Using Spectrophotometry to Determine Concentration

INTRODUCTION Spectrophotometry is a technique used for measuring the quantity of light that is absorbed or that passes through (is transmitted by a sample solution or mi!ture" #hile spectrophotometry techniques are a$ailable for all portions of the electromagnetic spectrum% in this lab% our spectrophotometers &ill detect electromagnetic &a$es that are bet&een '((nm and )((nm in &a$elength * the spectrum of $isible light" The data pro$ided by the spectrophotometer ta+es on t&o general forms, percent transmission and absorbance" Percent transmission is straight for&ard * it is simply the number of photons present after passage through a sample di$ided by the number of photons that entered the sample multiplied by -(( (i.e . transmission / (photons out0 photons in ! -(( " It relates to the quantity of absorbing component in a solution in an in$erse logarithmic proportionality" Absorbance (1 is a function of percent transmission (T % namely 1 / 2 log T" 3ecause absorbance is plotted on a logarithmic scale% there is a direct linear relationship bet&een the concentration of the absorbing substance and absorbance" 4ach substance &ithin a gi$en sample &ill absorb light at characteristic &a$elengths" The color displayed by a substance represents the &a$elengths reflected" 1n absorption spectrum can be generated by measuring the absorbance (after 5eroing on a blan+ o$er a range of &a$elengths" This is best depicted by a line graph such as the one sho&n here" 6ere you see the absorption spectra for three different pigments commonly found in plants"

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3y measuring the absorption spectrum of a substance (i.e. all the &a$elengths at &hich it absorbs it is possible to identify it or at least place it in a particular class of compounds" The &a$elength at &hich pea+ absorption occurs% the absorption maximum (: ma! % is $ery useful &hen trying to identify an un+no&n" 3y creating and measuring a series of standards (e.g. serial dilutions % it is possible to quantify the amount or concentration of a substance in a sample" 7or e!ample% in their pure form% the nucleic acids can be quantified by absorption measurements in the U; range" <ou &ill practice generating an absorption spectrum for food color dye using a Spec =( spectrophotometer in >art I of this lab" >art II of this lab allo&s you to apply spectrophotometry in order to measure chlorophyll concentrations in oli$e oil" Oli$e oil is made by pressing0e!tracting the rich oil from the oli$e fruit by $arious methods" There are $arious grades of oli$e oil? three common grades are, e!tra $irgin% regular% and light" 4!tra $irgin oli$e oil is considered the highest quality" It is the first pressing from freshly prepared oli$es" It has a greenish2yello& tint and a distincti$ely fruity aroma because of the high le$els of chlorophyll and other $olatile materials e!tracted from the fruit" Regular oli$e oil is collected &ith the help of a &arm &ater slurry to increase yield" It is pale yello& in color% &ith a slight aroma% because it contains fe&er $olatile compounds" @ight oli$e oil is $ery light in color and has no aroma because it has been processed under pressure to remo$e the chlorophyll and $olatile compounds" @ight oli$e oil is commonly used for frying because it does not affect the taste of fried foods and it is relati$ely ine!pensi$e" The absorbance spectrum% in the $isible light range% of chlorophyll gi$es interesting results" The chemistry of chlorophyll creates three blue absorbance pea+s at '-A% 'B'% and 'C= nm% and one red absorbance pea+ at A)( nm" That means that chlorophyll absorbs blue and red light &ell and transmits or reflects green light" This is &hy &e percei$e chlorophyll and the plant lea$es that contain it to be $arious shades of green" >lants tend to absorb blue and red light &ell * green light% not so &ell" The primary obDecti$e of this e!periment is to determine the concentration of $arious chlorophyll containing oli$e oils using their absorbance properties" <ou &ill use a ;ernier Spectrometer to measure the absorbance by chlorophyll in each oil sample" <ou &ill measure the absorbance of e!tra $irgin oli$e oil o$er the $isible light spectrum and select the &a$elength of ma!imum absorbance (: ma! " 1 higher concentration of the solution absorbs more light (and transmits less than a solution of lo&er concentration" The e!tra $irgin oli$e oil has a chlorophyll concentration (molarity of 9.82 x 10-6 mol/L" <ou &ill first calculate the chlorophyll concentration for each of the fi$e serial dilutions that &ere made from the e!tra $irgin oli$e oil" <ou &ill then use the ;ernier spectrometer to measure the absorbance and transmittance of each dilution at the : ma!" The graph of absorbance or transmittance vs" concentration for the serial dilutions &ill describe a direct relationship% +no&n as eer!s La"" Transmittance (T is defined as the fraction of incident light &hich is transmitted ( i.e" passes through a sample" Thus% T / I0Io% &here Io equals the intensity of light &hich stri+es the sample and I is the intensity of light after passing through the sample" Transmittance is usually e!pressed as a percentage, #$ % &'/'o( x 100

1bsorbance (1 % or optical density% is a logarithmic function of T and is e!pressed as, A % lo)10 &1/$( % lo)10 &'o/'( Note that absorbance has no units" The shorthand for absorbance is 1 !!!% &here &a$elength at &hich the measurement &as made (e.g" 1'=( "
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is the

So% for e!ample% at -((. transmittance% 1 / log -"( / (" 1t B(. transmittance% 1 / log (-0("B / ("E(" The Spec =( spectrophotometer has t&o scales% one calibrated from ( to -((. Transmittance and the other as 1bsorbance% ranging from infinity to (" Note that the highest calibrated unit of absorbance is ="(" Spectral data are usually plotted as absorbance (y2a!is $ersus &a$elength or concentration (!2a!is " The relationship bet&een absorbance and transmittance is illustrated in the follo&ing diagram,

7I8UR4 92= So% if all the light passes through a solution &ithout any absorption% then absorbance is 5ero% and percent transmittance is -((." If all the light is absorbed% then percent transmittance is 5ero% and absorption is infinite" Notice that absorbance is basically a log2transformed $ersion of transmittance &hich means that a plot of absorbance against concentration should result in a linear relationship" #e &ill test this by plotting absorbance and transmittance on both regular graph paper and semi2log graph paper" <ou &ill determine the concentration of chlorophyll in lesser grades of oli$e oil by measuring their absorbance &ith a spectrometer and using the best2fit line equation of the 3eerFs la& cur$e to calculate the lesser grade oilFs chlorophyll concentrations"

G1T4RI1@S ;ernier Spectrometer ;ernier @ogger Pro E soft&are Spec =( spectrophotometer e!tra $irgin% regular% and light oli$e oil e!tra $irgin oli$e oil serial dilutions (B regular graph paper >ROC4DUR4 Using a Spec =( The &a$elength on a Spec =( is selected by adDusting a prism &ithin the instrument such that only a narro& range of light &a$elengths are directed through the sample" In addition% the bulbs used to generate the light come in a $ariety of &a$elength ranges" 7or general purpose &or+% &e utili5e broad range bulbs &hich allo& absorbance to be measured o$er the entire $isible light range" In order to measure the absorbance of a particular substance in a reaction mi!ture% it is necessary to first H5ero outH the spectrophotometer such that only the absorbance of the substance of interest is measured" This is done &ith a blan+ 2 a cu$ette &hich contains the carrier sol$ent &ithout the substance of interest" *eroin) the Spec 20 computer food dye solution cu$ettes plastic pipettes semi2log graph paper

-" Turn on the Spec =( and allo& it to &arm up for B 2 -( minutes (left front +nob " Set &a$elength using the dial on top of the Spec =("

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=" >repare a blan+ cu$ette by adding the sol$ent (&ater &ithout the absorbing substance (the food dye " The blan+ is used to calibrate the Spec =( so that any absorbance attributable to the sol$ent and0or glass cu$ette can be compensated" 3y 5eroing the Spec =( to the blan+% you &ill measure only the absorbance due to the substance in question" E" #ith no tube in the holder% adDust the meter needle to read infinite absorbance (/ (. transmittance using the left front +nob" '" Using a Iim&ipe% &ipe off0polish the outside of the blan+ cu$ette to remo$e greasy finger smudges" Using a &a! pencil or Sharpie% ma+e a small $ertical mar+ at the top of each cu$ette for alignment in the sample holder"

7I8UR4 92' B" Raise the sample holder trapdoor and insert the cu$ette such that the line on the cu$ette lines up &ith the line on the sample holder" Close the lid"

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A" Using the right front +nob% adDust the meter needle to read absorbance / ("( (/ -(( . transmittance " This step is called setting the Hfull scaleH"

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The spectrophotometer is no& calibrated to this blan+ at this &a$elength" +easurin) Absorbance on the Spec 20 )" Remo$e the blan+ and insert the cu$ette containing the food dye solution" Close the lid" C" Read the absorbance (lo&er scale or transmittance (upper scale as appropriate for your sample" Since you are creating an absorption spectrum% you should note the absorbance at each &a$elength"

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9" Create an absorption spectrum for the food dye solution" Geasure the absorbance from '(( to )(( nm at -(nm inter$als" Remember that you must re25ero the Spec =( &ith the blan+ e$ery time you change the &a$elength" 4nter your absorbance $alues in the table pro$ided here"

,oo- .ye Solution Absorbance /alues J '(( '-( '=( 'E( ''( 'B( 'A( ')( 'C( '9( Absorbance J B(( B-( B=( BE( B'( BB( BA( B)( BC( B9( Absorbance J A(( A-( A=( AE( A'( AB( AA( A)( AC( A9( )(( T13@4 92-(" 8raph the absorbance on the y a!is against &a$elength on the ! a!is to create an absorption spectrum for the food dye solution" 1t the end of this lab% chec+ the absorption spectrum of the food dye solution using the ;ernier spectrometer" Is the spectrum similar to the one you produced &ith the Spec =(K Absorbance

Using a ;ernier Spectrometer to apply 3eerFs @a& -" Obtain a rac+ &ith the follo&ing prepared cu$ettes, e!tra $irgin oli$e oil (+no&n chlorophyll concentration fi$e serial dilutions of e!tra $irgin oli$e oil (calculated chlorophyll concentration regular oli$e oil (un+no&n chlorophyll concentration light oli$e oil (un+no&n chlorophyll concentration $egetable oil (used for dilutions * blan+

=" Calculate the chlorophyll concentration of the serial dilutions and enter those $alues in the results table" 4ach dilution is a -,- dilution% so the chlorophyll concentration is hal$ed from each dilution to the ne!t" Remember that the chlorophyll concentration in the e!tra $irgin oli$e oil is 9"C= ! -(2A mol0@" E" Start the @abLuest @ogger Pro E program" '" Calibrate the spectrometer" a Use the cu$ette &ith $egetable oil as the blan+" 3e careful not to touch the sides of the cu$ette so as not to get fingerprints on the surface" >lace the blan+ cu$ette in the spectrometer" 1lign the cu$ette so that the clear sides are facing the light source of the spectrometer" Select Calibrate M Spectrometer from the 4!periment menu" The calibration dialog bo! &ill display the message, N#aiting"""""""seconds for lamp to &arm upO" The minimum &arm up time is one minute" 7ollo& the instructions in the dialog bo! to complete the calibration" Clic+

B" Determine the &a$elength of ma!imum absorption (J Pma! for chlorophyll a in e!tra $irgin oli$e oil" a b Ta+e the cu$ette &ith the e!tra $irgin oli$e oil and place it in the spectrometer" Clic+ 1 full spectrum graph of the oil &ill be displayed" Note that one area of the graph contains a pea+ absorbance (: ma! " Clic+ to complete the analysis" Record the &a$elength of ma!imum absorbance (e"g" '-A "

A" Record all of the absorbance $alues for each serial dilution of the e!tra $irgin oli$e oil% the regular oli$e oil% and light oli$e oil at J ma!" )" To obtain transmittance $alues go to Q4!perimentF to Qchange unitsF" Select Q. transmittanceF" Record transmittance $alues for each serial dilution and the regular oli$e oil and light oli$e oil at J ma!"
Solution e!tra $irgin oli$e oil (4;OO serial dilution - (4;OO serial dilution = (4;OO serial dilution E (4;OO serial dilution ' (4;OO 0oncentration &mol/L( ! -(2A Absorbance at J max $ransmittance at J max

9"C=

serial dilution B (4;OO regular oli$e oil &un1no"n 1( light oli$e oil &un1no"n 2(

T13@4 92= C" 8raph absorbance and transmittance against concentration" 1pply 3eerFs @a& to estimate the concentrations of the regular and light oli$e oil" 4nter those $alues in the table" In addition to the table% also include graphs sho&ing the data and linear2regression equation for the serial dilutions" <our graphs are easy to create in 4!cel" 4!cel &ill automatically calculate the linear2regression equation for you" DISCUSSION LU4STIONS - #hat is the relationship bet&een percent transmission and absorbanceK

= #hy is absorbance data most frequently used to determine the concentration of a substance in a solutionK

E If an un+no&n substance has a green color% &hat &a$elengths &ould you e!pect that it -oes not absorbK

' 6o& did you use the standard cur$e that you generated for the serial dilutions to determine the quantity of chlorophyll a in the t&o lesser grades of oli$e oilK

1D1>T4D 7ROG, Spectroscopy with Vernier R 2006 Vernier Software & Technology