Extraction of Curcumin from

Turmeric and Spectroscopic Analysis

Introduction
Research Questions
What is a suitable method for extracting curcumin from turmeric? What variables impact this
extraction? How can you determine how much curcumin was extracted?

Learning Goals
This is the last experiment of the course, and you will use skills and concepts you have already
learned (plus some new techniques) to answer the research questions. In this experiment, you
will design an experiment to extract curcumin from turmeric and determine the concentration of
the resulting solution using visible spectroscopy and Beers Law. You will examine how changes
in sample concentrations result in changes in absorbance and transmission.

This experiment introduces you to spectroscopy and extraction. The solvents used are non-toxic,
making them Inherently Safer and allowing us to conduct the extraction using Safer Solvents.
Additionally, the extracted curcumin comes from Renewable Feedstocks.

Background: Natural Dye Molecules

All fruits and vegetables contain molecules that impart color. Some of these compounds can be
isolated and used as dyes for fabric or other foods. Some common natural dyes are shown below.
Today we will explore the dye molecule curcumin, which is one of the yellow compounds found
in turmeric. We will be extracting the curcumin using ethanol as a solvent. Extraction is the
separation of one or more components from a mixture. Making tea is an example of using water
to extract the flavoring and caffeine from tea leaves.

Name Plant Molecular structure

-
Carrots
Carotene

Curcumin Turmeric

Lycopene Tomatoes

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Review: Absorbance and Transmission

You were first introduced to the concepts of absorbance and transmission in Experiment 2: Light
Inquiry. In that procedure you observed the emission spectra of various gases through a
spectroscope. You also observed continuous light through colored filters and noted how the
spectrum of the continuous light source was impacted by the filters.

The number of photons a sample of material absorbs is directly related to the number of particles
in the path of the beam light. This important concept means that by measuring the change in
intensity or number of photons (I) of a light beam before (I0) and after (It) it passes through a
solution of molecules, we can measure the number of molecules in the solution (see Figure 1).
The intensity change is related to the number of molecules the beam encounters. More
technically, the log of the ratio of I0 to It is called the absorbance (A).
Red Red
Orange Orange
Yellow Yellow
Green IO It Green
Blue Blue
Indigo Indigo
Violet Violet
Blank
0.35
Red Red
0.30
Orange Orange
Absorbance (a.u.)

Yellow Yellow 0.25

Green IO It Green 0.20
Blue Blue
Indigo Indigo 0.15

Violet Violet 0.10

Sample 0.05

0.00
Light
Detector 400 500 600 700 800
Source Wavelength (nm)

I0
A = log
It

Figure 1: For the Red Dye #3 sample above, all of the red and some of the blue light is
transmitted (passes) through the sample, whereas the yellow, orange, and green light is absorbed.
Thus, the absorbance values of yellow, orange, and green light are high and the absorbance
values of red and blue are lower because red and blue light are transmitted.

Beers Law states that the absorbance of a sample is directly proportional to its concentration.
The absorbance (A) specifically depends on certain properties of the sample: the wavelength
() of the measurement, the extinction coefficient () at that wavelength, the path length (l) of the
sample, and the concentration (c) of the sample.

A = lc (Beers Law)

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Alternatively, we can also explain this phenomenon by quantifying the transmission of a sample,
I
or the number of photons that the sample does not absorb. Transmission, t , is related to
I0
absorbance:
It/I0 %T A
A I
10 = t =T 1 100 0
I0 0.1 10 1
0.01 1 2
In the example spectrum shown above, the transmission values of red and blue wavelengths of
light would be high but those of orange, green, and yellow would be low. Scientists use both
absorbance and transmission data to report the optical properties of a sample depending on what
information they want to convey.

Spectrometers

To explore these optical properties, youll be using some new equipment in this experiment: a
spectrometer. This instrument allows you to determine the absorbance and/or transmission of a
compound (Figure 2).

Spectrometers consist of a built-in light source, which produces a narrow beam of light that
passes through a sample. The light that passes through the sample is split into its component
wavelengths by a diffraction grating, and a specialized detector then measures the amount of
light that reaches it at each wavelength. A computer converts this into an absorbance reading at
each wavelength. Often, this data is then displayed as an absorbance spectruma curve showing
the absorbance of your sample at each wavelength (see your pre-lab for an example).

(The spectrometer used in this experiment is called a SpectroVis. The computer is a small
handheld device called a LabQuest. Spectrometers come in many shapes and sizes, and many are
used with actual desktop computers.)

Figure 2: Schematic of spectrometer (top view)

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Its important to note that your compound of interest isnt the only thing absorbing the light that
comes from the light source: your cuvette and any solvent(s) your compound is dissolved in can
also absorb light. Because of this, you must obtain an absorbance spectrum for a blank sample
before you take any absorbance measurements of your compound. A blank sample is made up of
the cuvette and any solvent used to dissolve your compound, like water or ethanol. The appendix
at the end of this experiment gives instructions on how to calibrate your spectrometer, but
essentially, you start by obtaining an absorbance spectrum of your blank sample. The computer
then stores this and essentially subtracts it from subsequent absorbance spectra, meaning the
resulting spectra show only absorbance values for your compound (and not the components of
your blank sample). This is similar to zeroing a balancing or taring a weigh boat before you
weigh a sample.

Quantitative Dilutions and Calibration Curves

No instrument will automatically determine the concentration of a sample. Instead, you must
calibrate the instrument using a known solution and compare the response of an unknown. By
measuring the absorbance values of a set of standard solutions of known concentrations, you can
create a calibration curve that allows you to determine the concentration of an unknown of the
same species. This calibration curve correlates to how the spectrometer responds to various
concentrations of standard solutions. It involves plotting the absorbance values of standard
solutions versus their concentrations and determining the equation of the line that most closely
represents the relationship among these points (see Figure 3 below). Based on Beers Law, youd
expect this relationship to be linear. This line is called a best-fit line (or a linear regression).

Beers Law may fail if a solution is too concentrated or too dilute. This means that one can only
accurately determine the concentration of an unknown if its absorbance falls within the linear
range of the dataset. Rather than trying to analyze really concentrated solutions, it is considered
good lab practice to quantitatively dilute solutions to concentrations that give absorbance values
below 1.5 absorbance units.

An example using Yellow Dye #5 is shown below. The max (wavelength with the highest
absorbance) for this compound is 420 nm (see Figure 3). Figure 4 shows the absorbance
measurements for a series of standard solutions of Yellow Dye #5 at 420 nm. A best-fit line has
been shown for the lowest six concentrations. Notice that at higher concentrations, the data no
longer appears linear: these points do not fall on the best-fit line. Therefore, the highest four
concentrations have absorbance values that do not fall within the linear range.

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 Absorbance Spectra for Yellow 5
1.80
1.60
1.40
Absorbance

1.20
1.00
0.80
0.60
0.40
0.20
0.00
400 450 500 550 600 650 700
Wavelength (nm)

Figure 3: Absorbance spectra for various concentrations of Yellow Dye #5

The extinction coefficient for Yellow Dye #5 at 420 nm can be determined from the slope of the
calibration curve using the Beers Law data. The absorbance (A) is unitless because units of the
extinction coefficient (in L mol1 cm-1), the path length l (in cm), and the concentration c (in
mol L-1) cancel each other out.

A plot of absorbance versus concentration will have a slope (m) of l . For Yellow Dye #5 the
equation for the calibration curve is y = 20444x as shown in Figure 4 (next page). For most
sample holders l=1 cm.
m = l
m L
= = 20444
l mol cm

This data can be used to determine the concentration of an unknown by simply measuring the
absorbance and correlating to the equation for the line. An unknown solution with absorbance of
0.9054 would correspond to a concentration of 4.4310-5 M of Yellow Dye #5.

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 Calibration Curve for Yellow #5
2.00
1.80
Absorbance at 420 nm

1.60
1.40
1.20
1.00
0.80 y = 20444x
0.60
0.40
0.20
0.00
0.00E+00 2.00E-05 4.00E-05 6.00E-05 8.00E-05 1.00E-04
Concentration of Yellow #5 (M)

Figure 4: Beers law plot for Yellow Dye #5 using full and linear portion of the data set at 420
nm

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Name________________________________ GSI_______________________

Prelab
1. Use the provided spectra to answer the following prelab question.

1.80

1.40
-Carotene
Concentrations
1.00
Absorbance

1.21E-05 M
8.09E-06 M
4.04E-06 M
0.60
2.02E-06 M

0.20

-0.20
350 400 450 500 550
Wavelength (nm)

Figure 5: Full spectrum of -carotene at various concentrations (M)

-carotene is a compound extracted from carrots. Well be using data for beta-carotene as a pre-
lab exercise to prepare for your experiment using curcumin.

a. From the full spectrum of the -carotene standards, what color of light is most strongly
absorbed? What colors are transmitted? Support your answers.

b. Record the wavelength of maximum absorption. (The wavelength of maximum absorption,

max, is the wavelength that the instrument is most sensitive to for a particular compound.)

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2. Use the data table for -carotene to answer the following questions.

Table 1: Beers Law data for -carotene standards at max

Concentration (M) Absorbance (max)

1.21 10-5 1.62
8.09 10-6 1.08
4.04 10-6 0.44
2.02 10-6 0.13

a. Plot the data points given in the table on the following graph. Use a ruler to draw a best-
fit line.

Calibration Curve for Beta-Carotene

2.50

2.00
Absorbance at _______ nm

1.50

1.00

0.50

0.00
0.00E+00 2.00E-06 4.00E-06 6.00E-06 8.00E-06 1.00E-05 1.20E-05 1.40E-05 1.60E-05 1.80E-05
Concentration (M)

b. Calculate the extinction coefficient for -carotene from the slope of your graph and
explain its significance. Assume a path length of 1 cm.

c. Your lab partner brings you another sample at a concentration of 1.6010-5 M. You
record the absorbance and the instrument reads 1.93. Do you think you should include
this data in your plot and resolve for the slope? Why or why not?

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 d. Suppose you conduct an extraction of -carotene from grated carrots. You record a
spectrum of your sample, and the resulting absorbance value is 0.80 at max. What is the
concentration of your extraction sample?

e. The sample you used in part b was actually diluted before you determined its absorbance
at max. If you took 1.0 mL of your extraction and diluted it in 5.0 mL of ethanol before
taking the absorbance measurement, what was the original concentration of your
extraction?

3. Table 1 shows -carotene concentration and absorbance values; note that the highest
absorbance value is around 1.5.
a. Why is it important to have absorbance values lower than 1.0 or 1.5 absorbance units?
What can happen at absorbance values greater than 1.5?

b. Suppose you have a -carotene solution with an absorbance of 3.0. You perform a 1:1
dilution (e.g. dilute 1 mL of your original -carotene solution with 1 mL solvent) that
then gives an absorbance of 2.0. Is this expected? Why or why not?

9
4. Complete the following table to help you prepare your curcumin standard solutions in order
to construct a calibration curve in lab.

Volume of 40 Final
Volume of Total Volume Final
M Stock Concentration
Ethanol (mL) (mL) Concentration (M)
Solution (mL) (M)
4.0 0.0 4 40.0 4.010-5
4 20.0 2.010-5
4 15.0 1.510-5
4 10.0 1.010-5
4 5.0 5.010-6

5. What are some variables that might impact the amount of curcumin that can be extracted
from a sample of turmeric? Refer to the Standard Extraction Procedure in Part B of the
experiment for ideas. List at least three variables that could be investigated.

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 Experimental
Dye Extraction and Quantification
Goal

Extract curcumin from turmeric using the standard procedure provided. Determine the
concentration of the dye extracted by comparing your solutions to known standards using a
Beers Law plot. Optimize this extraction by developing an experiment that tests one variable in
the standard extraction procedure.

Work in groups of four to complete this experiment.

A. Calibration Curve

Note: One pair of students should complete Part A, while the other pair works on Part B.

Obtain approximately 5 mL of stock curcumin solution (4.010-5 M).

Record the exact concentration of the stock solution.

You will need to create dilutions in order to create your calibration curve. Use the curcumin
stock solution (40 M) to make a set of four more standard samples containing 20, 15, 10, and 5
M curcumin.

Follow the scheme you set up in the table in the pre-lab to prepare standard solutions in test tubes
using plastic pipettes. First, determine the volume of stock solution and ethanol you will need to
use in order to prepare the final concentration. Then, use plastic pipettes to transfer the desired
volume of stock solution and ethanol. You can use the pipettes to help you mix your solutions.

Record the volumes you actually dispense in your report sheet and use those to calculate
your final concentrations of curcumin.

Dry the inside of the cuvettes with a KimWipe if needed. Place 2 mL of each solution (40, 20,
15, 10, and 5 M) in a clean, dry cuvette. Cap each cuvette. Do not write directly on cuvettes
or cuvette caps to label your samplesuse label tape and attach it to the cuvette caps.

Set up the LabQuest and SpectroVis (see Appendix A at the end of

this experiment for spectrometer operating instructions) and
calibrate it using a blank sample. Be sure to place the semi micro
cuvette into the spectrometer in the correct orientation so that the
light passes through the narrow channel at the bottom of the cuvette.

What should you use to make your blank sample? Think

about what solvent you used to create all your standard
Figure 6. Correct pathway
solutions.
for light through a semi
micro cuvette.

11
Record an absorbance versus wavelength spectrum for each curcumin standard.

Determine the wavelength of maximum absorbance (max) and record the absorbance at
that wavelength for each standard in the table in your report sheet.

Plot a calibration curve on the graph provided on your report sheet.

Identify the highest concentration that is part of the linear range.

Use a ruler to estimate the best-fit line for the linear portion of your data and draw it on
your graph. Calculate the slope of your line.

B. Standard Extraction Conditions

Each person in the pair completing this part should conduct an extraction. You should have two
absorbance measurements at the end of this part of the experiment.

Obtain 50 mg of turmeric which is 0.050 grams. Place it in a test tube, along with 5 mL of 95%
ethanol. Mix your sample thoroughly. Continue to mix your sample periodically for the next five
minutes.

If at the end of five minutes your sample has solid turmeric suspended in it, filter the sample
through the filter paper provided into a clean piece of glassware. Remove 1 mL of the filtered
sample and place it in a new test tube.

Compare the color of your sample with the range of colors of the curcumin standards made by
the other pair in your group. (Be sure you are comparing solutions in the same type of glassware
to keep the visual path length similar, i.e. both samples should be in test tubes).

If your sample is less intense in color than the curcumin standard sample that gives an
absorbance value at the top of your linear range (see the calibration curve being
constructed by your partners), then place up to 2 mL of your extracted sample in a
cuvette.
If it is more intense in color than this standard sample, dilute your extracted sample with
ethanol until the color is less intense in comparison with the standard sample (be sure the
diluted sample is thoroughly mixed). Place your diluted sample in a graduated cylinder to
measure the final volume, and then put up to 2 mL of your extracted sample in a cuvette.

Set up the LabQuest and SpectroVis (see Appendix A at the end of this experiment for
spectrometer operating instructions) and calibrate it using a blank sample.

What should you use to make your blank sample? Think about what solvent you used to
create your extracted sample.

Record an absorbance versus wavelength spectrum for each extracted sample.

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 Determine the wavelength of maximum absorbance (max) and record the absorbance at
that wavelength for each extracted sample in the table in your report sheet.

Use the calibration curve constructed by the other pair in your group to determine the
concentration of curcumin in your extraction samples. Remember to take into account
any dilutions you performed before taking your absorbance measurements.

C. Design Your Own Variable Testing

With your group, decide on one variable whose effect on curcumin extraction efficiency you
would like to investigate. Plan your investigation with the following guidelines in mind:

1. Your experiment should result in four different extraction samples that can be
quantitatively assessed to show a trend concerning your variable of interest.
2. Each person in the group should make one of the extraction samples.
3. Your plan must comply with the following material limits:
a. 100 mL total amount of solvent (methanol, ethanol, propanol)
b. Use a maximum of 50 mg of turmeric for each extraction. Ensure that the amount
of solid used is kept constant for each extraction sample.
4. Explicitly state what other variables you are keeping constant between experiments.

As a group, bring your written plan (see Proposed Procedure worksheet) to your GSI for
approval before you begin your experimental work.

Carry out your planned extractions with your group and analyze the samples using the
spectrometer and your calibration curve.

Record all of the materials and amounts of chemicals that you used and isolated during
your extraction process.

Obtain a full spectrum for each of your extracted solutions.

Record the absorbance values at max for each of your solutions. Make sure your
absorbance values are within the linear range determined from your calibration curve,
and keep track of any dilutions you make of your extracted sample.

From your previous lab work, you should have already constructed a calibration curve for
curcumin in your desired solvent.

Determine the concentration of curcumin in your four extraction samples.

Summarize your findings with your group. Generate a table and an accompanying graph
(concentration of extract versus experimental variable). Also, write 1-3 sentences
explaining your findings.

Clean Up: Rinse all used cuvettes and cuvette caps thoroughly and dry them with KimWipe.
Return cleaned and dried cuvettes and caps back to the GSI bench/TA station.

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Proposed Procedure for Experimental Curcumin Extraction from Turmeric:
1. What is the variable your group has chosen to investigate?

2. How will you investigate this? Use the Standard Extraction Procedure to help you
formulate your procedure.

3. What variables will you keep constant throughout the experiment?

4. Whats the hypothesis for your experiment?

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 Appendix A: SpectroVis Plus Instructions
Preparing the instrument
Plug the LabQuest2 in using the provided power cable. Turn it on and wait for it to fully power
up (this can take several minutes).

Connect the SpectroVis Plus Spectrophotometer to the LabQuest with the cable included (the
USB side of the cable goes into the LabQuest).

Spectrometer calibration

5. Click on (the Meter tab) to get to the correct screen.

6. Click on Sensors with the stylus.
7. Choose Calibrate
Spectrometer.
8. Place the Blank sample in the cuvette holder, making sure that the cuvette is placed so
that the light source passes through the clear sides. Make sure youve cleaned the sides of
the cuvette with a KimWipe to remove any debris or fingerprints.
9. Follow instructions in the dialog box to complete the calibration. DO NOT skip the
Lamp Warm-Up. (If you are recalibrating later, when the spectrometer has been on for a
while, you may skip the lamp warm-up.)
10. Click Finish Calibration.

Full Spectrum Collection

Click to generate a spectrum. Place the sample cuvette in the cuvette holder, (check the
alignment). The spectrum will update in real time. Click (Stop) to halt data collection.
Click on any point in the spectrum with the stylus to display the absorbance at the wavelength.

Correct pathway for light

through a semi micro
cuvette.

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 Report Sheet

Name__________________________________

Partner(s)_______________________________

Part A. Calibration Curve

1) Complete the following table. Record the max of curcumin. _______________ nm

Volume Stock Volume of Total Volume Final Absorbance

Solution (mL) Ethanol (mL) (mL) Concentration (M) at max
4.0 0.0 4 4.010-5
4 2.010-5
4 1.510-5
4 1.010-5
4 5.010-6

2) Record the absorbance spectrum for one of your standard samples. Indicate max on the
curve.

16
 3) Plot the absorbance and concentration data from your table on the following graph. Draw
an approximate best-fit line using a ruler. Be sure to only include points that fall in the
linear range of the spectrometer.

Calibration Curve for Curcumin

1.60

1.40

1.20
Absorbance at _____ nm

1.00

0.80

0.60

0.40

0.20

0.00
0.00E+00 5.00E-06 1.00E-05 1.50E-05 2.00E-05 2.50E-05 3.00E-05 3.50E-05 4.00E-05 4.50E-05
Concentration of Curcumin (M)

4) What is the slope of your line? Use it to calculate the extinction coefficient of curcumin.

5) Compare this extinction coefficient with the value you determined for -carotene in the
pre-lab. How do you make sense of these different values?

17
Part B. Standard Extraction Procedure

6) Draw the absorption spectrum for your extracted curcumin solution.

7) What color of light is most strongly absorbed? What colors are transmitted? Record the
wavelength of maximum absorption. Does this make sense given the color of the
solution?

8) Record the absorbance values of your two extraction samples. Calculate the
concentration of curcumin (M) that you isolated from turmeric based on your calibration
curve from Part A. Dont forget to account for any dilutions you made (refer to pre-lab
Question 2d).

Sample Absorbance at Initial Volume Final Volume Concentration

________ nm (mL) (mL) (M)
1 1

2 1

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 9) Show your concentration calculations here:

10) How do the two extraction values your group obtained compare? Can you think of
reasons why they might be different?

Part C. Design Your Own Variable Testing

Procedure:

11) Write down the procedure you followed to test your variable of choice. Be sure to
explicitly state which variables you are controlling (and how you are controlling them).

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Results:

12) Record your absorbance values at max for each of your extraction samples. Calculate the
concentration of curcumin (M) that you isolated from turmeric based on your calibration
curve from Part A. Dont forget to account for any dilutions you made (refer to pre-lab
Question 2d).

Sample Absorbance at Initial Volume Final Volume Concentration

________ nm (mL) (mL) (M)
1

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13) Make a graph of your extraction concentrations versus the experimental variable (be sure
to label your axes). What trend(s) do you see? What can you conclude about the impact
of your chosen variable on the extraction of curcumin from turmeric?

Graph 1

14) Trends and conclusion:

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