Experiment 8

Spectrophotometry

Determination of Chlorophyll-a
Concentration in Spinach
In this experiment, the concentration of chlorophyll-a in spinach will be determined using a
spectrophotometric method.
Learning Objectives: Students will
understand the distinction between transmittance and absorbance.
understand the relationship between absorbance and concentration (Beers law).
learn to construct a spectrophotometric calibration curve using a spreadsheet.
learn to use a calibration curve to determine the concentration of a sample.
Experimental Objectives: Students will gain experience in the following experimental
techniques:

Use of volumetric glassware

Preparation of dilute solutions
Use of a spectrophotometer
Determining the wavelength of maximum absorbance, or max, for a sample
Extraction of color pigments of chlorophyll-a from spinach using an organic solvent

Introduction
Context: Chlorophyll Concentrations in Plants and Algae
The concentration of chlorophyll, a green pigment essential to photosynthesis in plants and
algae, serves as an indicator of the health of plants or bodies of water. Farmers monitor
chlorophyll concentrations in their crops. Environmental scientists measure chlorophyll
concentrations in water samples to determine algal biomass, which is directly related to the
health of the local aquatic environment. Scientists in many different disciplines have therefore
developed methods for measuring chlorophyll concentrations. Although the methods vary
slightly from one discipline to another, all are based upon the spectrophotometric principles and
techniques introduced in this experiment.

Chemical Principles: Pigments, Light, and Concentration

Pigments and Light
Chlorophyll is the name given to molecules that absorb sunlight to provide energy to synthesize
carbo- hydrates from carbon dioxide and water. This process, photosynthesis, is the foundation
for sustaining living plants. The two main green pigmented molecules are chlorophyll-a and b. The structures of chlorophylls-a and -b are shown on the follo wing page.

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Spectrophotometry

When white light, which spans all visible wavelengths, shines on a plant containing
chlorophyll molecules, some wavelengths of light are absorbed by the molecules while others
are reflected. What we see are the wavelengths of light that are reflected back. If a molecule
absorbs in the visible range (400 nm700 nm), we see the complement of what is being
absorbed. Using the color wheel shown below, we can see that objects which appear green are
those things that absorb red light.
The Visible Spectrum
Red 620 nm750 nm
Red
Violet
Orange 590 nm620 nm
Orange
Blue
Yellow 570 nm590 nm
Green 495 nm570 nm
1 nm = 109 m
Yellow
Green
Blue 450 nm495 nm
Violet 380 nm450 nm

Chlorophylls strongly absorb light because of the conjugation and resonance of alternating
single and double carbon-carbon bonds, mainly in the nitrogen-containing ring that surrounds
and binds the Mg2+ ion, as shown in the figure above. Even the small difference in structure
that distinguishes chlorophylls a and b at the periphery of this large ring causes shifts in the
wavelengths of light that are absorbed by the two molecules.
Chlorophyll concentrations can be measured after the pigments are extracted from plants or
algae. Plant tissue or dried algae is typically ground and left to steep overnight in a 70%
isopropanol/water mixture. The sample is centrifuged or filtered to remove unwanted
particles. The concentration of the chlorophyll solution can then be measured.

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Experiment 8

Concentration
A solution is a homogeneous mixture composed of two or more components. The component in the
largest amount is called the solvent (isopropanol, for example). All other component(s) are solute(s)
(chlorophyll-a, for example). The composition of the solution varies the amount of solute dissolved
in solution. One way to express concentration is mass of solute per volume of solution, but chemists
prefer to express concentration as molarity,
Molarity, M

mol
L

abbreviated, M, which indicates the number of moles of solute per liter of solution. Engineers often
favor molality, abbreviated m, which describes the number of moles of solute per kilogram of solvent.
Environmental scientists favor ppm (parts per million) and ppb (parts per billion), which are weight
comparisons. In this experiment the millimoles (mM) chlorophyll-a per liter (L) solution will be
determined spectrophotometrically.

Analytical Technique: Spectrophotometry

Beers Law

700 nm 400

When light passes through a light-absorbing solution, a portion of the light is absorbed by the
solute and another portion is scattered by particles in solution and by the sample holder or cuvette
(Figure 1).

Absorbance

Hayden-McNeil, LLC

Figure 1. A solution will absorb light of specific wavelengths.

The amount of light that passes through a sample can be measured at a given wavelength,, by a
spectrophotometer as either transmittance or absorbance. Transmittance is calculated by dividing
the light intensity, I, that passes through the solution by the light intensity that passes through a
blank sample, I0. The blank sample usually contains the solvent from the solution and takes into
account the light scattering by the solvent and the cuvette. Transmittance is generally reported
as percent transmittance, %T.

% =

100
0

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Spectrophotometry

Absorbance is defined as the amount of light that is absorbed by the sample. The absorbance,
A, is calculated as the negative logarithm of the transmittance, T.

= log =

The absorbance and transmittance of light for a solution depend on the wavelength of light used
and the concentration of the absorbing solute.

Spectrophotometers

Figure 2. A SpectroVis Plus Spectrophotometer

Absorbance and transmittance are measured using a spectrophotometer. The absorbance and
transmittance of light for a solution depend on the wavelength of light used and the concentration of
the absorbing solute. The spectrophotometer we will use is a SpectroVis Plus. Light (from the LED
bulb light source) passes through the sample. Emerging light goes through a good-quality diffraction
grating, and then the diffracted light is collected and sorted by the CCD array detector and sent to the
computer (Figure 3).

LED source

Sample

Diffraction grating

Linear CCD array detector

Figure 3. Schematic diagram of a SpectroVis Plus.

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Experiment 8

The absorbance of a sample is directly proportional to the sample concentration, c, and the
distance traveled by the light through the sample or path length, b. To equate the absorbance to the
concentration and path length, the absorptivity constant, a, is used. The resulting expression is
known as Beers law.

A = abc

The absorptivity constant is both solute and wavelength specific. When we use molarity (mol/L) to
express concentration, the absorptivity is renamed the molar absorptivity, . Beers Law is then rewritten as

A = bc.
The absorbance of a mixture of substances at a given wavelength is equal to the sum of the individual
absorbances of the two substances. For instance, the absorbance of a solution is equal to the sum of
the absorbance of the solvent, Asolvent, and the absorbance of the solute, Asolute.
Asolution = Asolvent + Asolute = solventbcsolvent + solutebcsolute

When determining the absorbance of a particular solute in solution, a correction, the subtraction
of the absorbance due to other solutes and the solvent, is typically applied. It is also necessary to
correct any spectrophotometric measurement for absorption due to any particulate matter floating in
solution.

Sample Handling
Before loading the cuvette into the SpectroVis Plus, wipe the surface of the cuvette with a Chem-Wipe
or paper towel to remove fingerprints. Fingerprints will scatter light, changing the absorbance. The
cuvette must be filled close to 80% full with the sample solution. Make sure that the clear surface
of the curvet is aligned along the light path when placing it in the sample holder.
Instrument Calibration
Before running any samples, a blank sample containing the solvent being used for the experiment
must be run first. This is an essential step to allow the instrument to subtract the absorption of light by
the solvent. The procedure for calibration is in the Procedure section.

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Spectrophotometry

Selecting Maximum Absorbance Wavelength

Absorbance is wavelength specific, so the wavelength with maximum absorbance, max, must be
determined. Plotting absorbance versus wavelength will aid in determining max. For instance, in the
plot below the max, the highest point in the curve is 582 nm. All further sample data should then be
collected at 582 nm. This selection process, however, is automatically done for you using the
SpectroVis Plus instrument in conjunction with the Logger Pro software (see Procedure section).
Absorbance vs. Wavelength (nm)
0.9
0.8
0.7

Absorbance

0.6
0.5
0.4
0.3
0.2
0.1
0
530

540

550

560

570

580

590

600

610

620

630

Wavelength (nm)

Figure 4. Plot absorbance vs. wavelength to determine max.

Calibration Curves
Beers law, A = bc, directly relates the absorbance of a sample to the concentration, c, of the
sample and the path length, b, of the spectrophotometer. As b is constant throughout an
experiment, a plot of absorbance versus concentration for a series of samples with known
concentrations yields a straight line (Figure 5).
The data points representing solutions of known concentration are called a standard curve or
calibration curve. The standard curve allows a researcher to determine the concentration of a sample
of unknown concentration by recording the unknown samples absorbance and comparing the
measured absorbance to the standard curve. Either by dropping a vertical from the intersection of the
absorbance with the standard curve, or by using the equation describing the best-fit line, one can
determine the concentration of the unknown. Because spectrometers measure transmitted light, the
linear Beers law relationship is only apparent for samples with absorbances in the range from 0.20 to
1.0 and concentrations (c) less than 0.01 Mconditions under which (almost) all the light isnt
absorbed. If the measured absorbance exceeds the upper limit, the sample must be diluted to reduce
the concentration of the sample. Concentrations of the standard solutions should be prepared via a set of
serial dilutions so that their measured absorbances fall within the allowed absorbance range. All best-fit
lines pass through the origin as a sample with c = 0 will have A =0. The slope of the standard curve can
be used to determine the molar absorptivity of the sample, b = m, provided the path length is known.
Assuming a 1 cm path length for the example in Figure 5, the = 77 cm1M1.

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Experiment 8

Standard Curve
0.9000
0.8000

Absorbance

0.7000
0.6000
0.5000
0.4000
0.3000
0.2000
0.1000
0.0000
0.0000

0.0020

0.0040

0.0060

0.0080

0.0100

0.0120

Concentration (M)

Figure 5. The standard curve is used to determine the concentration of a sample with an absorbance
of 0.5390. Using Microsoft Excel, the best-fit equation is y = 76.876x. Solving for x, the molarity when
y 5 0.5390 is 0.0070. There is reasonable agreement between methods.

Focus
In this experiment, chlorophyll-a is extracted from a spinach leaf in isopropanol (well
actually use a 70:30 isopropanol/water solution, the usual blend sold in drug stores as
rubbing alcohol). The concentration of extracted chlorophyll-a in isopropanol solution is
determined spectrophotometrically, and the mg chlorophyll-a per g spinach is calculated.

Experiment
This is one of a handful of experiments in Chemistry 112 where a percentage of your grade
will be based on the quality of your results.
In todays experiment you will determine the wavelength of maximum absorbance of a pure
chlorophyll-a sample, prepare a calibration curve using the chlorophyll-a standard, extract
chlorophyll-a from a spinach leaf, and determine the concentration of the dilute spinach
extract.

Health, Safety, and Environmental (HS&E) Items

Chlorophyll solutions in isopropanol will be used in this experiment. Wear appropriate
personal protection equipment (PPE), such as a pair of goggles for eye protection, at all times.
Wash any area of skin that comes in contact with an isopropanol solution with running water for
at least 15 minutes. All organic liquid waste should be collected in an organic waste container.

Chemicals
70% isopropanol
0.300 mM chlorophyll-a in 70% isopropanol solution

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Spectrophotometry
Equipment
SpectroVis Plus Spectrophotometer (4 per lab)
6 cuvettes
2 100 mL beakers
25 mL volumetric flask
10 mL volumetric flask
5 mL volumetric pipet
2 mL transfer pipet
Parafilm
Kimwipes

Mortar and
pestle

Volumetric
flasks

spatula
mortar
pestle
labeling tape
weighing paper/boat
funnel
disposable transfer pipets
filter paper pipet pump

Volumetric Transfer
pipet
pipet

SpectroVis P l u s
spectrophotometer

Experimental Procedure
Determination of the Wavelength of Maximum Absorbance for Chlorophyll-a
Sample Preparations
1. Obtain two cuvettes and make sure they are clean. If necessary, wash the cuvettes with soap
and water and then rinse thoroughly with deionized water. Then rinse both cuvettes several
times with a small amount of 70% isopropanol before use. Collect the isopropanol rinses in
a 100 mL beaker and label it as isopropanol waste.
2. Fill one of the cuvettes with 70% isopropanol to the bottom of the white circle (about 3/4
full). Isopropanol is the solvent used in this experiment. It will therefore serve as the blank
sample.
3. Drain the other cuvette thoroughly, rinse with approximately 0.5 mL of chlorophyll-a solution,
and fill at least 80% full with the chlorophyll-a solution. This is your undiluted chlorophylla sample solution
4. Place a cap on the cuvette. Wipe the outside of each cuvette with a ChemWipe to make sure
that it is clean with no fingerprints.
Disposal of isopropanol: Do NOT pour any isopropanol down the sink. Use your waste beaker
to collect the isopropanol rinses as well as the used isopropanol extracts. When the beaker is
full and/or at the end of the lab period, dispose of your waste in the organic waste bottle
located inside the fume hood.

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Experiment 8
Starting up the SpectroVis Plus spectrophotometer
1. Connect the Spectrophotometer to a powered USB port on the computer or a
powered USB hub.
2. Start Logger Pro on your computer. The default data type is absorbance. Since we want
to measure the absorbance of a solution, proceed directly to the Calibrate section below.
Calibrating the SpectroVis Plus
1.

2.

3.

To calibrate the, choose Calibrate Spectrophotometer from the Experiment menu.

Note: For best results, allow the Spectrophotometer to warm up for a minimum of five
minutes.
After the Spectrophotometer has warmed up, place the blank cuvette in the
Spectrophotometer. Align the cuvette so the clear side of the cuvette is facing the light
source.
Follow the instructions in the dialog box to complete the calibration, and then Click
.

Obtain an absorption spectra for your undiluted chlorophyll-a sample and max
1. Place your undiluted chlorophyll-a sample in the sample slot. Then, click
. After
seeing the spectra appear, click
.
A
2. To get an enlarged view of the spectra, click the auto-scale icon,
.
3. To observe max, click the configure spectrophotometer icon,
. A pop up window
will appear. In most cases, the instrument will have already picked the max. The
wavelength selected is indicated by a checkmark in the square next to the wavelength.
Standard (Calibration) Curve Development
The quality of your results will depend heavily on the calibration curve you prepare. For this portion of
the experiment, it is essential that you use proper technique in preparing the diluted solutions. Refer to
the appendix section on glassware at the end of the lab manual for instructions on correct use of pipets
and volumetric glassware.
Preparation of chlorophyll-a calibration samples using successive dilution method.
1. Using a 5 mL volumetric pipet and a 10 mL volumetric flask, prepare solutions using a series of
three dilutions of the chlorophyll-a solution. The first diluted solution should be prepared from the
stock solution and will have a concentration equal to 50% of the stock solution of chlorophyll-a.
2. The second diluted solution should be prepared from the first diluted solution and will have a
concentration of 25% of the stock solution. Ultimately, you should have three solutions with
concentrations equal to 50.0%, 25.0%, and 12.5% of the original concentration of the stock
chlorophyll-a solution.
3. Be sure to label each diluted sample. Calculate the exact concentrations of each of the diluted
samples using the concentration of the stock chlorophyll solution.

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Spectrophotometry
Recording the absorbance of the calibration samples and obtaining the calibration plot
You have prepared 3 dilution samples. Including the original undiluted sample, you now have 4
chlorophyll-a solutions with known concentrations. To measure and record the absorbance data and
to obtain a calibration plot, do the following:
1.

Place the undiluted sample once again in the sample slot. Generate the spectra by clicking
and then
.

2. Click the icon and choose collection mode absorbance vs concentration and click OK.
[A dialog box will ask you whether to save the spectra. Either yes or no does not matter.]
3. Click
and then click
. A dialog box will ask you to enter the concentration of
the sample. Enter it. Even though the program records the absorbance and concentration for
you, you should record these data separately in your note book.
4. Remove the sample from the slot and put in your second most concentrated sample. After the
reading stabilizes, click
. And then enter the concentration. Record the data in your
notebook also.
5. Repeat step 4 for the next 2 samples.
6. After all samples are run, click
7. Click Linear Fit,

to end data collection.

, to see the best fit line equation for the standard solutions.

Chlorophyll Extraction
1.

Obtain a spinach leaf. Remove the stem and using weighing paper or a weighing boat,
weigh the leaf on an analytical balance. Tear the leaf into small pieces. You will need a
sample that weighs approximately 0.10.2 g. Record the exact weight of your sample in
your notebook.

2.

Place the spinach sample in a mortar and add 1 spatula of sand and 1 mL of isopropanol.
Grind the leaf with a pestle until it is thoroughly broken down. Add enough isopropanol
to cover the leaf and leave it steeping for 510 minutes.

3.

Set up a filter using filter paper and a funnel. Place the funnel into a 25 mL
volumetric flask. Filter the contents of the mortar into the 25 mL volumetric flask.
Rinse the mortar with 1 to 2 mL of isopropanol. Rinse the filter with 2 to 3 mL of
isopropanol. If the solution is not clear after the first filtration, filter the extract a
second time. Fill to the mark on the neck of the volumetric flask with isopropanol
(70%). Cap the flask and mix by gently rocking the flask back and forth.

Measuring the Chlorophyll Extract

1. Set the wavelength on the spectrophotometer to max for chlorophyll-a and measure the
transmittance of the extracted sample.
2. Some absorbing particulate matter from the spinach leaf may have migrated through

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Experiment 8
the filter paper. Therefore the transmittance measured at max for chlorophyll must be
corrected. This can be achieved by measuring the transmittance in a region where the
chlorophyll and other plant pigments do not absorb light. Reset the wavelength of the
spectrophotometer to 750 nm and measure the transmittance. Dont forget that you will
need to use the blank as you have changed the wavelength.
3. Dispose of your chlorophyll solutions in the organic waste containers in the lab.
Remove your cuvette from the SpectroVis Plus, clean all glassware and return it to the
appropriate bins in the back of the lab. Wipe down the benchtop at your work station with
a damp paper towel before leaving lab.

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Spectrophotometry

Post Lab Exercises

Data Reduction and Analysis
1. Export the tabulated data of absorbance vs wavelength (A vs ) and present the table in your report.
2. Present a copy of the spectra (graphical plot of A vs ). This can be accomplished by copy (from
Logger Pro) and paste (into MS Word). Identify and label max.
3. Present a copy of the plot of A vs Conc. Again, this can be accomplished by copy and paste from
Logger Pro to Word.
4. Finish the DRA following the instruction in the following page.

Reference
1. Gross, Jeana. Pigments in Vegetables: Chlorophylls and Carotenoids. New York: Van Nostrand
Reinhold, 1991.

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