Using Spectrophotometry to Determine Concentration(1)

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Using Spectrophotometry to Determine Concentration

INTRODUCTION

Spectrophotometry is a technique used for measuring the quantity of light that is absorbed orthat 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 thequantity 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 colordisplayed 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 absorptionspectra 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 inthe 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$efruit 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 fruityaroma 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 $olatile compounds" @ightoli$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 '= nm% and one

red absorbance pea+ at A) nm" That means that chlorophyll absorbs blue and red light &ell andtransmits 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 obecti$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 ofe!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 chlorophyllconcentration (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 eachdilution 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 sampleand 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 !!! is the

&a$elength at &hich the measurement &as made (e.g " 1'="

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,

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$e oil by measuringtheir absorbance &ith a spectrometer and using the best2fit line equation of the 3eerFs la& cur$e

to calculate the lesser grade oilFs chlorophyll concentrations"

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G1T4RI1@S

;ernier Spectrometer computer;ernier @ogger Pro E soft&are food dye solution

Spec = spectrophotometer cu$ettes

e!tra $irgin% regular% and light oli$e oil plastic pipettese!tra $irgin oli$e oil serial dilutions (B semi2log graph paper

regular graph paper

>ROC4DUR4

Using a Spec =

The &a$elength on a Spec = is selected by adusting 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+% &eutili5e 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 ofinterest 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

-" 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% adust the meter needle to read infinite absorbance (/ .transmittance using the left front +nob"

'" Using a im&ipe% &ipe off0polish the outside of the blan+ cu$ette to remo$e greasy fingersmudges" 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"

B" Raise the sample holder trapdoor and insert the cu$ette such that the line on the cu$ette linesup &ith the line on the sample holder" Close the lid"

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A" Using the right front +nob% adust the meter needle to read absorbance / " (/ - .

transmittance" This step is called setting the Hfull scaleH"

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"

" 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 ateach &a$elength"

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"

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,oo ye Solution Absorbance alues

J Absorbance J Absorbance J Absorbance

' B A

'- B- A-

'= B= A=

'E BE AE

'' B' A'

'B BB AB

'A BA AA

') B) A)

' B A

'9 B9 A9

)

-" 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

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+

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=" 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 fromeach dilution to the ne!t" Remember that the chlorophyll concentration in the e!tra $irgin

oli$e oil is 9"= ! -2A mol0@"

E" Start the @abLuest @ogger Pro E program"

'" Calibrate the spectrometer"

a Use the cu$ette &ith $egetable oil as the blan+" 3e careful not to touch the sides of thecu$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"

b Select Calibrate M Spectrometer from the 4!periment menu" The calibration dialog bo!

&ill display the message, #aiting"""""""seconds for lamp to &arm up" 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 Ta+e the cu$ette &ith the e!tra $irgin oli$e oil and place it in the spectrometer"

b Clic+ 1 full spectrum graph of the oil &ill be displayed" Note that one area of thegraph 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!"

Solutiononcentration

&mol/L( ! -2A

Absorbance

at J max

$ransmittance

at J max

e!tra $irgin oli$e oil (4;OO 9"=

serial dilution - (4;OO

serial dilution = (4;OO

serial dilution E (4;OO

serial dilution ' (4;OO



serial dilution B (4;OO

regular oli$e oil &unno"n 1(

light oli$e oil &unno"n 2(

" 8raph absorbance and transmittance against concentration" 1pply 3eerFs @a& to estimate theconcentrations of the regular and light oli$e oil" 4nter those $alues in the table" In addition tothe 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 oesnot absorbK

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

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1D1>T4D 7ROG,

Spectroscopy with Vernier 2006 Vernier Software & Technology

Using Spectrophotometry to Determine Concentration(1)

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