Quantitative Gas Chromatography:
Determination of D-Limonene in
Orange Peel by Liquid CO2 Extraction
Please read Harris and Lucy, Quantitative Chemical Analysis (10th Edition) Ch. 24 Gas
Chromatography.
Introduction
In this experiment, D-limonene (Figure 1), a natural product found in citrus peels, will be extracted
from orange rinds and quantified. D-limonene is the primary constituent of orange oil and a major
constituent of other citrus oils. It is used as an insecticide, fragrance, and solvent, and has been
considered among the candidates for use as a biofuel. It is typically extracted from waste orange
rinds, a byproduct of juice production.
Figure 1. D-limonene structural diagram
Liquid CO2 Extraction
As with any analytical technique, preparation of the analyte in a reasonable form for analysis is an
important first step. Direct analysis of a solid sample of orange rind is difficult, so the best
approach is to separate the limonene and other orange oils from the larger, more complex
biological structures of the orange rind. Commercially, this process is typically done by extraction
with an organic solvent or by steam distillation. However, an emerging technology for the
extraction of relatively non-polar small organic molecules is liquid CO2 extraction.
At atmospheric pressure, CO2 does not have a liquid phase, but sublimes (converts directly from
solid to gas) at -78 ºC. However, at pressures above the triple point (Figure 2), CO2 has solid,
liquid, and gaseous phases; if solid CO2 is exposed to room temperature at these pressures, liquid
CO2 is formed as the solid melts and then boils. The sample is boiled in the liquid CO2, which
dissolves small, non-polar molecules (e.g., limonene), extracting them from the sample. When the
pressure is relieved, the CO2 becomes a gas and diffuses away leaving neat (no solvent) orange
oil. In an industrial setting the orange oil may be ready to use at this point, demonstrating one of
Experiment 4
Chem 4B Spring 2023
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the distinct advantages of using liquid CO2 as a solvent, rather than an organic solvent that must
be removed by evaporation and collected for disposal.
Figure 2. The phase diagram for CO2 showing the triple point.
For analysis, we dilute this sample with a volatile solvent because lower concentrations are
required for much of the analytical instrumentation that is commonly used, including gas
chromatography.
You will determine the quantity of limonene present in orange rind by first extracting orange oil
with liquid CO2, and then analyzing it by gas chromatography and comparing the sample you have
prepared to a set of standard samples. You will construct simple calibration curves based on
limonene solutions of known concentrations and calibration curves based on the internal standard
anisole. Keep in mind that only a small fraction of your sample (1 µL) will be injected into the GC
for analysis; you will then use the data to estimate the D-limonene content of orange rind on a pergram basis.
Determining the best method for GC analysis takes considerable research and fine-tuning of the
instrument. The method for the gas chromatographic analysis of D-limonene has been determined
for you, following the principles outlined in Harris 10th ed. Section 24.5, with special care taken
to ensure that D-limonene from biological samples does not co-elute with other components of
orange oil, and that it is not thermally decomposed by the method selected. A Flame Ionization
Detector (FID) is used, and the analysis package provides both peak areas and peak heights for all
of the major peaks. The column is an Agilent HP-5, composed of a (5% Phenyl)methylpolysiloxane stationary phase, which is less polar than the solvents. A split injection with a
20:1 ratio is used; this means that only ~ 1/20th of the sample injected is analyzed on the column –
20 parts of the injected volume bypass the column, 1 part is separated on the column and detected.
Experiment 4
Chem 4B Spring 2023
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Limonene is the major analyte in all of these samples and so we anticipate that its concentration
will be fairly high.
Gas Chromatography
Chromatographic separations use variation in retention times of species in a column to separate
them for characterization. In gas chromatography, volatile liquids or gases are carried through a
column (usually a long, narrow, capillary) by an inert carrier gas (e.g., He) and separated primarily
based on their volatility. Comparison of retention times to standard samples run using the same
column conditions is an excellent method for determining identities of compounds in a complex
mixture (e.g., biological samples). A Flame Ionization Detector (FID) responds to ions that form
when hydrocarbons exiting the column combust in a hydrogen flame and a chromatogram of the
data is generated. The peaks in the chromatogram can be measured for peak height or integrated
to obtain peak area. Quantitative determination of the amount of a particular compound is done
through the construction of calibration curves (analyte concentration as a function of peak height
OR peak area) with or without the use of an internal standard, as is demonstrated in this
experiment.
Experiment 4
Chem 4B Spring 2023
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References
[1] D. C. Smith, S. Forland, E. Bachanos, M. Matejka and V. Barrett, "Qualitative Analysis of
Citrus Fruit Extracts by GC/MS: An Undergraduate Experiment," Chem. Educator, vol. 6, no.
1, pp. 28-31, 2001.
[2] K. R. Williams and R. E. Oierce, "The Analysis of Orange Oil and the Aqueous Solubility of
d-Limonene," J. Chem. Educ., vol. 75, no. 2, pp. 223-226, February 1998.
[3] J.-M. Lavoie and E. Chornet, "Identification of Secondary Metabolites in Citrus Fruit Using
Gas Chromatography and Mass Spectroscopy," J. Chem. Ed., vol. 85, no. 11, November 2008.
[4] "Phase
Diagrams
of
Pure
Substances,"
[Online].
Available:
https://www.chemguide.co.uk/physical/phaseeqia/phasediags.html. [Accessed 10 January
2020].
[5] H. L. Buckley, A. R. Beck, M. J. Mulvihill and M. C. Douskey, "Fitting It All In: Adapting a
Green Chemistry Extraction Experiment for Inclusion in an Undergraduate Analytical
Laboratory," J. Chem. Educ., vol. 90, no. 6, pp. 771-774, 2013.
[6] L. C. McKenzie, J. E. Thompson, R. Sullivan and J. E. Hutchison, "Green chemical processing
in the teaching laboratory: a convenient liquid CO2 extraction of natural products," Green
Chemistry, no. 8, pp. 355-358, 2004.
[7] D. C. Harris and C. A. Lucy, "Ch. 24 Gas Chromatography," in Quantitative Chemical
Analysis, 10th ed., New York, New York: Macmillan Learning, 2020, pp. 667-702.
[8] D. C. Harris and C. A. Lucy, "Ch. 23 Introduction to Analytical Separations," in Quantitative
Chemical Analysis, 10th ed., New York, New York: Mamillan Learning, 2020, pp. 637-666.
Experiment 4
Chem 4B Spring 2023
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Prelab Questions
1.
List the boiling points of anisole and D-limonene. Which one do you expect to elute first in
gas chromatography?
Anisole: __________°C
D-limonene: __________°C
2.
Complete Exercise 24-D in Harris 10th edition (24-D in 9th edition, 23-A in 8th edition).
3.
READING - Harris 10th ed, Chapter 24
Experiment 4
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Materials and Methods
Work in groups of 2.
Purpose
You will use liquid CO2 to extract limonene from an orange peel. You will then quantify the
amount of limonene per gram of orange peel using gas chromatography.
Materials
Equipment
•
•
•
•
•
•
•
orange or a sample of orange peel
a fine grater or citrus zester
15 mL polypropylene centrifuge tube with screw cap
15 to 20 cm of copper wire (20 AWG)
Mortar and pestle
Gas Chromatograph with FID detector, HP-5 column
KimWipes or other laboratory wipes
•
•
•
•
•
•
•
•
insulated gloves
glass Pasteur pipettes
metal scoopula
large plastic beaker
5 mL volumetric flask
1 mL syringe
0.44 µm syringe filter
GC autosampler vial with cap
Chemicals
Carbon Dioxide, dry ice,
CO2
liquid, and gas
Warning!
Ethyl acetate
Danger!
Experiment 4
CAS 124-38-9
100 g
May cause cryogenic burns or injury. May displace oxygen and
cause suffocation. May increase respiration and heart rate.
Contents under pressure should be handled with extreme care.
CH3COOC2H5
CAS 141-78-6
10 mL
Flammable liquid and vapor. Vapors and liquid can cause eye
irritation. May cause skin irritation, and allergic reaction.
Ingestion may cause irritation of the digestive tract, central
nervous system depression, headache, nausea, fatigue, and
dizziness. Inhalation may cause respiratory tract irritation,
narcotic effects, and may be otherwise harmful.
Chem 4B Spring 2023
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Anisole, 10 mg/mL in Ethyl
acetate
CH3OC6H5
CAS 100-66-3
1 mL
Warning!
Causes eye, skin, digestive tract and respiratory tract irritation.
Vapors may cause dizziness or suffocation.
D-limonene
C10H16
CAS 5989-27-5
<1 mg (in orange oil)
Danger!
Flammable liquid and vapor. May cause eye, skin, digestive tract
and respiratory tract irritation. May cause skin sensitization or an
allergic reaction; prolonged contact may cause defatting of the
skin and dermatitis. Target organs: Skin.
Part I: Limonene Extraction
Procedure
1.
Obtain a medium orange and a grater to collect the orange peel sample. Make sure the outside
of the orange is dry, then gently remove the thin outer layer of the rind from the orange (the
“zest”) with the fine grater, being careful to use only the colored portion of the peel. The
mass of your sample should be ~1 g; record the actual mass you use.
Note: you want to avoid introducing any water to your sample throughout this preparation
2.
Obtain a 15-mL polypropylene centrifuge tube with red screw cap. Clearly label the tube with
a name or code identifying your group.
3.
Prepare a “basket” for the orange rind using ~15-20 cm of copper wire (22 gauge). Coil the
end of the wire into a flat spiral about three or 4 times around, small enough to fit into the
centrifuge tube such that when lowered into the centrifuge tube, it forms a platform to support
the orange rind, with the residual wire serving as a handle that does not protrude from the end
of the tube when the lid is on (see Figure 3).
Experiment 4
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Figure 3. Assembly of copper wire, orange rind, and dry ice in centrifuge tube.
4.
Place the orange rind into the tube, gently packing it down but ensuring that none goes below
the copper wire “basket”. Be careful not to pack the rind too tightly; it must be compact, but
loose enough for liquid CO2 to flow through.
5.
Grind dry ice with a mortar and pestle to a coarse powder, then pack it on top of the orange
rind until your centrifuge tube is nearly full. If there is not enough dry ice packed into your
tube the pressure will not be high enough to form liquid CO2. Use weighing paper as a funnel
to help you get the dry ice into the tube.
Caution: Dry ice is extremely cold and the vessels containing it can give you frostbite! Use
insulated gloves.
6.
Screw on the cap of the tube just until it stops, not too tight. Submerge the tube in hot tap
water (~40-50°C) contained in either a clear plastic graduated cylinder or a large plastic
beaker. DO NOT use a glass beaker. Tongs can be used to help submerge the tube
underwater. Some bubbles will be released out the top of the tube and you should start to see
the CO2 melt and some loss of color in the orange rind.
Note: Excessive bubbling usually means the cap is too loose. No bubbling usually means the
cap is too tight and the tube may violently rupture or explode. Again, you want to avoid
introducing any water into your tube
If the liquid CO2 does not form quickly, gently tighten the cap a little tighter. It may be
necessary to add additional dry ice to the tube.
Experiment 4
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7.
Once you obtain liquid CO2, wait until the bubbling is slow and the amount of CO2 remaining
is minimal, then remove the tube from the water, and carefully remove the cap, slowly
releasing the pressure. Repeat the extraction two more times by adding more CO 2 in order to
isolate as much limonene as possible from the sample. A visible amount of orange oil should
collect in the bottom of the centrifuge tube.
Note: Between each extraction, check that the orange peel has not frozen into a solid plug.
Once the peel is frozen together, CO2 cannot flow through it, and no limonene can be
extracted. If peel is frozen, thaw by placing the centrifuge tube in warm water.
8.
Carefully remove the copper “basket” and all of the orange rind solid.
9.
Fold a nickel sized piece of KimWipes™ in quarters and use a wooden stick to push it into
the mouth of a Pasteur pipette, creating a course pipette filter. See Figure 4 for a diagram of
the apparatus.
10. Quantitatively transfer your extracted oil by using ~1 mL ethyl acetate to dissolve the residual
oil and filter it through the pipette filter into a 5 mL volumetric flask.
Caution: Ethyl acetate is harmful if swallowed or the vapors are inhaled and should be used
in a fume hood. Ethyl acetate is also flammable.
11. Rinse the centrifuge tube with a small volume (~1-2 mL) of ethyl acetate; filter this through
the same pipette filter into the volumetric flask.
12. Add 1.00 mL of the provided anisole standard solution (approximately 10 g/1000 mL; record
exact concentration) directly to the volumetric flask.
13. Dilute to the mark with ethyl acetate and mix thoroughly by inverting the flask end-over-end
several times (e.g., 10-15 inversions).
14. Prepare a GC sample vial of this solution using a 1-mL syringe and a syringe filter. Your GSI
will demonstrate proper technique for this. Carefully bring your GC vial to room 217 to be
crimp-capped and submitted to the ISF. (You may wear one glove to handle your vial in the
hall.)
Experiment 4
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Figure 4. Assembling a pipet filter
Cleanup and Waste Disposal
1.
Dispose of the orange peel in the designated compost bucket.
2.
Centrifuge tubes, and filter paper apparatus may be disposed of in the blue container for
chemically contaminated solid waste.
3.
Dispose of the excess ethyl acetate solution in the waste bottle in the fume hood.
4.
Return the copper wire (coiled) to the hood.
5.
Return the 5mL volumetric flask (Clean and Dry) to the ISF stockroom window.
Experiment 4
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Part II: GC Analysis
GC Settings:
Column Stationary Phase: Agilent J&W HP-5
(5% phenyl, 95% methylpolysiloxane), 0.25
µm film thickness
Injection Volume: 1.0 µL
Inlet Temperature: 200 ºC
Split Ratio: 20:1
Column Dimensions: 30 m long, 0.320 mm
diameter
Detector: Flame Ionization Detector (FID)
Detector Temperature: 350 ºC
Carrier Gas: Helium
Carrier Gas Flow Rate: 2.0 mL/min
Oven Program: start at 60 ºC, hold for 2.5
minutes, increase to 160 ºC at 25 ºC/min.
Procedure
Five standard solutions were prepared by the instructional staff. GC-FID traces were obtained for
Solutions 1-5 and are provided.
1.
In a well-formatted table, record the limonene concentrations in the standard samples.
2.
From GC-FID traces, determine the peak height and area for limonene and anisole peaks
3.
Calculate the concentration (using both peak height and peak area) of anisole in the sample
you prepared.
4.
Table 1. has been constructed and partially filled out to assist you. Complete the table with
solution concentrations provided by your instructors.
Table 1. GC Analysis of Standard Samples of D-Limonene in Ethyl Acetate
Limonene
(Retention time =
Anisole
min)
Solution
ID
Concentratio
n (g/mL)
LimStd1
5.00 × 10-4
2.00 × 10-3
LimStd2
1.00 × 10-3
2.00 × 10-3
LimStd3
3.00 × 10-3
2.00 × 10-3
LimStd4
5.00 × 10-3
2.00 × 10-3
LimStd5
7.00 × 10-3
2.00 × 10-3
Experiment 4
Peak Area
(pA·s)
Peak Height
(pA)
(Retention time =
Concentration
(g/mL)
Chem 4B Spring 2023
min)
Peak Area
(pA·s)
Peak Height
(pA)
Page 11 of 15
Report Sheet
Name__________________________________
Partner(s)_______________________________
Answer may not exceed 2 sentences.
Part I: Limonene Extraction
Analytical:
1.
Another method of determining the amount of limonene extracted might have been to preweigh the centrifuge tube and then weigh by differences to obtain the mass of the extracts.
What would be the disadvantage of this method?
2.
What errors or inherent limitations (list two) of the procedure used in this lab might have
changed the amount of limonene you have calculated to be present in orange rind, as compared
to the amount that would actually be extracted in an industrial facility?
1)
2)
Conceptual
3.
What causes the centrifuge tube to become pressurized?
Experiment 4
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Part II: GC Analysis
Data Analysis:
4.
In Table 1, report the concentration, peak height, and area for the limonene and anisole
standards.
Table 1: GC Analysis of Standard Samples of D-Limonene in Ethyl Acetate
Limonene
Solution
Number
______
(Retention time =
Concentrati
on (g/mL)
Anisole Standard
min)
Peak Area
(pA·s)
Peak
Height
(pA)
(Retention time =
Concentratio
n (g/mL)
Peak
Area
(pA·s)
min)
Peak
Height
(pA)
LimStd1
LimStd2
LimStd3
LimStd4
LimStd5
The following analyses can most easily be completed using Excel or a similar spreadsheet
program. Attach a copy of your all relevant sample calculations to the Report Sheet.
5.
Construct calibration curves ‘peak height vs limonene concentration’ and ‘peak area vs
limonene concentration’ using data from limonene standards. Attach well-formatted figures
to the Report Sheet.
6.
Write the linear regression equations (with correct units) for both calibration curves. Define
any variables you use.
Limonene:
Peak height vs
concentration
Peak area vs
concentration
Experiment 4
Chem 4B Spring 2023
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7.
Using your calibration curves and data obtained from the GC trace of the orange rind sample
you extracted, what is the D-limonene content of orange rind in milligrams of limonene per
gram orange rind (mg/g)? Include 95% confidence interval for your answers (you should have
an answer from each calibration curve). Show ALL steps in your calculations on a separate
sheet of paper or well-formatted spreadsheet. See Harris 10th ed, Section 4-8 “Calibration
Curves” pp. 84-87 for reference.
8.
Calculate the ratios of limonene to anisole concentration ([Lim]/[Ani]), peak area (A Lim/AAni),
and peak height (HLim/HAni) for the standard samples. Construct and attach a well-formatted
table to clearly present this data using labels and appropriate units.
9.
Calculate ALim/AAni and HLim/HAni for your sample.
10. Construct calibration curves on a computer (Excel or similar) of ‘peak area ratio vs.
concentration ratio’ (ALim/AAni vs. [Lim]/[Ani]) and ‘peak height ratio vs. concentration ratio’
(HLim/HAni vs. [Lim]/[Ani]). Attach a copy to the Report Sheet.
11. Using your calibration curves above from Q10, what is the D-limonene content of your sample
(report as mg D-limonene / g orange peel) with 95% confidence interval? (You should have
an answer from each calibration curve)
Experiment 4
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Analytical:
12. Is peak height or peak area a better method of quantifying limonene content of a sample?
Why?
13. Is a calibration curve of limonene peaks or comparison to an internal standard such as anisole
a better method of quantifying limonene content of a sample? Why?
14. Looking at the GC chromatogram for your sample, discuss the purity of your extract.
15. The data output for your GC trace only displays time from 3-8 minutes. What is happening in
the first three minutes?
Attachments
✓
✓
✓
✓
✓
Relevant calculations (calibration curve, propagation of uncertainty, etc.)
GC chromatograms
Calibration Curves (2) – Limonene Standards
Table – Internal Standards
Calibration Curves (2) – Anisole Internal Standard
Experiment 4
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