flask Briefly aescribe the procedure.

2' .Y:'.:13"_:T..-.r"d

rvaverengti. at maximum absorption, L* for the Kirdnoa solutioa? what i2":":l:^:r$wavetengthi(nctudethename, utie, page it-.t"i a" of thereference

2.

3.

4.

to get the answ*.) you used

QUESTTONS

I' why is glass not a suitabie cen material for use in w spectroscopy?

2' state one advantage of using the lrv-vis speckophotometer compared to a Spectonic 20 for thisanalysis.

t8

Table 2.1

Solution Concentration @pm) Absorbance

Standard I ao 0-qq1Standard 2 1q D-q-9 rStandard 3 Iu $ - 2s1

Standard 4 4 n . \-l-ov i'Jll\-Unknown a o - )\L{

DATASHEET EXPERTMENT 2

UV-YISIBLE DETERMINATION OF AN T.INKNOWNCONCENTRATION OF KMnO4 SOLUTION

Nam6 . F4uu - wArrr -Apn

r*

Student ID :

MassofKMnOo: 0'S\Show the sample calculation for ttre preparation of standard 3:

Coaceatration gf r rn kn 916,11;

Lectursr's signature,

Date :

Group:

L- = Se+-gtf ,.r^

19