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Determination of DEET Concentration in Bug Spray

Dates Performed: 6, 11, and 13 April 2016


Date Submitted: 29 April 2016
Performed By: Andrew Lindsay, Trevor Forsman, George Rotsides, Taylor Mauceri

Abstract
The concentration of DEET (N,N-diethyl-meta-toluamide) in
Off Deep Woods bug spray
was determined using gas chromatography. A gas chromatograph equipped with a thermal
conductivity detector was used to separate DEET from other components of the sample mixture
and an internal standard of dibutyl phthalate (DBP). To optimize flow rate in the gas
chromatograph, a Van Deemter curve was produced. However, due to a produced curve lacking
a well-defined minima, a flow rate of 1.2 ml/min was selected using a literature source. After the
optimization of the flow rate, resolution in the sample mixture was achieved by programming a
temperature gradient. The concentration of DEET was determined relative to the internal
standard DBP. It was determined

that Off Deep Woods bug spray contains 34.50 2.03 % DEET
weight by volume ath the 95% confidence level. Compared to the advertised DEET
concentration (wt./v) of 25%, there was a relative error of 38%. The high relative error can be
attributed to the low reproducibility of manual injections.

Introduction
In this study, the concentration of DEET (N,N-diethyl-meta-toluamide) in Off Deep
Woods bug spray was determined using gas chromatography (GC). DEET is the most common
active ingredient

in insect repellents, and Off Deep Woods bug spray is advertised to contain 25%
(wt./v) DEET. The concentration of DEET can be determined using an internal standard of
dibutyl phthalate (DBP). The internal standard is a compound that is very similar, but not
identical to the chemical species of interest in the samples. This is done because instrument
response may vary from one trial to another, but the response to both the analytes of interest and
the internal standard are affected to the same degree [1]. Equation 1 below allows for the
concentrations of analytes to be obtained relative to an internal standard,

Sr
Ss
C r = k ( C s ) (Equation 1)
where Sr represents the response to the analyte, Cr is the concentration of analyte (ppm), Ss is the
response to the standard, Cs the concentration of the standard (ppm), and k is the response factor
for the analyte relative to the internal standard.
For the separation and detection of components in prepared bug spray samples, a gas
chromatograph equipped with a thermal conductivity detector (TCD) was used. After a sample is
injected through the inlet of a gas chromatograph, the sample is vaporized and is carried through
the column by a carrier gas. The carrier gas is usually helium, however, unreactive gases such as
nitrogen and hydrogen may also be used. For analytes that are not easily resolved, hydrogen is
recommended to improve separation [1]. The column is housed in an oven where the temperature
can be controlled in order to vaporize the sample. In the column, gaseous compounds interact
with a thin liquid film stationary phase coated on the capillary walls or a support material. This

causes each compound to elute with a different retention time. Assuming good resolution, each
peak in the chromatogram represents a single compound.
While samples are often injected into a gas chromatograph via an autosampler, samples
can also be manually injected. Manual injections have associated disadvantages due to the
inconsistency in reproducing the injection volume and technique. Manually injecting samples
also requires constant oversight while analyzing samples. For the detection of components
separated by the gas chromatograph, a TCD was used, which senses changes in the thermal
conductivity of the column eluent compared to a reference flow of carrier gas. Changes in the
thermal conductivity of the column effluent will result in a temperature change, causing a
resistance change, which can be measured. Since all organic and inorganic compounds have a
thermal conductivity different than helium, all compounds can be detected using a TCD [1].
To achieve resolved peaks, a temperature program is important in GC since components
are separated primarily based on boiling point. However, before writing a temperature program,
it is important to select the optimal flow rate that gives the greatest column efficiency. The
efficiency of a column can be defined by the number of theoretical plates (N) of a column. The
more plates a column has, the more efficient the column is and the separation between two
solutes will be greater. The plate count for a column depends on the flow rate, the mobile phase
used, and the nature of the solute. From the number of theoretical plates, the height equivalent to
a theoretical plate (H) can be determined from the length of the column. The optimal flow rate is
where plate height is a minimum. A Van Deemter curve can be constructed to determine the
optimal flow rate by plotting theoretical plate height versus the flow rate from the injections of

an unretained solvent [1]. Equation 2 below was used to solve for the height equivalent to a
theoretical plate in the column,

L
(Equation 2)
N
where H is the height of the plates in the column, L (fixed) is the length of the column (cm), and

H=

N is the number of plates in a column.


The number of plates (N) was determined from equation 3,

N=

5.55t2r
W 21/2

(Equation 3)

where N is the number of plates in a column, tr is the retention time for each component, and
W1/2 is the peak width at half height.
Peak resolution is a measure of how well two adjacent peaks are separated from each
other. Peak resolution can be improved in GC by using a temperature gradient. The use of longer
and narrower columns also leads to greater resolution. To determine if resolution between two
peaks is greater than the desired resolution value of 1.5, equation 4 was used,

Rs =

1.18(t2t1)
W h1+W h2

(Equation 4)

where Rs is the resolution, t1 is the retention time of the first peak, t2 is the retention time of the
second peak, and w h1 and w h2 are the corresponding peak widths measured at half the peak
height.

Methods/Materials
Instrumentation
An Agilent Technologies 7890B Gas Chromatograph was used to analyze the bug spray
sample. A HP5 column housed in the GC oven, consisting of (5%-Phenyl)-methylpolysiloxane
with dimensions of 30 m length, 0.25 m film, and a 0.25 mm outer diameter was used for the
separation. This instrument utilizes a thermal conductivity detector to analyze the elutants. The
samples were injected manually. To determine the optimal flow rate, ethanol was analyzed at
flow rates from 1.0 mL/min to 2.8 mL/min in 0.2 mL/min intervals. The injector temperature for
this process was set to 200 o C and the column temperature was 100 o C. Using the data from these
analyses, a Van Deemter curve was plotted to determine the optimal flow rate which was 1.2
mL/min.
For the analysis of the standards and bug spray samples, the injector temperature was 200
C. The column temperature was 175 o C and held for 5 minutes. The temperature was then

ramped to 200 o C increasing at a rate of 3.5 o C/min.

Standard and Sample Preparation and Analysis


A 5 mg/mL DEET solution and a 5% (wt./vol.) DBP solution were prepared for analysis.
Then a 2.50 mL portion of the DEET solution was spiked with a known volume of a 5% DBP
solution. The unknown was prepared by diluting Off Deep Woods bug spray to a known volume
and then spiking it with a known volume of the 5% DBP solution. Using DBP as an internal
standard, the sample and standard were analyzed in triplicate.

Results/Discussion
To develop a method for the determination of DEET in Off Deep Woods bug spray by
GC, a Van Deemter curve was initially prepared to optimize the flow rate. A Van Deemter curve
as shown by Figure 1, is used to determine the optimal flow rate which is indicated by the
minima of the curve.

Figure 1. Van Deemter curve for ethanol used to determine the optimal flow rate.
However, the Van Deemter curve generated does not have a clear minima. The poor results can
be attributed to poor reproducibility in the manual injection of samples. Therefore, an optimal
flow rate could not be effectively determined using this method. To choose an optimal flow rate,
a similar analytical study involving the determination of DEET by GC analysis was researched.
Using the method from this study, a flow rate of 1.2 ml/min was selected [3].

To achieve sufficient resolution, a temperature program was written so that adjacent


peaks did not coelute. Peak resolution is important because if peaks are not resolved, quantitative
analysis is inaccurate. To verify that there were no errors due to poor separation, the resolution
between adjacent bands was determined. The resulting values of resolution are shown in Table
1.

Table 1. Resolution of adjacent peaks in the gas chromatograms of the standards and the
unknowns.
Trial

Resolution (peaks 1 & 2)

Resolution (2 & 3)

Standard 1

7.4

15.5

Standard 2

7.6

15.7

Standard 3

7.0

13.9

Unknown 1

9.6

20.2

Unknown 2

9.4

28.2

Unknown 3

9.1

27.4

Unknown 4

7.4

15.8

Since all resolution values were greater than 1.5, all peaks were fully resolved.
For the determination of DEET using an internal standard, it is important to calculate the
response factor for the analyte relative to the internal standard. Table 2 contains the resulting
peaks for the DEET standard containing 5 mg/mL DEET and a 5% (wt./vol.) DBP solution. The
response factor was determined using Equation 1.
Table 2. Gas chromatography results for the DEET standard and the calibration constant.
Trial

DEET (peak area)

DBP (peak area)

k (response factor)

237.0

246.1

0.01926

272.0

313.7

0.01734

287.8

319.1

0.01748

Mean

262.6

293.0

0.01803

Standard deviation

22.4

40.7

0.00107

The bug spray sample was then analyzed using DBP as an internal standard. The concentration
of DEET in each sample are shown in Table 3.
Table 3. Gas chromatography results for the analysis of DEET in a bug spray sample.
Trial

DEET (Peak
Area)

DBP (Peak
Area)

Sample
(mg/mL)

Bug Spray (%
wt./v)

4102.1

927.7

40.88

34.34

4183.5

258.0

149.91

125.93

3545.5

69.6

470.97

395.61

4815.2

1078.9

41.26

34.66

From this analysis, trials 2 and 3 of the unknown are outliers, therefore were not included for the
determination of DEET. One possible cause for outliers is that DBP in volatile. Due to the
volatility of DBP, the DBP in the analyte may have vaporized from the sample before injection
into the GC. The average concentration of DEET in bug spray is shown in Table 4.

Table 4. Gas chromatography results for the analysis of DEET in a bug spray sample after the
removal of outliers.
Trial

Bug Spray (% wt/v)

34.34

34.66

Mean

34.50

95% confidence interval

2.03

A HP-5 column which is composed of 5% phenyl-methylpolysiloxane, was used for the


analysis of DEET. This column is a fused silica column that features a low-polarity phase and
crossbond diphenyl, dimethyl, and polysiloxane groups. A HP-5 column is commonly used for
general purpose drugs, solvent impurities, pesticides, hydrocarbons, PCB congeners, essential
oils and semivolatiles. Other column choices that are used for similar analysis are DB-5, CP-Sil
8 CB, and ZB-5 columns. In addition to choosing the correct column, an appropriate detector
must be used. A TCD was used for the determination of DEET in a bug spray sample. TCDs
measure a bulk property of the mobile phase and sense thermal conductivity of a column eluent
and compares it to the reference carrier gas. One of the main disadvantages of using a TCD is
low sensitivity. To improve the sensitivity of detection, a flame ionization detector (FID) could
be used. Compared to a TCD, a FID is much more sensitive, has better linear and dynamic range,
does not respond to mobile phase impurities or inorganic compounds, and can be used for less
concentrated samples. Due to the greater sensitivity of FIDs, this detector has a lower detection
limit than TCDs.
When analyzing a sample using GC, the composition of the mobile phase is not as
important as in High Performance Liquid Chromatography (HPLC). For HPLC experiments, the

composition of the mobile phase is crucial because the components of a sample are separated by
polarity. Thus by adjusting the polarity of the mobile phase, the separation of the analytes is
increased. Contrary to HPLC, GC separates the components of a mixture by boiling point. Thus
to increase the separation of the components, a temperature gradient is more effective.

Conclusion
A method for determining the concentration of DEET in a bug spray sample was
successfully developed. Using a Van Deemter curve the optimal flow rate was unable to be
determined, thus a flow rate 1.2 mL/min was used. This value was obtained from a literature
source. A temperature program was developed and produced good resolution between adjacent
peaks ranging from 7.4 to 28.2, which shows complete peak resolution. Using this method, the
DEET concentration (wt./v) in bug spray was determined to be 34.50 + 2.03 % at the 95%
confidence level. There was a 38% relative error in the sample compared to the 25% DEET
reported in OFF Deep Woods bug spray. There was large variability in two of the trials
producing results which were unreasonable. This error could have been due to the volatilization
of the internal standard, DBP. To minimize the presence of outliers and reduce relative error, the
samples could be injected by an autosampler and the samples could be prepared on the same day
of analysis.

Citations:
[1] Hallock-Waters, Kristen, Gas Chromatography. 4/10/2016

[2] Skoog, D. A., West, D. M., & Holler, F. J. (2014). Fundamentals of analytical chemistry (9th
ed.). Cengage Learning.
[3] Cherstniakova, S. A.; Garcia, G. E.; Strong, J.; Bi, D.; Weitz, J.; Roy, M. J.; Cantilena, L. R.
Rapid Determination Of N,N-Diethyl-m-Toluamide and Permethrin in Human Plasma by Gas
Chromatography-Mass Spectrometry and Pyridostigmine Bromide by High-Performance Liquid
Chromatography. Journal of Analytical Toxicology. 2006, 30, 2126.