Phthalates in Dog Toys Gc/ms

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Procedure

This procedure was adapted from the procedure outlined by Dr. Michelle Ward for CHEM 1255 Instrumental Analysis.3 Phthalate standard solutions were prepared by diluting the phthalate stock solution (2000 μg/mL) with methylene chloride (CHCl2) to yield 10 mL of 5, 10, 15, 20, 25, and 50 ppm solutions. Individual phthalate solutions were prepared by diluting diisononyl phthalate and diisodecyl phthalate to 10 mL to yield separate 10 ppm solutions. The dog toy samples were prepared by slicing the dog toy into 7 mm pieces. These pieces were treated with liquid nitrogen until brittle and ground to a fine powder using an analytical mill. 20 mL of methylene chloride was added to 2.0027 g of ground toy and the mixture sonicated for 30 minutes to extract surface phthalates. Three sample solutions were made by diluting 1 mL extract to 25 mL, 100 mL, and 250 mL with methylene chloride. All solutions were filtered through a 0.45 mitron PTFE syringe into autosampler vials and subsequently loaded into the GC/MS. The solutions were analyzed using the “phthalates” method in the TuroMass Software.

Results

The 10mL standard phthalate solutions ranging from 5 to 50 ppm were prepared by diluting the 2000 ppm stock solution with methylene chloride. The amount of stock solution was calculated using M1V1:

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Example Calculation for 15ppm Solution:

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- The spectra were obtained for each standard solution. Figure 4 shows the spectrum for the 50 ppm solution. Although there were supposed to be 11 phthalates in the stock solution, only 7 showed up on the chromatogram. [pic 9][pic 10]

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- Surprisingly, there was an extra phthalate, cyclobutyl isobutyl phthalate, present in all the standard solutions that was not on the original stock solution list. Absent from the chromatograms were bis(2-chloroethyl)ether, bis(2-chloroisopropyl)ether, bis(2-chloroethoxy)methane, dimethyl phthalate, and DEHP. The peaks were identified by the software, which integrated each peak and compared the results with a known library. All chromatograms and integrations identifying peaks are attached.

Chromatograms for diisononyl phthalate and diisodecyl phthalate were also obtained and their peaks identified. (See attached) Three chromatograms were obtained for each of the dog toy dilutions. Figure 5 shows the chromatogram for the most dilute sample (1 mL extract in 250 mL methylene chloride).[pic 11][pic 12]

In all three spectra, the only phthalate found was DnOP, with a retention time of 17.20 min. To quantify the amount of phthalate present in the toy sample, a calibration curve of peak area vs. retention time was created (Fig 6).[pic 13][pic 14]

Using the equation for the best fit line (2),the amount of DnOP in the diluted sample was calculated.[pic 15]

[pic 16]

Then the original %weight/weight (%w/w) of phthalates was calculated using the dilution factor and the original weight of the sample. A summary of the calculations is in table x. The following example is a calculation for the 1 mL extract in 250 mL methylene chloride:

[pic 17][pic 18]

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The standard deviation was calculated by first calculating the standard deviation of the calibration curve was calculated using the following equation:

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Sr is the standard deviation of the regression (calculated using Excel), m is the slope, n is the total number of data points, y is the instrumentation output, is the mean value of y for the calibration data points, xi are the individual values of x and is the mean value of x for the calibration data points. The value for the standard deviation of the calibration curve was then divided by the concentration of the sample to yield the deviation in %w/w. These values were averaged to yield a final concentration and standard deviation.[pic 21][pic 22]

Table 1: Concentration of DnOP in Dog Toy

Extract Concentration (mL/mL CHCl2)

%w/w

Standard Deviation (%w/w)

1/25

-40.634%

0.127%

1/100

-37.796%

0.509%

1/250

-26.064%

1.272%

Average

-34.83%

0.64%

Discussion

This experiment was conducted to determine at what concentration phthalates, if any, were present in dog toys. The only phthalate present in the extract solution was found to be DnOP at a concentration of -34.83 ± 0.64 %w/w (Table 1). This, however, is physically impossible. The calibration curve (Fig 6) shows the peak area decreasing with increasing concentration, which is also not possible. A source for this error is that the concentrations written on the chromatograms were reversed, meaning that the calibration curve is backwards and incorrect. The R2 value for this curve (0.9363) implies that there is a relatively strong correlation between the data points; when reversed (ie 50ppm is now 5, and 5 ppm is now 50), the R2 value decreases to 0.7539, which still indicates a relationship, just not as strong of one. Thus, it is difficult to comment on the source of error for the calibration curve, but it is likely that the vial numbers were input incorrectly. The study must be conducted again to verify this hypothesis.

The calibration solutions only contained 6 of the phthalates in the original mix, and 1 extra phthalate, cyclobutyl isobutyl phthalate, not present in the original solution. It is unclear why some compounds were not present, or why there was an extra peak. The peaks for these compounds may have been obscured by noise; however, as shown in the chromatogram for the 50 ppm solution, all peaks appear strong with similar peak areas relative to one another. One possibility for the source of error may lie in sample preparation. This data was provided by another