You are on page 1of 9




Th9 word spectroscopy is used to refer to the broad area of science dealing with
the absorption,
emission, or scattering of erectromap.etic radiatiou by morecur"s, ionl,


Spcctroscopic techniques are some


of the most widely used aualytical methods in the world today.
Diffsrent regions of the eleckomap.etic spectrum such as infrared, visible,
ultraviolet, or, X-ray
radiation can be used to interact with matter. There are maoy importaat
inskumental methods of
chemicai analysis that rely on the interactiou betweeu lignt ana matter
to probe chemical skucture.


white light is visible light that contains all the colors of the rainbow.
If white ught hits a colored
solutio4 the eieckons in the solution'will absorb some of that ligh! while
the rest will be ransmitted
through tlie solution. It is thi trausmitted light that we see with iur eye".
lt" i*fort-t ti La"ot*a
t}at a red solution does not absorb red lig!! it actually absorbs blue-green lightThe solution appears
red because the intensity of the blue-green light in the original white
light has been decreased- The
follorving table shows the relationship between the color Ibsorbed by
a solution, and the color we

Table 1-1: Relatiouship Betq/eea Absorbed and Observed Colour
Absorbed'Wavelength in nrn (Absorbed Colour)


400 (violet)


450 (blue)




490 @lue-green)


570 (ye11ow-geen)


580 (yellow)

Dark blue

600 (orange)


650 (red)







=cl. The wavelength chosen is usually that one at which the absorbalce. The detector compares the two iatensities and reports the result as either percent transmittance (hD or absorbance (A). dimensionless. accurate aad seositive methods for measuring the concenkations of dilute absorption spectrophotomeky.ieaplaceriirti:'leambetx'een :.1.:^:l=omaaor asci the delestor.-.-\ .". s1 si'nply Beer's Law. will be less than t. Althoug! the instuments that arc used to measurc thc intcraction of various and operation" they all regions of clectoomapetic radiation with matter differ a great deal in dcsip 'nskument that is used to a simple coutain thc samc basic components. These parameters are combined into a mathematical relationship lmown as follows: .. solutions is by colorimetry or The Spectronic 20 inskument you will use can correctly bc called a colorimeter.::t).One of the most convcnient.-.-:a. Source 0etector Monochremator Sample Figure 1.rll y A: ebC Equation (3) / equation 3.It is important because it shows that absorbance is directly proportional to the concenkation of the absorbiag species. -.rc:5ilr. e (epsilon) b the molar absorpfivity..:::-i. and c is the concentration.Equation 3 is known as the Beer-Lambert Law. The -_:-:t:. A: fle t): 2 f---log (o/" T) Equation (2) amount of iight absorbed is dependeut on how well the substance absorbs ligh! the path length of the light.The5:-!leis-.Lr a n4r{iqq-rl7r'qrarrelenafh $i1qc fhe rrn.ator proCucieg abeam with a siagle eoergy or a narrow baad of energies.. becausc it measures the atrsorption of light in the visible spcctrum that wc perccive as color.i. These terms are defaed to be: Equation (1) (the fraction of I" that gets through the samples is called Trancnittan6.6 is the path length.1: Main Components of an Absorptioa Speckophotometer A]l the parts of the'nctru6ent work together as follows (FigUre 1. and the technique used is said to be colorimekic.1): Ligtrt from the source Passes '-:-:l:: --te . it tells us how much light is absorbed at .ifc nn a 2rp fimil:alltr usually given in ncr lifer Qf e-d f-he n3fh lensf}' ic -nlec are expressed as lvfl sm-t to make A the s most commonly rroits otr certimeters.. A schematic diagram of measure the absorption of visible light is shown in Figurc 1. f .:=a=I. The molar absorptivity is a characteristic of the absorbing species.'-ed:rctor. lf some of the iigbt is absorbed by the srtnple. the iatensity of the beam reachi-ng the detector. I.The tecb:rique is based upon the Ereasurement of thc amount of ligbt energy a solutiou absorbs from a bea"' of light of a certain wavelength.log T 7 Jog (U .'. of tbe species to be analyzed. and the concenhation. is at a maximum..

the if the concent-ation is described in units other than moles per liter. Quaatitative aualysis: to determine. lj:3.'ple from its absorplion speckumTo determine the waveleugth at maximum absorbance $'-J for a soft drink 5rmpleTo determine the udcnown concentration of a soft drink from the calibration curve. 3. 2. Equation (4) The picture below is a representation of a Siectronic 20-D colorimeter which is the instrument you will be using in this experimeul Use of the inskument is easy and results are excellent provided you follow all directions carefully- Sanrple Gomparbnent 1 N o/o Transm ission adjusbnen t Orr/Off and 0 % Tr:ans. the concentration ef 31 nnknown solutiou from the I 2- stqn dard calibrafr on curve.mission adjustment Figure 1. then e is replaced with a symbol or absorptivity.vsis caa be dooe with the visible absorption measurements: Qualitative aaalysis: To determine the wavelengtfu of maximum absorbance from the absorption spectmm as well as absorptivity value of a specific molecule.*^ lg^ .2: A Diagram Showing the Components of a Speckonic 20 Spectrophotometer T*'o t1'pes of aaal. To determine the origin of the colour of a soft drink sar. and Beer's I-aw is written as follows: However. APPARATUS Beaker Pipet Gla-ss rod \blumeiric flaslq 50 mT Dropper [1/l . OBJECTf\TES 1.

ofthesoftdrilkintoa50. Set ttre wavelength to 600 nrn3.Standard.arrk' /1ha 'fl2n1q' iq ']icilll''l 'r':+:: i:: r.. butit is made of special an obtain a cuvette.youwilllearnhgwalawofsPectoscopy. . t .trir-'r' Yr of water_ anrl. Repeat step 2 using 10 -T ' 15 mT 20 B. imtrument to warm up (minimrrm 15 minutes)' Turn on the spectonic 20 and wait for the 2. test tube.Retort stand CHT"I\.00rnT. ther fill f..solutionsofSoftDrink(KnownConcentrations) l. The cuvette may look like 9t9"ty tube a:rd rinse it with distilted the Clean expensive! trigh quatity glass aad i" -rrJi* tl-.*""*eot and then use properly to how learn valuc will fi. Pour soft drink into a beaker aod stir to remove thc carbonation- 2. Adjust 0% transmittan ce (h T") (Adjustinc dark current - uothing should be in the sample comPartment' i.puolp""t knownasBcer'sLaw... ott"iora by mcasuring vs' (1-_).Pipet5."t uv a soft Drink 'olotioo has a maximum absorbanqJrignt which at (t) wavelength find the the absorbance of a sample . of spectroscopy' You wi-ll be inkoduced 16 1fos fuadarnentals expcrim*t this Y1u of part fust In the you will use the instrumeot to a Spectro*"iO . "f'o'u*"" 'h"the unlcnown solution can then be measured at the fte absorlrocJof single wavel"rr* . A plot of absorbaacc *ur"l*gtu"^ airo"ot Drink Soft the of "t wavelength rryill show You ?'*' Inthesecondpart. piecc of scieutific equipment and should be Treated Thcspectronic 20 is a moderately e:pensive witl all due care and resPect' Instrument: Thermo Spectronic/Geuesys 20 OP erating Instructions same wavelength *d its conceltration' compared to the calibratio[ curse to deterEitre A.canbeusedtodeterminethcconccntrationofan'unknownsolution-A of differe8t dilutions of Soft Drhk at a calibration curvc will be preparcd by -"*. grea'yellow PROCEDI]RE.. The "uro.'l.lre firhe ehnrrt thisexperiment). spectrophotometer.rrtt sot. Cover ald shake to 3. Operation of the Spectronic ' mr aud 25 mL of soft dri-nk- 20 and Determination of ]'-. ted..flCATS or or:lnge in colour) A can of soft drink (blue.Carefullywipeawayanysolutionandfingerprintsfromtheoutsideof the tube using 1c a KimwiPe' .termedBeer-I-ambert.rm of a soft Drhk i.Preparationof.0'.lvolrrmeticflaslcanddilutetothemarkwith Store ia 2 5 rall beaker' rnake a homogeneous solution' distilled #ater.

Read the absorbaace aad record the rearlings in Table 12. Cleaa alother cuvetie and rinse it with a small arnount of the 'stendard' soft drink whose absorbalce is to be measured (i. 12.5. set the Spectronic 20 to the wavelength maximum 2. Measure and record the absorbance of the 'uuktrowr' soft drink solution- (1"*) obtained from part B. close the top aud change the wavelength to a setting which is 20 nm lower. drilk solution.'aekic flask a:id dilute =e s. Put the 'unknowu srmple' into a cuvette not darker than 1fos most concentrated until it is about % firll_ (Note: Alternately. a t c . Read aad record the absorbalce.mple holder. iO. Measwe and record the absorbance of each 5 'standard' soft 4. Remove the cuvette.use any one of the diluted Soft Drink solutions in Part A). taking absorbance readi:rgs at each 20 nm interval. wipe it with a Kimwipe and place it in the sample holder with hash marks aliped.i o rle rca:t *ith distilied wrater. Make sore the colour of the rmkova soiutiou you prepared is standard-sol:rtiou- 3. ?:'. in the procedure above. QuantitativeA-nalysis Of The Soft Drink Solution 1. 6.Stoppcr aod shake the flask to homogeaize the soiution. Remove the the cuvette and set it aside without emptyiag the distilled water.: r )sing a graph paper. 8. Pour some of the soft drink ilto papt dril* and determire the )*. Then fill it about % fiil with solution. the 'ilnknown samplz' may be assigned to you fu the lab instructor. 1. for"rt the cuvette coutaining the same Soft Drink solutiou you used in Step (6). to the lab!) Drhk $ample a beaker and stir to remove {he carbonation- l.e. . Set 0 and 100%T as given 3. Repeat Steps 8 through 11 until 360 nm. Adjust 0 absorbance aad 100 % transmittatrce with the cuvette 6sntzining g6s'!!:nk' ia the s. Reset 0 % transmittarce 9. Refer to instructions proided by the lab instnrctor during the laboratory bnefng-) D.: i-:-k witlout measuri-ng tie volume iato a 50 lT volr'.Remove the cuvette- if it has chaaged (sample comparhent must be empry). i 1. Preparation of 'Unknown' Soft 1 . plot the absorption spectrum of your scft Q'[ote: Remember to bring a graph C. Tnsert the cuvette of distilled water and reset the 100 %T.

3: J- t Red orange Al Example of an Absorption Spectrum and The Corresponding Absorbed Colours.@O NOT use a tcst tubc brush on thc cuvettes. the straight line through thc points and the title oa the top.on plot to determine thc concentration of your u. 2. where four opeu circles indicate the measurements of the four hown coucentrations.The plot which is made of absorbance vs. Note the equally spaced divisions.Find the concenhatiou of your unknown by detcrmining where its absorbauce crosses the calibration Iine on your graph. Plot the absorption spectrum of thc soft drink ou a graph paper.known soft drhk solution.Now you cao use this calibrati.a i 0) o d . As cxample is showu below. Next make a plot of the absorbaace of the diluted soft drink solutions versus their concentrations. Usc thc following diagram to erplain the origin of the color of your soft drink in the discussion! I t t . Clean and dry all cuvettes. Include the diagram indicating the wavele. One single shaight line is drawn that comes closest to all these circles.o 4Sm rnranreEngth (nm) grccn yel Figure 1. Since you have ouly used soft ddnk in this experinen! the waste catr be poured down the drain.concentation.) DATA ANALYSIS 1.a olot like the qrarrh below is called a "Beer's Law Plot"- 6 . Cleaning Up l. aI pphs should contain a title and appropriate labels on the axes. it will scratch them. Sec below for an exam. Your graph should Ell the entire page and use accurate narkings.1le.D l< o {)) .E.lgth regions and the correspouding colors of visiblc lig[t.

for the unknown whieh reads 0. this was 25 %).o-5 I Absorbance 0-4 al sOC nm o-3 I valu=fotmy z/ unknornn grrye ) -a 02 *olut'pn # 123 I :516lor 0_1 10 I 20 :o 40 Corcerdraticn Figure 1. 5- between the fwo ternos.sming to lab. how wil-l the following be affected (i. no change)? ab. Following the line straight down from this point.the one with the square . when the concentration of an aualyte increases. State Beer's Lawmathematically and defi-ue each term- Define 1"--. the concenbation for the unknown # L23 !s determiraf'(for the sxarnple.Vn * 123 50 [96] Iaw Plot" Note iu ttre example.eral l6gas imFortaaf f6 aspects of the experiments. il I I QIIESTIONS 1. Give the equation which shows how you can mathematically conyert I I l_ 4.What lrpe of electomapetic radiation will be used in this experiment? 3.crease. .They are intended to introduce you to sev. Report yow value in your 'conclrrsions-. lc] I PRE. b.Transmittance and absortance.31.-D"fio".LABORATORY QITESTTONS I The follow'ing preparation questions should.4: An Example of I a Beer's unknc\. the student created a fifth point . be answered before c. decrease. You must turn il the answers to vour in<tructorbefore you will be allowed to begin the experimenl l. What is the purpose of using the'blank' solutioa? 4. 'Based on Beer's Iaw. Absorbance Transmittance I 2- I 3. What colouds) is/are being absorbed in your soft drink I I I Why is i1 imFortant to first obtain the absoqption spectrum of the soft drink before paking calibration curve? a .DEfiEeqpe{troscqpy2.

lq 0']q 0-fq1 0 o-9a1 O'7?]o d-33-9 bG o.rt 6R t f d}l (Stfaf.2: The wavelength and absorbance of soft drink 500 580 540 s o'olo o-@7 o.308 480 o-13? o-13+ o"f. oGh o-obb o.l oJ o.rgq 560 hyeraqa Absorbance Waveleueth [nm) I o t -fBq 500 o'?to 6-3oB a2oA o..ls3 440 o.olo o'ol6 o.fLeyfS) arowof softdrink: C+s Po) Bed Table 1.t o-osg o.ntID: : 1 VISIBLE SPECTRA OF SOFT DRTNKS : l9 Ju0e Pt3 t\ur Sarah Hanaq Et thlrrrad fftir Date 2A0qqlqD5 Group.os.}7 460 o-152 0-lE3 o'a*r o.o o.oSB 360 L-: 5fO .018 6 -oAl 380 o-ob+ o'o+o o-ob5 o'6bb o-obo o. Nameofsofta.DATASHEET EXPERTMENT TI{F'.105 O-lo3 6-lo3 420 o'oBo o'qb o -oq5 o'Dot o-cn-3 o-o?-3 D.153 400 6.t 0q o-lq + o.oGb s. Nrme Stude.221 520 .

) l'os5 'Ern{ 2 l' 3 l.T &nti Sampie calculation forthe concert-atioa of solution 2: I I 9.".'e. flvyla". * L c*'*Y q'* c -o+L[ > t-bYo **(o o.t^' I I I I ? +l 0V\ 1-330 l-?+o z-yE t-b 4D *-{.Ofl hb Lecturer's signature..Srn{. - -t b*[ 00cw Concentration of ualnowu: I I Unknovn sauple A 9-ao I I 3-?o v9 LOu.{tt)tt)(U /- L- .Table 13: Table of soft drink conceotration (vohune Concenhation (volume %") Solutions Yo) afi absorbance Iuat Absorbance €olta-ti 0O $vaph Tu?iiild"^eI o'1qo o'tr?Ll' tom{ l-51+ 0.r" Or C= lo /" . I I I -T I '".T l5r$/ a.r. 4 :'$o 2t)rn-t I 5 It'.