Experiment VII
Analysis of Fragrance Compounds in Water and Soil
Purpose
To demonstrate the procedures for separation of organic contaminants from
environmental matrices and to illustrate some of the steps necessary for their
preparation for chromatographic analysis.
References
1. Sawyer, C. N., McCarty, P. L., and Parkin, G.F. Chemistry for Environmental
Engineering, 5th ed., McGraw Hill, 2003.
2. Simonich, S. L.; Begley, W. M.; Debaere, G.; Eckhoff, W. S. Environ. Sci. Technol.
2000, 34, 959-965.
3. A.M. DiFrancesco, P.C. Chiu, L.J. Standley, H.E. Allen, and D.T. Salvito. Environ.
Sci. Technol. (in press).
Background
Fragrance materials (FMs) are a group of over 3,000 structurally diverse compounds
that are widely used in consumer products, such as laundry detergent, soap, and
shampoo. Most FMs are used at low concentrations and have global industry volumes
less than 1 metric tons per year (mT/y), while a small number of FMs have production
volume exceeding 3,000 mT/y. Through down-the-drain disposal of consumer
products, FMs are discharged into sewage and enter municipal wastewater treatment
plants (WWTPs).
In recent years, there has been growing concerns about the potential impact of organic
micro-pollutants, such as pharmaceuticals and personal care products, in the
environment, and WWTPs effluent is considered the primary source of input. Several
FMs, particularly polycyclic and nitro musks and their metabolites, have been detected
in surface waters downstream of WWTP discharge points as well as in aquatic
environments and aquatic organisms. The polycyclic musks AHTN (7-acetyl1,1,3,4,4,6-hexamethyl-1,2,3,4-tetrahydro-naphthalene) and HHCB (1,3,4,6,7,8hexahydro-4,6,6,7,8,8-hexamethylcyclopenta-gamma-2-benzopyran) have been used
as molecular tracers to track the transport of WWTP organic matter in surface waters.
The fragrance compounds that have been studied at the University of Delaware are the
following:
O
O
O
O
O
AH TN
OH
O
O
Diphenyl Ether
Benzyl
Acetate
Acetyl Cedrene
O
o
O
DPMI
p-t-Bucinal
O
O
OH
O
Eugenol
Hexylcinnamaldehyde
Hexyl Salicylate
O
HHCB
Isobornyl Acetate
O
O
O
OH
O
d-Limonene
Linalool
Methyl Dihydrojasmonate
 - Methyl Ionone
O
OH
Methyl
Salicylate
O
NO 2
O2 N
NO2
O2 N
O
Phenyl Ethyl
Alcohol
NO 2
OTNE
Musk Ketone
Musk Xylene
HO
OH
-Pinene
Terpineol
Procedure
Spiking cocktails have been prepared by dissolving weighed amounts of a subgroup of
the FMs in methanol (99.5% Aldrich). The cocktails have been stored at 5°C in
borosilicate vials capped with Teflon®-lined closures and sealed with low-permeability
vinyl tape (3M). FM standard solutions for gas chromatography-mass spectrometry
(GC/MS) calibration were prepared by dissolving a pre-weighed amount of each
chemical in methanol. The standard stock solution was then transferred to a bottle
sealed with Teflon® tape and stored at -30°C. Calibration standards were made
through dilution of the stock solution using methanol and were stored in a similar
fashion.
Calibration Standards
1. Calibration standards will be prepared and the calibration data will be provided. This
will, for each calibration standard, consist of the concentration and instrument
response for each of the compounds that it contains.
2. You will be provided a chromatogram of a standard solution containing a number of
FMs. These will be identified so that you can compare the retention time for your
compound(s) to those of the standard.
Samples
Take one set of samples consisting of a FM spiking solution and 3 soil samples. Each
of the 5 g air dried soil samples has been previously spiked with an equivalent of 1 mL
of your FM solution. Be sure you note the letter designation of your FM on all samples
you turn in for analysis.
Analysis of FM spiking solution
1. Transfer a portion of your FM spiking solution to a 4-mL borosilicate vial for analysis
by GC-MS. This is sample 1.
Extraction of water sample
1. Place 100 mL tap water into your separatory funnel.
2. Add 1 mL of your FM spiking solution to the water in the separatory funnel.
3. Add 5 mL DCM.
4. Stopper funnel and shake once.
5. Being careful to have the stopper pressed tightly into the palm of your hand, tip the
funnel so the stem is nearly vertical.
6. Slowly open the stopcock so that the vapor inside the flask will equilibrate in
pressure with that of the atmosphere.
7. Close the stopcock.
8. Shake 15 – 20 times to ensure good mixing of the DCM with the water and thus
transfer of the FMs from the water to the DCM. Check for pressure equalization
several times during this process (steps 5 – 7). This is a good place to ensure that
each person gets some experience.
9. With the separatory funnel in its conventional orientation with the stem down,
remove the stopper. Allow the DCM to settle. If some water is commingled or
droplets appear stuck, gentle tap.
10. Carefully drain the DCM into a vial or test tube leaving the water in the separatory
funnel.
11. Run the DCM phase into a test tube or vial and add approximately 1 g anhydrous
sodium sulfate to remove water. (If you have visible droplets of water, it may be
necessary to transfer a portion of the DCM to another tube before adding the
sodium sulfate.)
12. Transfer a portion to a 4-mL vial for analysis by GC-MS. This is sample 2.
Extraction of soil sample
1. Two of the three soil samples will be extracted with DCM in an ultrasonic shaking
bath. The third sample is an extra to be used only if you have a problem. Add 10
mL DCM to each of the 3 soil samples. Place 2 of the samples into the ultrasonic
shaker. Remove one after 5 min and the other after 45 min.
2. Transfer the DCM phase into a test tube or vial and add approximately 1 g
anhydrous sodium sulfate to remove water. (If you have visible droplets of water, it
may be necessary to transfer a portion of the DCM to another tube before adding
the sodium sulfate.)
3. Transfer a portion to a 4-mL vial for analysis by GC-MS. These are samples 3 and
4.
Analysis
Analyze the samples for FMs using GC-MS. Data will include the retention time and
response. For your sample 1, a mass spectrum will also be provided.
Data Analysis
1. Prepare calibration curves for your FMs and plot the regression line with the best fit
linear equation for the data obtained.
2. Determine the concentration (g/L for all samples and also g/g for the soils that
were extracted) of your FMs for all samples using best fit equations determined
in “Data Analysis No. 1 and present in a neat, easy to read table. Be careful that
you have correctly accounted for the volumes of solvent used.
3. Compute, and include in your table, the recovery of your FMs from the water and
soil samples, based on the analysis of the FM spiking solution.
Questions
1. How would you propose that the recovery of the FMs in your sample might be
improved?
2. For your FMs show on your mass spectrum the molecular ion. What is its MW?
How does that compare to the calculated MW?
3. Extra Credit. Indicate the MW for each of the 3 most abundant peaks that are not
the parent. Propose the moiety that has been lost and the formula for the resulting
ion.
Apparatus
4 mL sample vials
Separatory funnel
Ultrasonic shaker
Pipettes (volumetric and Pasteur)
Gas Chromatograph-Mass Spectrometer (GC-MS)
Reagents
FM standards in methanol
Sodium sulfate
Dichloromethane