The Introduction Of Phthalates Biology Essay

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Phthalates are clear liquid form in room temperature and have no odor. Phthalates are the most commonly used plasticizers which are added into products to increase the polymer flexibility and durability of the product.

This is due to their function as intermolecular lubricants. They are additives and not reagents, thus they are not chemically bound in the polymer and are therefore able to leach from the matrix (Zou & Cai, 2011). Phthalates are lipophilic and therefore tend to concentrate in the lipid phase of food (Zhu et al., 2010). Their industrial applications are related to the length of the ester chain. The long chain or high molecular weight phthalates are such as DEHP, DnOP, DiNP, DiDP. They are primarily used in polyvinyl chloride (PVC) polymer and construction materials (Yen et al., 2011).

Ingestion of food containing phthalates is an important route of exposure. Dietary intake of phthalates per day must be regulated. According to MAFF, the maximal daily intake of DBP is 0.48 µg/kg/day, butyl benzyl phthalate (BBP) is 0.11-0.29 µg/kg/day (MAFF, 1996), while DEHP is 4.9-18.0 µg/kg/day (Meek & Chan, 1994). The Bundersverband NaturKost Naturwaren (BNN) has also published recommended value for phthalates in edible oils, for DEHP the recommended value is 3 mg/kg, while for BBP, DiNP, DiDP and other is 5 mg/kg (BNN, 2006).

Healthcare applications such as blood-bags and medical tubing, such that hospitalised patients undergoing haemodialysis and respiratory therapy can be exposed to high levels of phthalates leaching out of these devices (Schettler, 2006). Medical devices made of PVC and softened with DEHP, usually made in tubing for administering purpose result in a maximal daily DEHP exposure of 9.5 mg/day or 0.14 mg/kg/day in adults. Infants may be exposed to 2.5 mg/kg/day via this pathway.

Ingestion of phthalates can also occur via their use in the coatings of pharmaceuticals (drugs) and nutritional supplements (Schettler, 2006). This is because phthalates are coated in polymer to influences the time and location of drug delivery in the gastrointestinal tract. The most commonly used phthalates in this field are DBP and DEP.

Polymer toys softened with phthalates is also an important exposure route in children. Children can be exposed by mouthing or chewing older PVC toys, as phthalates can leach out into their saliva. In December 1999, the European Union restricted on six phthalates with maximum concentration of total phthalate contents in toys, not exceeding 0.1 % (w/w). The six phthalate esters are DBP, BBP, DEHP, DnOP, DiNP and DiDP. Subsequently, Consumer Product Safety Improvement Act (CPSIA, 2008) also implemented 0.1 % (w/w) limit for these six phthalate esters.

Inhalation

Inhalation of household dust containing phthalates (particularly DEHP) from PVC flooring and building materials is another exposure route (Rudel et al., 2003). They also reported that the total phthalate concentration in dust ranging from 0.3 to 524 µg/g of dust. The phthalate air concentration range from samples to other locations is from 0.005 to 28µg/m3. Inhalation of contaminated dust will be more serious in larger inhalation exposure.

Adibi et al. (2003) reported that there is an important pathway of exposure for lower molecular weight phthalates due to the identification of significant correlation in DEP, DBP and BBP.

In Norway, DEHP was found to be the largest contributor of phthalate contamination since the mean of DEHP was 640µg/g of dust from a 960µg of total phthalates/g of dust. Thus, this showed an estimated mean of 0.76µg/day of DEHP to adult inhalation exposure (Oie et al., 1997).

In Tokyo, phthalate levels in indoor air of 27 houses were analyzed and reported that the median concentrations of DEP, DBP, BBP and DEHP were 0.10, 0.39, 0.01 and 0.11 µg/m3 respectively. As a result, adult that breathing 20 m3/day, will result in inhalation exposures of 2, 7.8, 0.2, 2.2 µg/day respectively (Otake et al., 2004).

Intravenous

Since variety of medical devices is made of PVC plasticized or softened with DEHP to deliver medical care for medical used, thus the leaching of phthalate is hardly avoided.

Recent studies on DEHP metabolites in urine of infants receiving intensive medical therapy documented that the median of metabolite MEHP was 129 ng/mL. It was pretty much higher compared to the median in the National Health and Nutrition Examination Survey (NHANES) study which is 2.7 ng/mL (Calafat et al., 2004)

Moreover, other studies which also measured DEHP metabolites in urine on infants reported that increasing intensity of care and more frequent use of DEHP containing devices resulting in higher levels of urinary metabolites of DEHP. The mean of MEHP levels in low, medium and high intensity care infants were 9.3, 41, 139 ng/mL respectively (Green et al., 2005)

Skin adsorption

Phthalates can also be absorbed through the skin following the use of perfumes, cosmetics, clothing, sunscreens and other personal care products containing them (Blount et al., 2000) such as nail polishes and hair sprays (Koo & Lee, 2004). Skin absorption of chemicals from the face may be up to 10 fold higher than the arm.

Studies using rodent skin had shown that the absorption of phthalates is relatively less permeable in human skin compared to rat skin (Scott et al., 1987). DMP and DBP are usually used for insect repellants. However, US Environmental Protection Agency (EPA) included that DBP, DEP, DMP and DnOP are potentially toxic inerts and may use along with other ingredients in insecticides or repellants causing dermal or inhalation exposures.

Effects of phthalates towards people

People have high possibilities exposed to phthalate esters since they are bulky, largely and widely in used on industrial to make various type products include households, detergents, bottles, skin care, cosmetics, medical care and even food. Since all these are daily requirements, thus people are exposed to high risk of phthalates every day. Phthalate esters have endocrine disrupting properties, and have adverse effect of male reproductive development. Studies have found that DEHP can cause malformations of the reproductive system (testicular toxicant) in male rats through an endocrine disrupting mechanism (Gray et al., 1999) and may increase the liver weight in rats.

Exposure to hormone disrupting chemicals like phthalates are being one of the factors contributing to observed trends of declining fertility, increased incidence of testicular cancer and falling sperm counts in European men based on the studies of their effects in rodent (Foster et al., 2000). In addition, DBP has anti androgenic properties (male hormone suppressing) which inhibit the production of testosterone (Foster et al., 2000).

Elevated levels of phthalates in blood have also been implicated in premature breast development in Puerto Rican girls (Colon et al., 2000). It was also found that phthalates may persist in human tissues for longer time.

Phthalates in indoor air and dust have been associated with asthma, eczema, and rhinitis in children (Bornehag et al., 2004). This is because children are more easily exposed to phthalate esters compare to adults. Exposure of children to phthalates via PVC toys may disrupt the hormonal development of children and can lead to early puberty, reproductive defects and other health problems such as chronic effects on the kidney and liver (Zou & Cai, 2011).This problem occurs due to the characteristics of phthalates which are not covalently bound to polymeric material and can leach into their mouth through saliva.

Objective

The aim of this present study is to develop a method for determining phthalates esters in edible corn oil as well as to investigate phthalate contamination in corn oil at retail level and also to compare the results with those from the previous study.

2.0 LITERATURE REVIEW

2.1 Case study 1

Headspace solid-phase microextraction of phthalic acid esters from vegetable oil employing solvent based matrix modification (Holadova et al., 2007)

This study was to investigate the applicability of Headspace solid phase microextraction for the simple and fast analysis of phthalates in vegetable oil samples. This was a new solvent free analytical procedure based on headspace solid phase microextraction (SPME) coupled to gas chromatography employing a mass spectrometric detector (GC/MSD) for the determination of phthalic acid esters in vegetable oils (Holadova et al., 2007).

Table 1. Summary of Optimized Condition for different parameters of case study 1.

Parameter

Optimized condition

Type of dissolving agent

Methanol & n-hexane stored at 4°c

Solvent used for sample matrix modification agents

Acetonitrile & methanol

Methanol gives the highest yield of extracted phthalates

Types of SPME fibers

Polydimethylsiloxane with a thickness of 100µm

Best extraction of phthalates

Setting of GC analysis

Manual SPME

Oven program

5 min at 45°c then increased to 130° at 20°c/min, then increased to 240°c at 3°c/min and finally increased to 270°c at 20°c/min

Nitrogen carrier gas flow rate

I mL/min

Detector and Injector temperature

300°c and 250°c

Automated SPME

Oven program

5 min at 50°c then increased to 180° at 20°c/min, then increased to 250°c at 5°c/min and finally increased to 270°c at 10°c/min

Helium carrier gas flow rate

1 mL/min

Injector and transfer line temperatures

250°c and 270°c

Detector

Segment scan and SIM mode

For the preparation of standard mixtures, methanol and n-hexane were used. Various solvents were tested as sample matrix modification agents with the aim to facilitate the transfer of esters from oil matrix into headspace. The best solvents that had been found for the used of matrix modification were acetonitrile and methanol. However, the best results, the highest yields of extracted phthalates, were achieved when 1mL methanol was added to 1 g of oil sample. Four types of SPME fibers [silica fibers coated with polydimethylsiloxane (thickness 100µm, PDMS 100), carboxen/polydimethylsiloxane (thickness 75µm, CX/PDMS), polyacrylate (thickness 85µm, PA) and polydimethylsiloxane/divinylbenzene (thickness 65µm, PDMS/DVB) were tested. Polydimethylsiloxane with a thickness of 100µm seem to be the most efficient in headspace analyses of various phthalates in fat containing samples.

SPME of phthalates was carried out manually using a polydimethylsiloxane 100 fiber together with methanol as the matrix modifier and 20 minutes phthalate extraction from the headspace above 1 g of magnetically stirred oil sample modified with 1ml of methanol enables headspace phthalate determination in vegetable oil samples. The temperature was maintained at 40 â-¦C (measured in headspace); the sample was incubated for 60 min at the same temperature prior to extraction.

In the GC analysis, it was divided into two parts, Manual SPME and Automated SPME whereby the manual SPME combined with gas chromatography coupled to a mass spectrometric detector for automated SPME. In manual SPME, the initial oven temperature was set at 45°c at 5 minutes then increased to 130°c at 20°c/min, then increased to 240°c at 3°c/min and finally increased to 270°c at 20°c/min. Nitrogen was used as carrier gas at flow rates 1mL/min. The injector and detector temperatures were 250°c and 300°c respectively.

In automated SPME, the initial oven temperature was set at 50°c at 5 minutes then increased to 180°c at 20°c/min, then increased to 250°c at 5°c/min and finally increased to 270°c at 10°c/min. Helium was used as carrier gas at flow rates 1mL/min. The injector and transfer line temperatures were sent at 250°c and 270°c respectively. The detector was operated simultaneously in a Segment Scan and a Selected Ion Monitoring mode.

2.2 Case study 2

Phthalate, adipate and sebacate residues by HRGC-MS in olive oils from Sicily and Molise (Italy) (Dugo et al., 2011).

The aim of this study was to analyze Sicilian extra virgin olive oils from crop years of 2006-2009 for the presence of phthalates, adipates and sebacates to compare the results with those from previous study and also to compare the contamination of the samples produced in Sicily with those produced from Molise (Dugo et al., 2011).

Table 2. Summary of Optimized Condition for different parameters of case study 2.

Parameters

Optimized values

Type of dissolving agent

n-hexane stored in 4°c

Type of extracting agent

Acetonitrile

2g of olive oil mix with 1mL of acetonitrile in a closed vial

The phase were separated, the surnatant (acetonitrile) was discharged and the oily phase extracted again

The acetonitrile phase was collected, centrifuged at 5000rpm for 10 min and used for GC analysis

Settings of HRGC-MS

Column

Supelco SPB-5MS (5% diphenylsiloxane, 95% polydimethylsiloxane)

Oven program

0 min at 60°c then increased to 275°c at 15°c/min, isothermal for 14 min

Helium carrier gas flow rate

40cm/s

Transfer line temperature

230°c

Injector temperature

250°c

Full scan EI mode from 40 to 400amu and SIM mode

The standard solutions at different concentrations containing mixtures of all the plasticizer in analysis were prepared in n-hexane. Acetonitrile was used to extract phthalate, adipate and sebacate residues from olive oil samples. 2g of olive oil were mixed with 1 mL of acetonitrile in a closed vial and shaked. The phases were separated, the acetonitrile was discharged and the oily phase extracted again. The acetonitrile phases were collected, centrifuged at 5000rpm for 10minutes and used for GC analysis.

For the quantification and separation of phthalates, adipates and sebacates, Supelco SPB-5MS (5%diphenylsiloxane, 95% polydimethylsiloxane) capillary column was used. The oven temperature was set at initial temperature 60°C, and then increased to 275°C at 15°C/min and isothermal for 14minutes. The carrier gas was helium at constant rate 40cm/s. The transfer line temperature was 230°C, the injector temperature was 250°C. Injection volume was 1µL. The acquisition was performed in a full scan EI mode from 40 to 400amu and in single ion monitoring (SIM) mode.

2.3 Case study 3

Transfer of eight phthalates through the milk chain - A case study (Fierens et al., 2013).

The objective of this study was to investigate the occurrence of phthalates in milk and dairy products according to the principle from farm to fork (farm, industry and retail level) (Fierens et al., 2013).

Table 3. Summary of Optimized Condition for different parameters of case study 3.

Parameters

Optimized values

Type of extracting agent

Acetone/n-hexane (1:1)

Extraction of 5 to 80g of milk or diary product to at least 0.5g of fat

n-hexane Dichloromethane

Extraction of packaging materials

Flow rates of GPC

Dichloromethane as mobile phase

4 mL/min

Settings of GC-EI-MS

250°c in splitless mode with non polar stationary phase

DB-XLB column

Oven program

1 min at 50°c then increased to 320°c at 15°c/min, held constant at 320°c for 15min

SIM mode

Amount of phthalates added into sunflower oil (reference sample for analysis of high-fat food products)

250µg/kg

5 to 80g of milk or dairy product sample was extracted with acetone/n-hexane (1:1) to obtain at least 0.5g of fat. For the extraction of packaging materials, n-hexane followed by a solvent exchange to dichloromethane was used. Gel permeation chromatography (GPC) was applied to purify all fat extracts. Dichloromethane acted as mobile phase with flow rate of 4mL/min.

The instrumental analysis of the phthalates was performed by the means of gas chromatography-low resolution-mass spectrometry with electron impact ionization (GC-EI-MS). Sample injection occurred at 250°c in splitless mode and phthalates were separated on a DB-XLB column with a non polar stationary phase. The oven temperature was set at 50°c at 1 minutes, then increased to 320°c at 15°c/min and was then held constant at 320°c for 15minutes. Detection of phthalates took place in selected ion monitoring (SIM) mode.

250µg/kg of phthalates was added into sunflower oil to act as a reference sample for analysis of high fat food products.

3.0 MATERIALS

3.1 Chemicals

Dimethyl phthalate, DMP [C6H4(COOCH3)2, 99% purity], Diethyl phthalate, DEP [C6H4(COOC2H5)2, 99% purity] and Dibutyl phthalate, DBP [C6H4(COO(CH2)3CH3)2, 99% purity] were purchased from ACROS organic (USA). Bis(2-ethylhexyl) phthalate, DEHP [C6H4(COOCH2CH(C2H5)(C3H6)CH3)2, 99% purity] and Di-isodecyl phthalate, DiDP [C6H4(COO(CH2)9CH3)2, 99% purity] were purchased from FLUKA Analytical. Di-isononyl phthalate, DiNP [C6H4(COO(CH2)8CH3)2, 99% purity] and Di-n-octyl phthalate, DnOP [C6H4(COO(CH2)7CH3)2, 99% purity] were purchased from ALDRICH Chemical (USA). HPLC Grade Dichloromethane (DCM, CH2Cl2) was purchased from DUKSAN Reagents while Analytical Reagent Grade Dichloromethane (DCM, CH2Cl2) was purchased from Fisher Scientific Chemicals. HPLC Grade Methanol was purchased from MERCK ´KGaA, Darmstadt, Germany.

3.2 Instrumentation

All the weight of phthalates were weight by analytical balance (Sartorius ± 0.0001) manufactured by Germany. The gel permeation chromatography (GPC) was used to purify corn oil and to separate phthalates from corn oil. The GPC systems manufactured by Waters (USA) consisted of a 1515 Isocratic HPLC pump, an autosampler, a 2487 Dual λ Absorbance Detector, and a Waters fraction collector. Separation occurs on Water Envirogel column and runs by Breeze software. The instrumental analysis of phthalates ester were also performed using Gas Chromatography Mass Spectrometry (GCMS) - Agilent Tehnologies 5975C (Inert XL MSD) and Agilent Technologies 7890A (GC system) with 30m column manufactured by Agilent Technologies, Inc (USA). Data analysis was performed by Chemstation software.

4.0 METHODOLOGY

4.1 Identify the retention time of phthalates using GPC

In order to identify the retention time of phthalates using GPC, 50ppm of each DMP, DEP, DBP, DEHP, DnOP, DiNP and DiDP were prepared. Each of them was diluted with AR grade Dichloromethane in 250mL volumetric flask. All the glass wares used were rinsed three times with Dichloromethane to remove possible contaminants or impurities. Each sample was injected two times for determination of retention time by GPC.

DiDP

DiNP

DnOP

DEHP

DBP

DEP

DMP20130123_135128.jpg

Figure 8. Phthalates that being used. From left which is the lowest molecular weight phthalates, DMP, followed by DEP, DBP, DEHP, DnOP, DiNP and DiDP, which is the highest molecular weight of phthalates.

Fraction Collector

Autosampler

Waters Envirogel column

Dual λ absorbance detector

Isocratic HPLC pump20130123_140351.jpg

Figure 9. The components of the GPC system.

An overview of GPC parameters was shown in Table 4. The wavelength is set at 230nm. The mobile phase used in GPC is HPLC grade Dichloromethane at a flow rate of 3mL/min. The pressure now is around 94-118 psi. The columns used are Envirogel column with 1 short and 1 long column at 19x150mm and 19x300mm respectively. The Envirogel columns are packed with high performance, fully porous, highly cross-linked styrene divinylbenzene copolymer particles. The injection volume is 500µL with total run time for 30 minutes.

Table 4. Gel Permeation Chromatography parameters.

GPC parameters

Settings

Wavelength

230 nm

Mobile phase

HPLC grade Dichloromethane

Flow rate

3 mL/min

Pressure

94 -118 psi

Column

1 short + 1 long

Short - 19 x 150mm

Long - 19 x 300mm

Envirogel column - styrene divinylbenzene copolymer particles

Injection volume

500 µL

Total run time

30 minutes

4.2 Determination of dilution factor and retention time of corn oil

Mazola corn oil was used. A series of dilution factor of corn oil was prepared. 10x, 30x, 60x 80x and 100x dilution were made by dissolving 1ml, 0.33ml, 0.17ml, 0.13ml, and 0.1ml respectively with AR grade Dichloromethane in 10mL volumetric flask. The density of corn oil was found to be 0.922. All the glass wares used were rinsed with Dichloromethane at least three times to remove impurities. The samples were injected two times and run under the same GPC parameters.

20130123_152536.jpg

Figure 10. Corn oil sample taken from Mazola.

Table 5. Concentration of corn oil from a series of dilution factor.

Dilution Factor

Amount of corn oil added

Concentration (ppm)

10 x

1.00 mL

~ 92200 ppm

30 x

0.33 mL

~ 30426 ppm

60 x

0.17 mL

~ 15674 ppm

80 x

0.13 mL

~ 11986 ppm

100 x

0.10 mL

~ 9220 ppm

4.3 Determination of the LOD of phthalates in GPC

A series of standard solutions containing of 7 phthalates were prepared. 0.5ppm, 1ppm, 2ppm, 3ppm, 4ppm, 5ppm, 6ppm, 7ppm, 8ppm, 9ppm and 10ppm of solutions containing 7 phthalates were made by dissolving in AR grade Dichloromethane to 10mL of volumetric flask. The samples were run under the same GPC parameters except that the injection volume is 8000 µL.

Detection limits are defined as the amount or concentration of analyte that enough to give a response (YDL) that is significantly different (three standard deviation (SDBR)) from the background response (OSHA, 1993).

YDL - YBR = 3(SDBR)

According to OSHA (1993), SDBR and data precision about the curve if are similar, the standard error of estimate (SEE) can be substituted for SDBR in the above equation. The following equations derive a formula for DL.

Where,

Yobs = Observed response

Yest = estimated response from regression curve

n = total number of data point

Thus, at point YDL on the regression curve

YDL = A(DL) + YBR

A = slope

Therefore,

DL = (YDL - YBR) / A

Substituting 3(SEE) + YBR for YDL, as a result

DL = 3(SEE) / A

4.4 Determination of the retention time of phthalates and construction of calibration curve of phthalates analyzed by GCMS

To identify the retention time of phthalates using GCMS, 50ppm of each DMP, DEP, DBP, DEHP, DnOP, DiNP and DiDP prepared stock solutions were used. Different concentration of standard phthalates mixture 0.5ppm, 1ppm, 5ppm, 11ppm, 15ppm, 20ppm and 25ppm were prepared. Micropipette was used to transfer required amount of phthalates from 50ppm phthalates stock solution and were further dissolved using AR grade Dichloromethane to 10mL of volumetric flask. All the glass wares used were rinsed at least three times with Dichloromethane to remove unnecessary impurities. The samples to be analyzed were filled into blue cap vial before GCMS analysis. The retention time of each phthalates was obtained and calibration curves were drawn from the data obtained after the analysis.

Correlation was calculated for each of the phthalate esters to determine whether the there is a linear dependence between y and x axis (peak area and concentration). If the correlation calculated is closely or equal to 1, it shows that there is a strong linear dependence. The correlation was calculated as follow.

pearsons.gif

An overview of GCMS parameters was shown in Table 6. The mass spectrometry's (MS) ion source and quadrupole analyzer temperature were maintained at 230°c and 150°c respectively. The detection of different phthalates compounds in MS took place in selected ion monitoring (SIM) mode. In the SIM mode, target ions of phthalates were monitored, maintaining at a dwell time of 30 ms for each ion.

Table 6. Gas Chromatography-Mass Spectrometry parameters.

GCMS Parameters

Settings

Injection Port Temperature

300°c

Front PTV Inlet

Splitless mode

Injection volume

1µL

Transfer Line Temperature

(Auxillary temperature)

325°c

Carrier gas-helium flow

1 mL/min

Column (Capillary Column)

DB-XLB

Column Diameter

30m (length) x 0.25mm (internal diameter) x 0.25µm (film thickness)

Oven program

100°c for 0 minutes

35°c/min to 300°c for 5 minutes

Total run time

10.714 minutes

MS Acquisition mode

SIM mode (Selected Ion Monitoring)

Solvent delay

2.5 minutes

EMV mode

Gain Factor

Table 7. The Quantification Ions and Identification Ions for each Standard Phthalate.

Phthalates

Quantification Ions (m/z)

Identification Ions (m/z)

DMP

163

194

DEP

149

177,222

DBP

149

205,223

DEHP

149

167,279

DnOP

149

167,279

DiNP

149

167,293

DiDP

149

167,307

4.5 Determination of the minimum detection limit of phthalates in GCMS

The purpose of determining the minimum detection limit (MDL) of phthalates is to identify the lowest concentration of phthalates that give no response in GCMS where peak area = 0. Calibration curve of each phthalates obtained from GCMS was referred. By using 'trend' function from excel, minimum detection limit of phthalates was determined. Figure 10 show that minimum detection limit is obtained from the x-interception.

Peak Area

Minimum Detection Limit

Concentration

Figure 11. Sketched graph shows where MDL can be obtained.

4.6 Determination of the LOD of phthalates in GCMS

A series of standard solution containing 7 phthalates were prepared. 0.5ppm, 1ppm, 2ppm, 3ppm, 4ppm, 5ppm, 6ppm, 7ppm, 8ppm, 9ppm and 10ppm of 7 phthalates mixture were prepared and dissolved in AR grade Dichloromethane to 10mL of volumetric flask. Each sample injected for six times under same GCMS parameters.

4.7 Determination of suitable spiked phthalate concentration in corn oil

1000mg/L of phthalates solution containing 7 phthalates was prepared by dissolving 0.1g of each phthalate in 100mL of methanol. The weight of each phthalate was measured using analytical balance to 0.10 ± 0.02g. After adding all the phthalates, methanol was added to 100mL of volumetric flask. The concentration of all the phthalates were around 1000ppm.

Table 8. Concentration of each phthalate in mixture.

Phthalates

Weight (g)

Concentration in 100mL Methanol (ppm)

DMP

0.1000

1000

DEP

0.1178

1178

DBP

0.1139

1139

DEHP

0.1063

1063

DnOP

0.1064

1064

DiNP

0.1010

1010

DiDP

0.1045

1045

2mg/kg, 6mg/kg, 10mg/kg, 16mg/kg, 20mg/kg of spiked phthalates in corn oil were prepared by micropipette 100µL, 300µL, 500µL, 800µL and 1000µL of phthalates respectively to 50g of corn oil in blue cap bottles.

20mg/kg

16mg/kg

10mg/kg

6mg/kg

2mg/kg20130123_160044.jpg

Figure 12. Spiked phthalate concentration in corn oil.

10x dilution was made with AR grade Dichloromethane for each concentration. And the concentration of spiked phthalates in corn oil is now 0.2mg/kg, 0.6mg/kg, 1.0mg/kg, 1.6mg/kg and 2.0mg/kg.

4.8 Separation of corn oil from phthalates using GPC clean up

The samples were injected to GPC with injection volume of 8000µL, to separate corn oil from phthalates under the same GPC parameters. Then, the eluted sample was collected, kept in COD tubes and stored in refrigerator. Each sample was repeated for three times in this procedure.

4.9 Preconcentration of samples blown with nitrogen gas using Visidry Drying Attachment

Each of the eluted samples was blown with nitrogen gas using Visidry Drying Attachment to 1mL under 10psig. After preconcentration, the sample was total transferred to blue cap vial to prevent any sample remaining in the COD tube by rinsing with AR grade Dichloromethane few times. The samples were stored in refrigerator for further analysis.

4.10 Determination of recovery

A series of control sample was prepared with same amount of phthalates (as spiked in corn oil) spiked into 50mL methanol. Thus, 2mg/L, 6mg/L, 10mg/L, 16mg/L and 20mg/L of spiked phthalates in methanol were prepared by micropipette 100µL, 300µL, 500µL, 800µL and 1000µL of phthalates respectively into 50mL methanol in blue cap bottles.

20mg/L

16mg/L

10mg/L

6mg/L

2mg/L20130123_160447.jpg

Figure 13. Spiked phthalate concentration in methanol.

All the control samples were then diluted half and become 1mg/L, 3mg/L, 5mg/L, 8mg/L, 10mg/L respectively. Then, it is proceed with GCMS analysis of preconc samples and control samples. The recovery is calculated as followed.

100%

B

A

x

Recovery =

Where,

B = data obtained from corn oil

A = data obtained from methanol

under each concentration

5.0 RESULTS AND DISCUSSION

Phthalates are very important and they are widely used in industry. The major application of phthalates is they serve as plasticizer to soften plastic. They are found mainly in PVC and of course in others consumer products even found in food. They are lipophilic and leach into environment inattentively because they are not covalently bound to plastic. Moreover, phthalates may cause a lots of health problem, it may cause reproductive defects on both male and female, breathing difficulties, chronic effects on kidney and liver. As a result, this showed that people are actually exposed to higher risk of phthalates since they exist everywhere, in the consumer products which are our daily requirement actually.

In this analysis, the standard phthalates that were being used are DMP, DEP, DBP, DEHP, DnOP, DiNP and DiDP. However, DiNP and DiDP were unable to be detected using GCMS even higher concentrations were applied. The analysis was started with determination of phthalates in GPC as well as GCMS, followed with separation of phthalates from corn oil by GPC clean up, the eluted sample was then blown with nitrogen gas to preconcentration the sample and lastly followed by GCMS analysis.

5.1 Identify the retention time of phthalates using GPC

An overview of retention time and peak area of each phthalate ester is shown in Table 9. Lower molecular weight phthalate esters have a longer retention time compared to higher molecular weight. The lowest molecular weight phthalate, DMP has a longer retention time, followed by DEP, DBP, DEHP, DnOP, DiNP and lastly by largest molecular weight phthalate, DiDP. The retention time range of phthalate esters falls in between 21-24.5 minutes. Thus, compound that is not fallen between this range is assumed not a phthalate ester.

Table 9. Retention time and peak area of phthalates by GPC.

Phthalates

Molecular Weight

Concentration (ppm)

Retention time, tR (min)

Peak Area

DMP

194.19

50

24.396

24.261

6184717

6420710

DEP

222.24

50

23.761

23.710

5983353

6059991

DBP

278.34

50

22.412

(ii) 22.462

6513320

6296764

DEHP

390.56

50

21.424

(ii) 21.642

5375390

5079493

DnOP

390.56

50

21.135

(ii) 21.374

4583404

4403730

DiNP

418.62

50

21.027

(ii) 21.053

5100366

5077065

DiDP

446.66

50

20.905

(ii) 20.906

4662185

4800366

GPC is a size exclusion chromatography, separates sample molecules based upon their relative size in solution. The larger molecular weight tends to retain in the column at the shorter time, thus eluted out fast while low molecular weight is in the reverse way. Therefore, this results support the principle of GPC since DiDP, the largest phthalate has a shorter retention time because it tends to retain in the packing materials of column for a shorter period and thus elute out faster from the column than that of lower molecular weight phthalates. It also removes non-volatile, high molecular weight components such as lipids, proteins, natural resins and cellular components. It is also appropriate for a broad range of analytes, polar and non polar (Joe Romano, 2005).

A recent study reported that the pore of the packing materials is much more effectively accessible for small molecule than for large molecule. Therefore, larger molecules have shorter retention time compared to that of smaller molecule and thus eluted out from the column earlier and reach the detector (Kostanski et al., 2004).

In terms of GPC parameter, wavelength of 230nm was used due to the occurrence of phthalates absorption. All phthalates consists of a benzene ring. This is believed that absorption occur at 230nm is due to the presence of benzene ring. The flow rate of 3mL/min instead of 5mL/min was used is to reduce the consumption of HPLC grade Dichloromethane since 3mL/min is also able to show a satisfied result although a larger flow rate may show a better separation. 1 short and 1 long column were used to have a better separation of phthalates because the peaks of phthalates tend to overlap within each other. Total run time of each sample was set to be 30 minutes since the retention time range of phthalate esters falls between the ranges of 21.0 - 24.5 minutes.

5.2 Determination of dilution factor and retention time of corn oil

A dilution factor of oil is made to ensure that the peak area of oil do not overlap or do not cover up the phthalates. Table 10 shows the retention time and peak area of corn oil. This is believed that the retention time of corn oil fall between 18.4 - 19.1 minutes. Since phthalates and corn oil fall between different range of retention time (phthalates = 21.0 - 24.5 minutes, corn oil = 18.4 - 19.1 minutes), this assumes that phthalates are able to be separated from corn oil by GPC clean up.

The higher the concentration, the larger the peak area. Thus, to ensure that the phthalate esters able to be separated from the corn oil, 100x dilution factor is chosen since it able to show result significantly and to reduce the overlapping of corn oil's peak area from phthalates' peak area. A reduced concentration can also be taken into consideration as long as retention time of corn oil is identified.

Table 10. Retention time and peak area of corn oil by GPC.

Dilution Factor

Concentration (ppm)

Retention time, tR (min)

Peak Area

10x

~ 92200ppm

18.410

18.411

73137480

73266590

30x

~30426ppm

18.207

18.183

26644954

26066253

60x

~15674ppm

18.122

18.122

12944727

12453795

80x

~11986ppm

18.093

18.083

9508489

9359980

100x

~9220ppm

19.043

19.006

8614628

8936637

5.3 Determination of the LOD of phthalates in GPC

An overview of data for determination of LOD as well as retention time of phthalates in mixture is shown in Table 11. From the data obtained, the peak area becomes larger corresponding to increasing concentration. The graph has a correlation of 0.995522 which is very close to 1. Thus, this shows that there is linear dependence between peak area and concentration.

The SEE is stand for standard error of estimate according to OSHA (1993). The SEE is calculated. The SEE calculated is 3676538, and thus LOD is calculated from the SEE obtained by using formula, DL = 3(SEE) / A.

LOD calculated is as low as 0.07821ppm. This indicates that in a change of 0.07821ppm of phthalates, a response that is significantly different from the background response can be observed. In other words, a significant change in peak area can be observed if there is a change of 0.07821ppm of phthalates.

Table 11. Data to determine LOD and retention time of phthalates in mixture by GPC.

Sample

Concentration (ppm)

Retention time, tR (min)

Peak area

1

0.5

24.200

5335052

2

1

22.434

8261662

3

2

23.782

18066580

4

3

23.824

33170811

5

4

23.838

48588960

6

5

23.911

57906061

7

6

23.981

71708277

8

7

23.723

82653536

9

8

23.818

93132647

10

9

23.786

97699245

11

10

23.793

107785186

Correlation

0.995522

STEYX (SEE)

3676538

Slope

11323812

LOD

0.07821

5.4 Determination of the retention time of phthalates and construction of calibration curve of phthalates analyzed by GCMS

An overview of retention time of each phthalate by GCMS is shown in Table 12. The retention time of DMP is around 3.74min, DEP is around 4.21min, DBP is around 5.36min, DEHP is around 7.06min and DnOP is around 7.89 min.

Table 12. Retention time of each phthalate by GCMS.

Phthalates

Retention time, tR (min)

DMP

3.74

DEP

4.21

DBP

5.36

DEHP

7.06

DnOP

7.89

Compound which has lower molecular weight will be the first to be ignited by the flame of GC compared to those of higher molecular weight. Sample introduced into GC inlet vaporized in gas phase and swept onto the column by Helium carrier gas and thus separation of compound occurs on column. Sample compounds emerge from the column is directed into the mass spectrometer and thus spectrum of each compound is determined as it emerges from the column. The identification of compound on mass spectrum relies on its own unique fragmentation pattern. As a result, DMP, which is the lowest molecular weight phthalate was first detected, shortest retention time compared to larger molecular weight phthalates.

Table 13. Correlation of each phthalate by GCMS.

Phthalates

Correlation

DMP

0.999178

DEP

0.997181

DBP

0.997269

DEHP

0.994822

DnOP

0.997496

From the results shown in Table 13, the calibration data for DMP, DEP, DBP, DEHP and DnOP have a correlation very close to 1. Thus, this indicates that there is a linear dependence between peak area and concentration. The peak area and concentration is closely related.

5.5 Determination of the minimum detection limit of phthalates in GCMS

The calibration data of each phthalate obtained from GCMS was referred. The minimum detection limit is believed to fall at x-intercept. The minimum detection limit is the lowest concentration of phthalates that give no response in GCMS whereby the peak area is zero. In other words, the minimum detection limit is the minimum concentration that can be determined by GCMS, beneath or below the minimum detection limit, GCMS unable to determine anything, gives no response. Thus, from the calibration data that have plotted, the MDL of DMP is 0.140697ppm, concentration that below 0.140697ppm, GCMS gives no response; while DEP is 0.407296ppm , DBP is 0.268514ppm , DEHP is 0.850789ppm , DnOP is 0.858423ppm, as tabulated in Table 14.

Table 14. Minimum detection limit of each phthalate by GCMS.

Phthalates

Minimum detection limit (ppm)

DMP

0.140697

DEP

0.407296

DBP

0.268514

DEHP

0.850789

DnOP

0.858423

5.6 Determination of the LOD of phthalates in GCMS

The sample was injected six times, thus average of data is calculated to determine the LOD of phthalates in GCMS. From the data obtained, SEE of each phthalate is calculated from the plotted graph. The SEE of DMP is 596688.6, DEP is 473004.9, DBP is 497646.3, DEHP is 95302.96 and DnOP is 95367.52.

An overview of data for determination of LOD of phthalates is shown in Table 15. LOD calculated for DMP is as low as 0.013874ppm. This indicates that in a change of 0.013874ppm of phthalates, a response that is significantly different from the background response can be observed. While LOD for DEP is 0.269017ppm, DBP is 0.322732ppm, DEHP is 0.126286ppm and DnOP is 0.429182ppm.

From the data obtained, the peak area becomes larger corresponding to increasing concentration. The graph of each phthalate has a correlation very close to 1. Thus, this shows that there is linear dependence between peak area and concentration.

Table 15. LOD of each phthalate by GCMS.

Phthalates

LOD

DMP

0.013874

DEP

0.269017

DBP

0.322732

DEHP

0.126286

DnOP

0.429182

5.7 Determination of recovery

In order to determine recovery, various procedures were taken. Firstly, is to determine the suitable spiked phthalate concentration in corn oil. A 1000mg/L containing 7 phthalates was prepared as a stock solution, to spike a suitable amount into corn oil.

1mg/L, 3mg/L, 5mg/L, 8mg/L and 10mg/L were the concentration that needed to be determined for recovery test. As a result, 2mg/kg, 6mg/kg, 10mg/kg, 16mg/kg and 20mg/kg of spiked phthalates in 50g corn oil were prepared. A greater amount of corn oil (>50g) was not taken into account to reduce the wastage of oil, while a smaller amount of corn oil was not chosen due to difficulties of transfer spiked phthalates into corn oil as the amount of spiked phthalates will be really small and to avoid any errors that might occur.

A 10x dilution was done to dilute 2mg/kg, 6mg/kg, 10mg/kg, 16mg/kg and 20mg/kg into 0.2mg/L, 0.6mg/L, 1mg/kg, 1.6mg/L and 2.0mg/L. Then, the samples were injected into GPC for the purpose of separate corn oil from phthalates. The flow rate of mobile phase is 3mL/min. And the total time for the phthalates peak to elute out is 8.233 minutes. Thus, the eluted sample will be approximately 24.699mL. The eluted sample is believed to dilute the injected samples by half. As a result, after the treatment of GPC clean up, the samples now are believed to have 0.1mg/L, 0.3mg/L, 0.5mg/L, 0.8mg/L and 1.0mg/L. In order to achieve 1mg/L, 3mg/L, 5mg/L, 8mg/L and 10mg/L for recovery test, thus the samples were subjected to preconcentration using Visidry Drying Attachment blown with nitrogen gas at 10psig to 1mL.

Besides that, a series of control samples was also prepared with same amount of phthalates spiked into 50mL methanol as corresponding to corn oil. They were also diluted half before undergoing GCMS analysis. . Since both corn oil preconc samples and methanol control samples are in same concentration, therefore, the recovery is calculated for each phthalate after undergoing GCMS analysis.

The recovery of each phthalate is required to be high. This is because high recovery validates the accuracy of the analytical technique for each phthalate. If the recovery is low, there is many possibilities bring up to this problem. For example, the phthalates may retain in the GPC column without clean up properly, the phthalate esters may also lost when transferring into COD tubes or transferring into blue cap vial for GCMS analysis, imperfect of total transfer may also arise the problem. There is also a tendency whereby the phthalates remain in the GCMS column or PTV inlet during analysis.

6.0 FUTURE STUDIES

In future studies, the recoveries test as mentioned as well as the determination of the real sample under the same treatment without spiking any phthalates have to be completed. Studies reported that DBP, DEHP, DnOP, DiNP and DiDP had high recoveries and relative standard deviations (RSD) value which were treated with GCMS analysis (Zou & Cai, 2011). According to Zou & Cai (2011), the DiNP and DiDP were determined at concentration level (spiked amount) of 50mg/L and 100mg/L, while other phthalates were determined at concentration level (spiked amount) of 5mg/L and 10mg/L with excellent recoveries ranging from 92% to 107% and RSD less than 7%. Besides that, U.S. Consumer Product Safety Commission reported that the permissible level (maximum contaminant level) of phthalate esters in food products should not more than 0.1%. (CPSC, 2009).

7.0 CONCLUSION

Phthalates play an important role in industry level. It is most commonly used as a plasticizer to soften plastic, increase flexibility of plastic. The consumer products nowadays are mostly made of plastic due to high durability with low cost, bulk production and wide application. Thus, since most of the consumer products are our daily requirement, people are hardly avoided from exposure to phthalates.

In this research project, the standard phthalates that were being used are DMP, DEP, DBP, DEHP, DnOP, DiNP and DiDP. The analysis was started with determination of phthalates in GPC. GPC is a size exclusion chromatography, which separates sample molecules based upon their relative size in solution. The larger molecular weight tends to retain in the column at the shorter time, thus eluted out fast while low molecular weight is in the reverse way. DiDP elutes out first followed by DiNP, DnOP, DEHP, DBP, DEP and lastly DMP. The retention time range of phthalates is between 21-24.5minutes, while retention time range of corn oil is between 18.4-19.1minutes. Since both phthalate esters and corn oil have different retention time and the peak area are not overlapping each other, thus phthalates are successfully separated from corn oil by GPC clean up. The LOD of phthalates in GPC is 0.07821ppm.

GCMS analysis was also started with determination of retention time of each phthalates. However, DiNP and DiDP were unable to be detected using GCMS even higher concentrations were applied. The principle of GCMS is in the opposite of GPC whereby compound with lower molecular weight will be the first to be detected compared to those of higher molecular weight. Thus, DMP has the shortest retention time, followed by DEP, DBP, DEHP and lastly DnOP. The minimum detection limit of phthalates falls between 0.140697 - 0.858423ppm. Therefore, below the range of minimum detection limit (0.140697 - 0.858423ppm), GCMS unable to determine anything, and gives no response. The LOD of phthalates falls between 0.013874 - 0.429182ppm.

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