Determination of the Concentration of Chlorophyll
in Olive Oil using Spectrophotometry
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 mixture. While
spectrophotometry techniques are available for all portions of the electromagnetic
spectrum, in this lab, our spectrophotometers will detect electromagnetic waves that are
between 400nm and 700nm in wavelength – the spectrum of visible light.
The data provided by the spectrophotometer takes on two general forms: percent
transmission and absorbance. Percent transmission is straight forward – it is simply the
number of photons present after passage through a sample divided by the number of
photons that entered the sample multiplied by 100 (i.e % transmission = (photons out/
photons in) x 100). It relates to the quantity of absorbing component in a solution in an
inverse logarithmic proportionality. Absorbance (A) is a function of percent transmission
(T), namely A = - log T. Because absorbance is plotted on a logarithmic scale, there is a
direct linear relationship between the concentration of the absorbing substance and
absorbance.
Each substance within a given sample will absorb light at characteristic wavelengths. The
color displayed by a substance represents the wavelengths reflected. An absorption
spectrum can be generated by measuring the absorbance (after zeroing on a blank) over a
range of wavelengths. This is best depicted by a line graph such as the one shown here.
Here you see the absorption spectra for three different pigments commonly found in
plants.
1
By measuring the absorption spectrum of a substance (i.e. all the wavelengths at which it
absorbs) it is possible to identify it or at least place it in a particular class of compounds.
The wavelength at which peak absorption occurs, the absorption maximum (λ max), is
very useful when trying to identify an unknown. By 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. For example, in their pure form, the nucleic acids can be
quantified by absorption measurements in the UV range. You will practice generating an
absorption spectrum for food color dye using a Spec 20 spectrophotometer in Part I of
this lab.
Part II of this lab allows you to apply spectrophotometry in order to measure chlorophyll
concentrations in olive oil. Olive oil is made by pressing/extracting the rich oil from the
olive fruit by various methods. There are various grades of olive oil; three common
grades are: extra virgin, regular, and light. Extra virgin olive oil is considered the highest
quality. It is the first pressing from freshly prepared olives. It has a greenish-yellow tint
and a distinctively fruity aroma because of the high levels of chlorophyll and other
volatile materials extracted from the fruit. Regular olive oil is collected with the help of a
warm water slurry to increase yield. It is pale yellow in color, with a slight aroma,
because it contains fewer volatile compounds. Light olive oil is very light in color and
has no aroma because it has been processed under pressure to remove the chlorophyll and
volatile compounds. Light olive oil is commonly used for frying because it does not
affect the taste of fried foods and it is relatively inexpensive.
The absorbance spectrum, in the visible light range, of chlorophyll gives interesting
results. The chemistry of chlorophyll creates three blue absorbance peaks at 416, 454, and
482 nm, and one red absorbance peak at 670 nm. The combination of these wavelengths
is green to the human eye, but different sources of chlorophylls will have different ratios
of these peaks, which create various shades of green. Thus, in the world of chlorophyll,
all greens are not the same.
The primary objective of this experiment is to determine the concentration of various
chlorophyll containing olive oils using their absorbance properties. You will use a
Vernier Spectrometer to measure the absorbance by chlorophyll in each oil sample. You
will measure the absorbance of extra virgin olive oil over the visible light spectrum and
select the wavelength of maximum absorbance (λ max). A higher concentration of the
solution absorbs more light (and transmits less) than a solution of lower concentration.
The extra virgin olive oil has a chlorophyll concentration (molarity) of 9.82 x 10-6 mol/L.
You will first calculate the chlorophyll concentration for each of the five serial dilutions
that were made from the extra virgin olive oil. You will then use the Vernier spectrometer
to measure the absorbance and transmittance of each dilution at the λ max. The graph of
absorbance or transmittance vs. concentration for the serial dilutions will describe a direct
relationship, known as Beer’s Law.
2
Transmittance (T) is defined as the fraction of incident light which is transmitted (i.e.
passes through) a sample. Thus, T = I/Io, where Io equals the intensity of light which
strikes the sample and I is the intensity of light after passing through the sample.
Transmittance is usually expressed as a percentage:
%T = (I/Io) x 100
Absorbance (A), or optical density, is a logarithmic function of T and is expressed as:
A = log10 (1/T) = log10 (Io/I)
Note that absorbance has no units. The shorthand for absorbance is Axxx, where xxx is the
wavelength at which the measurement was made (e.g. A420).
So, for example, at 100% transmittance, A = log 1.0 = 0. At 50% transmittance, A = log
(1/0.5) = 0.30. The Spec 20 spectrophotometer has two scales, one calibrated from 0 to
100% Transmittance and the other as Absorbance, ranging from infinity to 0. Note that
the highest calibrated unit of absorbance is 2.0. Spectral data are usually plotted as
absorbance (Y-axis) vs wavelength or concentration (X-axis).
The relationship between absorbance and transmittance is illustrated in the following
diagram:
So, if all the light passes through a solution without any absorption, then absorbance is
zero, and percent transmittance is 100%. If all the light is absorbed, then percent
transmittance is zero, and absorption is infinite.
Notice that absorbance is basically a log-transformed version of transmittance which
means that a plot of absorbance against concentration should result in a linear
relationship. We will test this by plotting absorbance and transmittance on both regular
graph paper and semi-log graph paper.
You will determine the concentration of chlorophyll in lesser grades of olive oil by
measuring their absorbance with a spectrometer and using the best-fit line equation of the
Beer’s law curve to calculate the lesser grade oil’s chlorophyll concentrations.
3
MATERIALS
Vernier Spectrometer
Vernier Logger Pro 3 software
Spec 20 spectrophotometer
extra virgin, regular, and light olive oil
extra virgin olive oil serial dilutions (5)
regular graph paper
computer
food dye solution
cuvettes
plastic pipettes
semi-log graph paper
PROCEDURE
PART 1 – Using a Spec 20
The wavelength on a Spec 20 is selected by adjusting a prism within the instrument such
that only a narrow range of light wavelengths are directed through the sample. In
addition, the bulbs used to generate the light come in a variety of wavelength ranges. For
general purpose work, we utilize broad range bulbs which allow absorbance to be
measured over the entire visible light range.
In order to measure the absorbance of a particular substance in a reaction mixture, it is
necessary to first "zero out" the spectrophotometer such that only the absorbance of the
substance of interest is measured. This is done with a blank - a cuvette which contains the
carrier solvent without the substance of interest.
Zeroing the Spec 20
1. Turn on the Spec 20 and allow it to warm
up for 5 - 10 minutes (left front knob). Set
wavelength using the dial on top of the
Spec 20.
4
2. Prepare a blank cuvette by adding the solvent (water) without the absorbing substance
(the food dye). The blank is used to calibrate the Spec 20 so that any absorbance
attributable to the solvent and/or glass cuvette can be compensated. By zeroing the
Spec 20 to the blank, you will measure only the absorbance due to the substance in
question.
3. With no tube in the holder, adjust the meter needle to read infinite absorbance (= 0%
transmittance) using the left front knob.
4. Using a Kimwipe, wipe off/polish the outside of the blank cuvette to remove greasy
finger smudges. Using a wax pencil or Sharpie, make a small vertical mark at the top
of each cuvette for alignment in the sample holder.
5. Raise the sample holder trapdoor and insert the cuvette such that the line on the
cuvette lines up with the line on the sample holder. Close the lid.
5
6. Using the right front knob, adjust the meter needle to read absorbance = 0.0 (= 100 %
transmittance). This step is called setting the "full scale".
The spectrophotometer is now calibrated to this blank at this wavelength.
Measuring Absorbance on the Spec 20
7. Remove the blank and insert the cuvette containing the food dye solution. Close the
lid.
8. Read the absorbance (lower scale) or transmittance (upper scale) as appropriate for
your sample. Since you are creating an absorption spectrum, you should note the
absorbance at each wavelength.
9. Create an absorption spectrum for the food dye solution. Measure the absorbance from
400 to 700 nm at 10nm intervals. Remember that you must re-zero the Spec 20 with
the blank every time you change the wavelength. Enter your absorbance values in the
table provided here.
6
Food Dye Solution Absorbance Values
‫ג‬
Absorbance
‫ג‬
Absorbance
‫ג‬
400
500
600
410
510
610
420
520
620
430
530
630
440
540
640
450
550
650
460
560
660
470
570
670
480
580
680
490
590
690
Absorbance
700
10. Graph the absorbance on the y axis against wavelength on the x axis to create an
absorption spectrum for the food dye solution. At the end of this lab, check the
absorption spectrum of the food dye solution using the Vernier spectrometer. Is the
spectrum similar to the one you produced with the Spec 20?
PART 2 – Using a Vernier Spectrometer to apply Beer’s Law
1. Obtain a rack with the following prepared cuvettes:





extra virgin olive oil (known chlorophyll concentration)
five serial dilutions of extra virgin olive oil (calculated chlorophyll concentration)
regular olive oil (unknown chlorophyll concentration)
light olive oil (unknown chlorophyll concentration)
vegetable oil (used for dilutions – blank)
2) Calculate the chlorophyll concentration of the serial dilutions and enter those values in
the results table. Each dilution is a 2:1 dilution, so the chlorophyll concentration is
halved from each dilution to the next. Remember that the chlorophyll concentration in
the extra virgin olive oil is 9.82 x 10-6 mol/L.
3. Start the Logger Pro 3 (version 3.6 or newer) program on your computer.
7
4. Calibrate the spectrometer.
a) Use the cuvette with vegetable oil as the blank. Place the blank cuvette in the
spectrometer. Align the cuvette so that the clear sides are facing the light source
of the spectrometer.
b) Select Calibrate ► Spectrometer from the Experiment menu. The calibration
dialog box will display the message: “Waiting.......seconds for lamp to warm up”.
The minimum warm up time is one minute. Follow the instructions in the dialog
box to complete the calibration. Click
5. Determine the maximum wavelength for chlorophyll a in extra virgin olive oil and set
up the data collection mode.
a) Take the cuvette with the extra virgin olive oil and place it in the spectrometer.
b) Click
A full spectrum graph of the oil will be displayed. Note that one area
of the graph contains a peak absorbance. Click
to complete the analysis.
c) To save your graph of absorbance vs. wavelength, select Store Latest Run from
the Experiment menu.
d) Click the Configure Spectrometer Data Collection icon,
dialog box will appear.
, on the toolbar. A
e) Select Abs vs. Concentration under Set Collection Mode. The wavelength of peak
absorbance (‫ג‬
will be automatically selected. If you wish to select a new
wavelength (414 or 416), click on the graph or check the box next to the desired
wavelength. Click
to proceed.
6. Collect absorbance-transmittance data for the five serial dilutions of extra virgin olive
oil. If the cuvettes are smudged, you should clean them with a Kimwipe before use.
a) Leave the extra virgin olive oil cuvette in the spectrometer. Click
When
the absorbance reading stabilizes, click
Enter the concentration of the
extra virgin olive as the concentration of the solution and click
b) Remove the cuvette containg the extra virgin olive oil from the spectrophotometer
and replace it with the cuvette containg the first serial dilution of excxtra virgin
olive oil. When the absorbance reading stabilizes, click
Enter the
concentration of the first serial dilution as the concentration. Remove the cuvette
with the first serial dilution from the spectrophotometer.
c) Repeat Step 6b for the remaining serial dilutions of the extra virgin olive oil.
When you have finished testing the serial dilutions, click
.
8
7. To determine the best-fit line equation for the serial dilutions of the extra virgin olive
oil, click the linear fit button,
, on the toolbar. Make sure that the bottom graph is
selected. Write down the equation for the standard solutions in your data table or lab
book.
8. Determine the chlorophyll concentrations of the lesser grades of olive oil (regular and
light).
a) Place a cuvette containing the regular olive oil of unknown concentration in the
spectrophotometer.
b) Select Interpolation Calculator from the Analyze menu. A dialog box will appear
that displays the concentration of the regular olive oil at the measured absorbance.
c) Click
Write down the absorbance and the concentration of the regular
olive oil in your data table.
d) Repeat steps 8a-c for the light olive oil.
e) Make sure you write down all of the absorbance values for each serial dilution of
the extra virgin olive oil, the regular olive oil, and light olive oil at ‫ ג‬max.
f) To obtain transmittance values go to ‘Experiment’ to change units. Select ‘%
transmittance’. Record transmittance values for each serial dilution and the
regular olive oil and light olive oil at ‫ ג‬max.
9. Use the graph paper and semi-log graph paper to graph absorbance and transmittance
against concentration. Use your hand-plotted graphs to see if the concentrations of the
regular and light olive oil match those calculated by the Vernier program.
RESULTS
Solution
extra virgin olive oil (EVOO)
serial dilution 1 (EVOO)
serial dilution 2 (EVOO)
serial dilution 3 (EVOO)
serial dilution 4 (EVOO)
serial dilution 5 (EVOO)
regular olive oil
light olive oil
Concentration
(mol/L) x 10-6
Absorbance
at ‫ ג‬max
Transmittance
at ‫ ג‬max
9.82
In addition to this table, also include your graphs showing the data and linearregression equation for the serial dilutions.
9
DISCUSSION QUESTIONS
1) What is the relationship between percent transmission and absorbance?
2) Why is absorbance data most frequently used to determine the concentration of a
substance in a solution?
3) If an unknown substance has a green color, what wavelengths would you expect that it
does not absorb?
4) How did you use the standard curve that you generated for the serial dilutions to
determine the quantity of chlorophyll a in the two lesser grades of olive oil?
5) The introduction of this experiment identified four visible light wavelengths of peak
absorbance for chlorophyll a. Describe each of the olive oils tested in terms of these
expected peaks. Are there any noticeable differences in the actual peaks (not just their
absorbance values)?
ADAPTED FROM:
Spectroscopy with Vernier © 2006 Vernier Software & Technology
10
11
Absorption Spectrum for Food Dye Solution
Absorbance vs. Concentration (mol/L) x 10-6
12
% Transmission vs. Concentration (mol/L) x 10-6
13
% Transmission vs. Concentration (mol/L) x 10-6
14