Analysis of mineral oil hydrocarbons in consumer products using … · 2020-03-13 · Aromatic Hydrocarbons (MOAH), with regards to their toxicity, an individual analysis is needed

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Bachelor Thesis Chemistry

Analysis of mineral oil hydrocarbons in consumer

products using silver-phase liquid chromatography-gas

chromatography

author

Roxane Biersteker

30th of June 2017

Studentnumber 10808272

Research institute Supervisors

Van ‘t Hoff Institute for Dr. W.T. Kok & Prof. dr. ir. J.G.M.

Molecular Sciences Janssen

Research group Daily Supervisor

Analytical Chemistry Research Group Mr. A. R. García MSc

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Index

1. Abstract 3

2. Introduction 4

3. Experimental section

3.1 Equipment 9

3.2 Characterization

3.2A HPLC separation 9

3.2B Analysis by GC-FID 11

3.3 Quantification

3.3A Solid Phase Extraction 11

3.3B HPLC separation 11

3.3C Analysis by GC-FID 12

4. Results


4.1A Separation between MOSH and MOAH internal standards 13

4.1B Characterization of the mineral oils 14

4.2 Quantification 18

5. Discussion

5.1 Characterization: separation between MOSH and MOAH

5.1A Ag/Silica column 19

5.1B Ag/Polymer column 19

5.1C Comparison columns 20

5.2 Characterization: classification aromatic compounds

5.2A Ag/Silica column 21

5.2B Ag/Polymer column 21

5.2C Comparison columns 22

5.3 Quantification 22

6. Conclusion 23

7. Outlook 24

8. References 25

9. Appendices 27

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1. Abstract

Mineral oil hydrocarbons are present in almost all food as a consequence of contamination and

numerous intentional uses in the production process. Due to the difference between the main

components of mineral oil, Mineral Oil Saturated Hydrocarbons (MOSH) and Mineral Oil

Aromatic Hydrocarbons (MOAH), with regards to their toxicity, an individual analysis is needed.

In this research the first steps were taken towards the development of a new method, based on

silver-phase liquid chromatography- gas chromatography, for the quantification and

characterization of mineral oil hydrocarbons in consumer products. Analysis of internal standards

using comprehensive LCxGC-FID, showed that a separation of around 1 minute between MOSH

and MOAH markers was established. However, analysis of the LCxGC-FID chromatograms of

three mineral oil samples with different grades of refinement showed that compounds elute in the

timeframe between the MOSH and MOAH markers. Regarding the mineral oil characterization, a

Ag/Polymer column was more suited to separate aromatics into groups than a Ag/Silica column,

which is possibly a result of the length of the column. Finally, results showed that the LC-GC

method is reliable for determining the amount of aromatics in mineral oils for both columns.

Further research should be undertaken to determine whether a complete separation between MOSH

and MOAH was established, for example by analyzing the fractions by GC-VUV.

In bijna elk voedsel zitten koolwaterstoffen die afkomstig zijn uit minerale olie. Deze kunnen slecht

voor de gezondheid zijn en daarom is een analyse methode nodig die deze koolwaterstoffen

detecteert. De koolwaterstoffen kunnen verdeeld worden in twee groepen, genaamd MOSH en

MOAH. MOSH bevat onverzadigde koolwaterstoffen en MOAH bevat verbindingen met

aromatische ringen. Aangezien de MOAH kankerverwekkend kunnen zijn, is het belangrijk dat

deze apart van de MOSH gedetecteerd kunnen worden. In dit onderzoek zijn drie minerale oliën

gekarakteriseerd en is het gehalte MOAH bepaald aan de hand van vloeistofchromatografie en

gaschromatografie. Door het gebruiken van zilver in de stationaire fase van de

vloeistofchromatografie kolom, was de verwachting dat een betere scheiding tussen MOSH en

MOAH bereikt zou kunnen worden dan eerder mogelijk was. Een betere scheiding tussen simpele

alkanen en aromatische verbindingen was mogelijk, maar bij het analyseren van de minerale oliën

bleek dat er maar een kleine scheiding tussen de MOSH en de MOAH zat. De resultaten van de

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drie methodes die gebruikt zijn voor het vaststellen van het gehalte MOAH in de minerale oliën,

kwamen sterk overeen. Dit suggereert dat de methodes betrouwbaar zijn, maar meer onderzoek

moet gedaan worden om vast te stellen dat de MOSH en MOAH compleet gescheiden zijn en om

de scheidingsmethode te verbeteren. Zodra de methode geoptimaliseerd is, zullen voedsel en

cosmetica producten geanalyseerd worden.

2. Introduction

Mineral oil hydrocarbons (MOH) are present in almost all foods as a consequence of contamination

and numerous intentional uses in the production process.1 MOH are mixtures of hydrocarbons

consisting of thousands of chemical compounds of varying size and structures. They are primarily

derived by physical separation and chemical conversion processes such as hydrogenation and

alkylation from crude oils, but also from synthetic products that are derived from liquefaction of

natural gas, biomass and coal. MOH consists of three classes of compounds: paraffins, naphthenes

and aromatics (Fig. 1), and they are usually highly alkylated. Two types of MOH are distinguished,

namely mineral oil saturated hydrocarbons (MOSH) and mineral oil aromatic hydrocarbons

(MOAH). The former consists of linear and branched alkanes as well as alkyl-substituted

cycloalkanes (paraffins and naphthenes), the latter include alkyl-substituted polyaromatic

hydrocarbons (aromatics).

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Figure 1: Alkanes (paraffins), naphthenes and aromatics1

Foods that contain the highest mean concentration of MOH include fish products, oilseeds,

confectionery and vegetable oil.2–5 To illustrate, in fresh- and sea-water fish MOSH concentrations

up to 1200 mg/kg fat were found, with a mean value around 200 mg/kg fat.6 Analysis of edible

oils showed that a large proportion were contaminated with more than 10 mg/kg mineral paraffins

with a maximum concentration of around 400 mg/kg.7,8 For rice a mean MOSH concentration is

132 mg/kg and the expected percentages of MOAH in MOH 15–30%. Other possible sources for

MOH in rice and the expected proportion affected are depicted in Figure 2.1

Figure 2: Sources of MOH which potentially contaminate rice: percentages of samples affected against the expected concentration1

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As shown in Figure 2, a source for MOH in food is packaging materials, especially those that are

made from recycled board and paper. Moreover, printing inks and MOH that are used as additives

in plastics contribute to MOSH levels, together with additives that are directly used in the

processing of food. Further uses of MOH are as release agents for sugar products and bakery ware

or surface treatment of foods such as confectionery.

The above mentioned sources of MOH result in an estimated dietary exposure of MOSH between

0.03 and 0.3 mg/kg b.w. per day.1 The background exposure to MOAH is estimated at 20%, with

respect to MOSH, although there has been little quantitative analysis of this group of hydrocarbons.

These estimations do not take into account that lip care products and lipsticks are also a possible

source of exposure, since a large part of these products end up being ingested. A study that was

conducted in 2015 showed that 68% of the products that were tested contained at least 5% MOSH

and synthetic hydrocarbons (POSH), and 31% contained more than 32% of these compounds.9 As

a consequence, it is likely that regular application of these products result in a significant increase

in exposure to MOH.

There is little information available on the toxicity and absorbance of MOH in mammals, especially

data on MOAH are scarce. Hydrocarbons from about ten to fifty carbon atoms are considered in

toxicology studies, since hydrocarbons with less than 10 carbon atoms are highly volatile, and

hydrocarbons with more than fifty carbon atoms are improbable to be absorbed following ingestion.

Previously published studies on the effect of MOSH are not consistent, although there is

concordance with the fact that they are mostly absorbed from the gut into the lymphatic system.10–

13 In rats, the estimated absorption of n- and cycloalkanes varies from 25% (C26-C29) to 90% (C14-

C18).14–18 In humans, mineral oil has been found in the spleen, liver and lymph nodes as well as in

body fat collected during Caesarian section.19,20 Surprisingly, the mineral paraffins of all body fat

samples that were analyzed had a highly similar molecular mass distribution; they are centered on

n-C23/n-C24 and range from n-C16 to n-C30. Since this pattern does not resemble a particular mineral

oil product, it suggests that selection takes place regarding absorbance of hydrocarbons. This could

be by effective elimination of certain hydrocarbons and limited absorption of heavier compounds.

Figure 3 shows the chromatogram of a typical sample, in which MOSH is mainly represented by

the broad hump (unresolved peaks).

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Figure 3: Chromatogram of MOSH from human abdominal body fat (85 mg/kg).20

Without specific removal of MOAH, all MOH are mutagenic. Especially three to seven ring

MOAH cause the mutagenicity, even more so when they have short side chains.21,22 They can be

activated by P450 enzymes into genotoxic carcinogens, which can be followed by the formation of

DNA adducts.23,24 Although MOSH are not carcinogenic, long chains in high amounts could act as

tumor promoters.25–29 Furthermore, some uncomplicated MOAH, e.g. naphthalene, are

carcinogenic by a non-genotoxic mode of action. Due to these differences between the MOSH and

MOAH, with regards to their toxicity, individual analysis is needed.

The determination of MOSH and MOAH has been proven to be challenging, since they form

irregular humps of unresolved peaks in gas chromatograms.30 The first on-line coupled

high performance liquid chromatography–gas chromatography method was developed in 1991 and

was improved throughout the years.31 A huge drawback of these methods was that they were not

able to detect MOAH separately. The first simple method for determining MOSH and MOAH

separately appeared in 2009.32 GC with flame ionization detection (GC-FID) is used since it

provides practically the same response per mass for components of structures that are similar. As

a consequence, quantification is possible without pure standards. MOSH and MOAH are of the

same mineral oil fraction and thus of the same volatility range, meaning that selectivity must come

from preseparation. For this, high performance liquid chromatography (HPLC) with a silica gel

column is used, since silica provides strong retention for aromatic hydrocarbons. Internal standards

are used to establish and check the fraction windows. Analysis showed that a gap of thirty seconds

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was established between MOSH and MOAH. One of the main obstacles is the limit of

quantification of 1 mg/kg, since for polycyclic aromatic hydrocarbons more than 100 times lower

legal limits are enforced. This means that if the MOAH consists of 1% carcinogenic material, a

detection limit of 1 mg/kg is not satisfactory. Therefore, it is fundamental to develop a method to

detect MOAH below this limit. Moreover, the current method presents some issues regarding to

the separation between MOSH and MOAH. Due to the small separation window between MOSH

and MOAH, the analysis becomes problematic when the content of the MOAH fraction is less than

1 %, in relation to the total amount of mineral oil hydrocarbons.

Using a comprehensive LCxGC method for the analysis of mineral oil solves the issues mentioned

before. This method is based on the collection of many fractions during the LC separation and their

subsequent GC analysis. This allows to confirm whether the separation window is sufficient for

the MOSH-MOAH separation of real samples and, with the appropriate LC conditions, to do a

characterization of the mineral oil hydrocarbons based on polarity and molecular weight

distribution. This information is useful to know which type of aromatic compounds, regarding to

the number of rings and alkylation level, are present in the sample. The standard quantitative

separation of MOSH and MOAH using solid phase extraction uses silver ion-silica as a solid

phase.33,34 This is because silver has strong favorable interactions with double bonds, and thus

aromatics are more retained that aliphatic compounds. However, no previous studies have been

reported that apply this stationary phase in LC-GC-FID.

In this research the first steps are taken to develop a new method, based on silver-phase liquid

chromatography- gas chromatography, for the quantification and characterization of mineral oil

hydrocarbons in consumer products. The main objective of this study is to establish a separation

between Mineral Oil Saturated Hydrocarbons and Mineral Oil Aromatic Hydrocarbons, and to

classify aromatic compounds in terms of the number of rings. Furthermore, the aliphatic and

aromatic content of the mineral oil samples is determined and compared to the standard method

based on Solid Phase Extraction. Two different silver-phase LC columns, one silica bonded and

one ion-exchange, are tested, due to their specific interaction with π-bonded compounds. The

method is applied to the analysis of three mineral oils with different grades of refinement.

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3. Experimental section

3.1 Equipment

- HPLC Waters 2696

- Photodiode Array Detector Waters 996

- Ag/Silica column Agilent Chromsep SS (length 10 cm, diameter 4.6 mm)

- Ag/Polymer column Chrompack Chromspher pi (length 25 cm, diameter 4.6 mm)

- GC-FID Agilent 6890N

- DB- 5HT column J&W Scientific (length 15 m, diameter 0.32 mm, film 0.1 μm)


3.2A HPLC separation

In all samples internal standards (Sigma Aldrich) were used for establishing and checking the

fraction windows (Fig. 4).30 5-α-Cholestane was used to establish the end of the MOSH fraction

and tri-tert-butyl benzene marked the beginning of the MOAH fraction. 1-Methylnapthalene and

perylene were used to mark the end of the MOAH fraction. Perylene was checked by UV detection,

but was not added to the samples that were analyzed due to solubility problems in hexane. Besides

these markers, internal standards were added for the verification of adequate method performance

and for the quantification (Appendix 3). The internal standards were injected in the LC to establish

the separation between MOSH and MOAH.

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Figure 4: Markers that were used to establish and check the fraction windows.30

For the characterization of and separation between MOSH and MOAH three mineral oils were

analyzed, for which two different LC columns were used. The samples were dissolved in hexane

and injected in the LC, and fractions of twenty seconds were collected. For both columns, Table 1

shows the concentrations of the samples, the method and injection volume as well as the collection

time. The eluents that were used are hexane and dichloromethane (DCM) (Biosolve Chimie

SARL); details of the LC methods are given in Appendix 1.

Table 1: LC methods and concentrations of samples analyzed for the characterization

Column conc.

MO

(mg/mL)

method injection

volume

(µl)

collection

time

(min)

Ag/Silica 50 9 40 4.00 – 25.00

Ag/Polymer 50 21 40 4.00 – 25.00

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3.2B Analysis by GC-FID

After the fraction collection the samples were injected (1 μL) into the GC-FID (splitless technique,

2 min., split 100:1). The oven temperature was programmed at 15 °C /min from 60 °C (3 min) to

350 °C (3 min). The carrier gas was helium at a flow rate of 2 mL/min. The data was processed by

the program Wolfram Mathematica.

3.3 Quantification

3.3A Solid Phase Extraction

Quantitative separation of aliphatic and aromatic hydrocarbons was carried out using silver ion-

silica solid-phase extraction.34,35 Ag/Silica (0.5 g) (Sigma Aldrich) was loaded into an cartridge

using two retaining frits and placed in an oven (120 °C, 2 h) for activiation. The Ag/Silica was

washed using dichloromethane (10 mL) followed by a conditioning step with hexane (4 mL). The

sample (0.5 mL, 20 mg/mL) including internal standards (0.006 mg/mL) was loaded onto the

column. The aliphatic hydrocarbons are eluted with hexane (5 mL) and the aromatic hydrocarbons

with hexane/dichloromethane (1:1, 6 mL) followed by dichloromethane (2 mL). The MOSH and

MOAH fractions were analyzed using GC-FID (section 3.2B).

3.3B HPLC separation

For the quantification three mineral oils (MO) were analyzed, for which two LC columns were

used. The samples were dissolved in hexane and injected in the LC, after which the MOSH and

MOAH fractions were collected. Table 2 (Ag/Silica column) and Table 3 (Ag/Polymer column)

show the concentrations of the samples and internal standards, the method and injection volume

as well as the collection time of the fractions. The eluents that were used are hexane and

dichloromethane (DCM); details about the LC methods are listed in Appendix 1.

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Table 2: LC methods and concentrations of samples analyzed for the quantification - Ag/Silica

column

Sample conc.

MO

(mg/mL)

conc.

I.S.

(mg/mL)

method injection

volume

(µl)

collection

time MOSH

(min)

collection

time MOAH

(min)

MO A 100 0.150 12 20 3.6 – 5.5 5.5 – 15.00

MO E 100 0.150 12 20 3.6 – 5.5 5.5 – 15.00

MO H 100 0.150 12 20 3.6 – 5.5 5.5 – 15.00

Table 3: LC methods and concentrations of samples analyzed for the quantification - Ag/Polymer

column

Sample conc.

MO

(mg/mL)

conc.

I.S.

(mg/mL)

method injection

volume

(µl)

collection

time MOSH

(min)

collection

time MOAH I

(min)

collection

time MOAH

II (min)

MO A 100 0.150 24 20 4.00 – 6.66 6.66 – 8.33 8.33 – 30.00

MO E 100 0.150 24 20 4.00 – 6.66 6.66 – 8.33 8.33 – 30.00

MO H 100 0.150 21 20 4.00 – 6.66 6.66 – 8.33 8.33 – 30.00

3.3C Analysis by GC-FID

After the MOSH and MOAH fraction collection the samples were evaporated to 0.5 mL and 1 μL

was injected into the GC-FID. The same program was used for the GC-FID as described in section

3.2B. The quantification was done using the internal standards (‘I.S. method’) and by calculating

the following ratio: area MOAH : area MOSH+MOAH (‘area method’).

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4. Results


4.1A Separation between MOSH and MOAH internal standards

Table 4 shows the fractions in which the MOSH and MOAH internal standards elute as well as the

separation time between these standards. The same methods are used as for the characterization.

The corresponding LC chromatograms are shown in Figure 5 (Ag/Silica column) and Figure 6

(Ag/Polymer column). The fractions are each 20 seconds and starting from 4 minutes (section

3.2A).

Table 4: Separation MOSH and MOAH internal standards

Figure 5: LC-UV chromatogram Ag/Silica column, I.S. 0.150 mg/mL + perylene, method 9.

Column fractions MOSH

I.S.

start fraction

MOAH I.S.

separation

time (min)

Ag/Silica 2 – 4 9 1.33

Ag/Polymer 5 – 8 12 1.00

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Figure 6: LC-UV chromatogram Ag/Polymer column, I.S. 0.150 mg/mL , method 21.

4.1B Characterization of the mineral oils

In the Figures 7 – 12 the LCxGC-FID chromatograms of mineral oil A, E and H are shown. All

mineral oils were analyzed using the Ag/Polymer column as well as the Ag/Silica column. In

Appendix 2 the LC chromatograms of MO A, E and H are shown.

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Figure 8: 2-D LCxGC-FID chromatogram of MO A (50 mg/mL) Ag/Polymer column

Figure 7: 2-D LCxGC-FID chromatogram of MO A (50 mg/mL) Ag/Silica column

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Figure 6: 2-D LCxGC-FID chromatogram of MO E (50 mg/mL) Ag/Silica column

Figure 7: 2-D LC-GC-FID spectrum of MO E (50 mg/mL) Ag/Polymer column

Figure 9: 2-D LCxGC-FID chromatogram of MO E (50 mg/mL) Ag/Silica column

Figure 10: 2-D LCxGC-FID chromatogram of MO E (50 mg/mL) Ag/Polymer column

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Figure 11: 2-D LCxGC-FID chromatogram of MO H (50 mg/mL) Ag/Silica column

Figure 12: 2-D LCxGC-FID chromatogram of MO H (50 mg/mL) Ag/Polymer column

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4.2 Quantification

In Table 5 the results of the quantification of MOAH in MOH are set out. Solid phase extraction

was used for the separation of MOSH and MOAH. The chromatograms and corresponding tables

that were used for the calculations are shown in Appendix 3.

Table 5: Ratio MOAH in MOH (SPE)

MO area method I.S. method

A 0.26 0.23

E 0.46 0.38

H 0.31 0.20

In Table 6 the results of the quantification of MOAH in MOH are set out. The Ag/Silica column


that were used for the calculations are shown in Appendix 3

Table 6: Ratio MOAH in MOH (Ag/Silica column)


A 0.26 0.3

E 0.47 0.48

H 0.34 0.35

In Table 7 the results of the quantification of MOAH in MOH is shown. The Ag/Polymer column


that were used for the calculations are shown in Appendix 3.

Table 7: Ratio MOAH in MOH (Ag/Polymer column)


A 0.25 0.27

E 0.43 0.46

H 0.30 0.31

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5. Discussion

5.1 Characterization: separation between MOSH and MOAH

5.1A Ag/Silica column

As shown in Table 4, the internal standards that establish the MOSH window elute until fraction 4

and the first MOAH marker starts at fraction 9. According to previous studies, all MOSH and

MOAH should elute in these windows. 30,32 The LCxGC chromatograms of MO A, E and H (Fig.

7, 9, 11) show a gap after fraction 4. This possibly means that all MOSH elute as indicated by the

marker. However, GC-VUV detection should be used to establish whether indeed no MOAH is

present in the given MOSH window. Aromatics absorb at a higher wavelength and thus can be

detected if they are present in the fractions. If analysis shows that the MOSH fractions only contain

aliphatic compounds and the fractions after only aromatic compounds, 5-α-cholestane is effective

as a marker to establish and check the end of the MOSH window. On the contrary, 1,3,5-tri-tert-

butylbenzene would not be useful as a marker to establish the start of the MOAH window, since in

all spectra MOAH seems to start at fraction 6 or 7 instead of fraction 9. A possible explanation of

this could be that the analyzed mineral oils contain highly alkylated aromatic compounds, which

due to steric hindrance do not interact as much with the silver stationary phase as less-alkylated

aromatics. As a consequence, these compounds elute before than the MOAH marker, which is only

moderately alkylated.

5.1B Ag/Polymer column

As shown in Table 4, the internal standards that establish the MOSH window elute until fraction

8. This should mean that all MOSH elute until this fraction as well, according to previous studies.

The start of the MOAH standards is at fraction 12, meaning that all MOAH should elute starting

from this fraction. The LCxGC chromatograms of MO A (Fig. 8) shows a gap at fraction 11. This

could mean that in fraction 12 the first MOAH elutes, and in fraction 10 the last MOSH. Important

to note, between fraction 6 and 10 a different kind of pattern can be observed. Possibly, these are

aromatic compounds that are highly alkylated. To establish whether these fractions contain

aromatic or aliphatic compounds or both, GC-VUV should be used.

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The 2-D LCxGC chromatogram of MO E (Fig. 10) shows a small gap at fraction 10, which possible

means that MOSH elutes before and MOAH after this fraction. The 2-D LCxGC chromatogram of

MO H (Fig. 12) shows that possibly MOSH elutes before fraction 9 and MOAH after fraction 11,

which is exactly as the internal standards indicated.

For the Ag/Polymer column, the 2-D LCxGC chromatograms of MO A and E show that between

the expected MOSH and MOAH windows, which are established using markers, compounds elute.

This probably is a consequence of the presence of highly alkylated aromatic rings. If analysis by

GC-VUV shows that these compounds are aromatics, a higher alkylated benzene molecule needs

to be used as a marker to establish the MOAH window.

5.1C Comparison columns

The main objective of this study was to establish a separation between Mineral Oil Saturated

Hydrocarbons and Mineral Oil Aromatic Hydrocarbons. Analysis of the internal standards using

comprehensive LCxGC-FID, showed that a separation of 1 minute (Ag/Polymer column) and 1

minute and 20 seconds (Ag/Silica column) between the MOSH and MOAH markers was

established. However, analysis of the LCxGC chromatograms of the samples showed that

compounds elute in the timeframe between the MOSH and MOAH markers. The findings in this

study are contrary to previous studies, which have suggested that the markers should correctly

establish the MOSH and MOAH windows. This result may be explained by the fact that in previous

studies, a silica-phase column was used opposed to silver-phase columns in this study. However,

in the previous study a separation of 30 seconds was established between the markers, whereas in

this study this separation was (more than) 1 minute. This would suggest that the separation

between MOSH and MOAH in this study is better. A possible explanation is that in the previous

study also compounds eluted in between the two windows, but because the analyzed concentration

of the mineral oil hydrocarbons is lower than in this study, these compounds cannot be detected in

the GC-FID. Another possible explanation for the compounds eluting in between the windows is

the high concentration of mineral oil that is analyzed in this study, which might result in an

overloaded column. Furthermore, highly alkylated benzenes that might be present in the samples

could elute at similar retention times as MOSH due to steric hindrance.

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Although the expected separation between MOSH and MOAH around 1 minute was not established

for the mineral oil samples, the separation seems to be slightly better when using the Ag/Silica

column. This could possibly be explained by the favorable interactions between silica and

aromatics. Moreover, the silica and silver are less tightly bound than the polymer and the silver,

leaving more room for the strong π-interactions between silver and the aromatics.

5.2 Characterization: classification aromatic compounds

5.2A Ag/Silica column

The LC chromatogram of the internal standards (Fig. 5) on the Ag/Silica column show that

the mono-ring aromatics elute together, and the di-ring aromatics separately. The LCxGC

chromatograms also show, although not clearly, two bands in the aromatic part. This could be a

slight separation between the mono and poly-ring aromatics, but to be certain GC-VUV could be

used, since different absorbance spectra are expected for mono- and poly-ring aromatics.

5.2B Ag/Polymer column

The LC chromatogram of the internal standards (Fig. 6) on the Ag/Polymer column shows that the

aromatics are divided into classes, according to the number of rings and degree of alkylation. The

internal standards appear as individual peaks, in which the first peaks are single aromatic ring

species that are sterically protected by a high degree of alkylation. These are followed by MOAH

with more rings and/or less alkylation. This effect is also observed in the LCxGC chromatograms

of the mineral oils (Fig. 8, 10, 12), in which bands of alternating intensity are visible. A higher

intensity color responds to a higher responds in the GC-FID, and thus a higher concentration of

MOH. According to the internal standards, the di-ring aromatics elute after fraction 30 (14

minutes), and the mono-ring aromatics before this fraction. The high intensity of fraction 31 is

caused by the breakthrough of dichloromethane, since this eluent reduces the strong retention

power of the column to retain MOAH.32 Since mineral oil E has the highest intensity band after

fraction 30, it probably has the most poly-ring aromatics. Mineral oil H has the lowest intensity

band after fraction 30, meaning that it has the least multi-ring aromatics. To classify these

compounds into sub-groups time-of-flight mass detection could be explored, since compounds can

be distinguished by selecting their unique masses.35 Another method of detection of aromatic

classes could be GC-VUV.

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Regarding the retention times of the mineral oil hydrocarbons in the GC-FID, a downwards shift

can be observed for mineral oil H compared to the other samples. This means that the average

boiling point of these hydrocarbons is lower, meaning that they have a lower average molecular

mass.

5.2C Comparison columns

In this study, one of the objectives was to classify aromatic compounds in terms of the number of

rings. For this, three mineral oils were analyzed using comprehensive LCxGC-FID. The results

showed that the Ag/Polymer column is most suited to separate aromatics into groups. This might

be explained by the length of the column, which is twice as long as the Ag/Silica column. An

Ag/Silica column of similar length should be used to conclude which stationary phase is more

suitable to separate aromatics into classes. Moreover, time-of-flight mass detection could be used

as well as GC-VUV, to determine whether the separation into classes was established.

5.3 Quantification

One of the aims of this study was to determine the aliphatic and aromatic content of the mineral oil

samples and to compare them to the standard method based on Solid Phase Extraction. Analysis of

the samples that were separated using SPE, showed that MO A contains about 26% MOAH, MO

E 46% and MO H 31% (Table 5). This was determined using the ratio area MOAH: area

MOSH+MOAH, since the internal standard method was not as accurate due to overlapping peaks

in the GC chromatograms. To determine the aromatic contents using the internal standards, analysis

should be done with different internal standards, or a lower concentration mineral oil.

The samples that were separated by LC were analyzed using a slightly shorter GC columns and

had a lower concentration, which resulted in less or no overlapping of the internal standards. Hence,

the area method as well as the internal standard method can be used to determine the amount of

aromatics in the mineral oils.

The MOSH and MOAH fraction collection time for the Ag/Silica column was determined using

the marker 5-α-cholestane. The LCxGC chromatograms show that a separation of MOSH and

MOAH supposedly happens after the fourth fraction. Analysis of the MOSH and MOAH fractions

showed that MO A contains about 28% aromatics, MO E 48% and MO H 35% (Table 6).

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The fraction collection time for the Ag/Polymer column was determined by analysis of the 2-D

chromatograms of the samples. As mentioned previously, the 2-D chromatograms show that

between the expected MOSH and MOAH windows, compounds elute. Since these are most likely

highly alkylated aromatics, this fraction was treated as MOAH. Analysis of the fractions by GC-

FID showed that MO A contains around 26% aromatics, MO E 45% and MO H 31% (Table 7).

When these determined percentages of aromatics in mineral oils are compared, they appear highly

similar for each method. This could mean that the LC-GC method is reliable for determining the

amount of aromatics in mineral oils using both columns. However, these findings may be somewhat

limited by the uncertainty of separation between MOSH and MOAH. The collected MOSH and

MOAH fractions should be analyzed by GC-VUV to determine whether a complete separation was

established. Moreover, since MOSH and MOAH appear as a huge hump in the GC chromatograms,

a baseline uncertainty should be taken into account.

6. Conclusion

The main objective of this study was to establish a separation between Mineral Oil Saturated

Hydrocarbons and Mineral Oil Aromatic Hydrocarbons. Analysis of the internal standards using

comprehensive LCxGC-FID, showed that a separation of 1 minute (Ag/Polymer column) and 1

minute and 20 seconds (Ag/Silica column) between the MOSH and MOAH markers was

established. However, analysis of the LCxGC-FID chromatograms of three mineral oil samples

with different grades of refinement showed that compounds elute in the timeframe between the

MOSH and MOAH markers. These compounds could be highly alkylated benzenes, which due to

steric hindrance can interact less with the stationary phase and thus elute at similar retention times

as MOSH.

The second aim of this study was to classify aromatic compounds in terms of the number of rings.

The results showed that the Ag/Polymer column is most suited to separate aromatics into groups,

which is possibly a result of the length of the column. To establish whether the difference in

separation between the aromatics is due to the difference in stationary phase or the length, a silver-

silica phase column of equal length should be used in the analysis of the mineral oils.

Another objective of this study was to determine the aliphatic and aromatic content of the mineral

24

oil samples and to compare them to the standard method based on Solid Phase Extraction. When

these determined percentages of aromatics in mineral oils were compared, they appeared highly

similar for each method. This could mean that the LC-GC method is reliable for determining the

amount of aromatics in mineral oils using both columns. However, a base-line uncertainty as well

as the uncertainty in the separation between MOSH and MOAH should be taken into account.

Further research should be undertaken to determine whether a separation between MOSH and

MOAH was established, for example by analyzing the fractions by GC-VUV.

7. Outlook

Further research should be carried out to establish whether a separation was obtained between

MOSH and MOAH. The fractions could be analyzed by GC-VUV to determine what kind of

compounds elute between the expected MOSH and MOAH windows. Another method to

determine this is to analyze the three mineral oil samples that are separated using solid phase

extraction into MOSH and MOAH. These fractions have already been analyzed using the LC-GC-

FID method. However, these results have yet to be analyzed. A further study could also classify

the groups of aromatics regarding the number of rings or even into subgroups by using GC-MS-

ToF. In order to establish a difference in retention time between MOSH and MOAH in the GC-

FID, a 50% phenyl–50% dimethylpolysiloxane column could be used. Since this column has a

high Kovat’s retention index for benzene, it is expected that the aromatic compounds will be

retained longer than the aliphatic compounds. As a consequence, a shift upwards in the LCxGC

chromatograms of the mineral oil might be visible for the aromatic part.

Ultimately, consumer products will be analyzed to characterize the mineral oil hydrocarbons and

to quantify the mineral oil aromatic hydrocarbons.

25

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27

9. Appendices

Appendix 1: LC methods

Table 8: LC method 21 (gradient)

Time

(min)

hexane

(%)

DCM

(%)

Flow

(mL/min)

0.00 100 0 0.5

5.90 100 0 0.5

6.00 50 50 0.5


Time

(min)

hexane

(%)

DCM

(%)

Flow

(mL/min)

0.00 100 0 0.5

5.90 100 0 0.5

6.00 30 70 0.5

Table 10: LC method 9 (isocratic)

Time

(min)

hexane

(%)

DCM

(%)

Flow

(mL/min)

0.00 100 0 0.3


Time

(min)

hexane

(%)

DCM

(%)

Flow

(mL/min)

0.00 100 0 0.3

3.0 100 0 0.3

3.5 50 50 0.3

28

Appendix 2: Characterization chromatograms

Figure 13: MO A 50 mg/mL Ag/Silica column

Figure 14: MO A 50 mg/mL Ag/Polymer column

29

Figure 15: MO E 50 mg/mL Ag/Silica column

Figure 16: MO E 50 mg/mL Ag/Polymer column

30


31

Figure 18: MO H 50 mg/mL Ag/Polymer column

Appendix 3: Quantification chromatograms

Figure 19: MO A 100 mg/mL Ag/Silica column

32

Figure 20: MO A 100 mg/mL Ag/Polymer column


33

Figure 22: MO E 100 mg/mL Ag/Polymer column

Figure 23: MO H 100 mg/mL Ag/Silica column

34

Figure 24: MO H 100 mg/mL Ag/Polymer column

Appendix 4: GC-FID chromatograms quantification

Figure 25: GC-FID chromatogram Ag/Silica column MOSH A2 100 mg/mL

35

Table 12: GC-FID Ag/Silica column MOSH A2 100 mg/mL

Peak Retention time Area Ratio Concentration

(mg/mL)

undecane 3.932883 1751903

bicyclohexyl 6.20558 1711231 1 0.150

tridecane 6.31775 2074898

MOSH 14.6053 554237859 323,88 48.58

5-α-cholestane 15.5762 2672122

Figure 26: Ag/Silica column MOAH A2 100 mg/mL

36

Table 13: GC-FID Ag/Silica column MOAH A2 100 mg/mL

Peak Retention

time

Area Ratio Concentration

(mg/mL)

hexylbenzene 5.82717 1564508

1-methylnaphthalene 6.23725 1550019 1 0.150

biphenyl 6.90308 2861023

1,3,5-tri-tert-

butylbenzene

7.50617 1734828

nonylbenzene 8.5305 1682918

MOAH 13.6331 184899958 119.29 17.89

Figure 27: Ag/Silica column MOSH E2 100 mg/mL

37

Table 14: GC-FID Ag/Silica column MOSH E2 100 mg/mL

Peak Retention

time

Area Ratio Concentration (mg/mL)

undecane 3.92533 1861746

bicyclohexyl 6.20183 1879537 1 0.150

tridecane 6.31675 1991460

MOSH 15.2425 741146168 394.32 59.15

5-α-cholestane 15.5865 1419054

Figure 28: Ag/Silica column MOAH E2 100 mg/mL

38

Table 15: GC-FID Ag/Silica column MOAH E2 100 mg/mL

Peak Retention

time


(mg/mL)



biphenyl 6.89942 3134947

1,3,5-tri-tert-

butylbenzene

7.504 1750914


MOAH 14.6131 557036339 341.38 51.21

Figure 29: Ag/Silica column MOSH H2 100 mg/mL

39

Table 16: GC-FID Ag/Silica column MOSH H2 100 mg/mL


(mg/mL)

bicyclohexyl 6.19742 1678621 1 0.150

tridecane 6.31183 2249088

MOSH 15.0675 1001065047 596.36 89.45

5-α-Cholestane 15.5638 1931779

Figure 30: Ag/Silica column MOAH H2 100 mg/mL

40

Table 17: GC-FID Ag/Silica column MOAH H2 100 mg/mL

Peak Retention

time


(mg/mL)



biphenyl 6.8945 2864792

1,3,5-tri-tert-

butylbenzene

7.49908 2118379


MOAH 13.6351 418983573 272.50 40.88

Figure 31: Ag/Polymer column MOSH A 100 mg/mL

41

Table 18: GC-FID Ag/Polymer column MOSH A 100 mg/mL


(mg/mL)

undecane 3.80975 168643

undecane 3.866 1337306

bicyclohexyl 6.16367 1555108 1 0.150

tridecane 6.28142 1673189

5-α-cholestane 15.5551 1508170

MOSH 15.9532 496911916 314.60 47.19

Figure 31: Ag/Polymer column MOAH I A 100 mg/mL

42

Table 19: GC-FID Ag/Polymer column MOAH I E 100 mg/mL

Peak Retention time Area Concentration

(mg/mL)

bicyclohexyl 6.16858 24414

MOAH 14,2743 33733097 2.244943712

Figure 32: Ag/Polymer column MOAH II A 100 mg/mL

43

Table 20: GC-FID Ag/Polymer column MOAH II A 100 mg/mL


(mg/mL)


1-

methylnaphthalene

6.2045 1302254

biphenyl 6.8745 1323919 1 0.150

1,3,5-tri-tert-

butylbenzene

7.47825 1399619


MOAH 8.75158 145253563 109.71 16.46

Figure 33: Ag/Polymer column MOSH E 100 mg/mL

44

Table 21: GC-FID Ag/Polymer column MOSH E 100 mg/mL


(mg/mL)

undecane 3.88175 1542899

bicyclohexyl 6.16758 1505222 1 0.150

tridecane 6.28467 1627921

5-α-cholestane 15.5582 1007296

MOSH 15.9235 318337985 211.49 31.72

Figure 34: Ag/Polymer column MOAH I E 100 mg/mL

Table 22: GC-FID Ag/Polymer column MOAH I E 100 mg/mL


(mg/mL)

MOAH 14.96 19709201 2.10

45

Figure 35: Ag/Polymer column MOAH II E 100 mg/mL

Table 23: GC-FID Ag/Polymer column MOAH II E 100 mg/mL


(mg/mL)


1-

methylnaphthalene

6.20308 1371503

biphenyl 6.87442 1407345 1 0.150

1,3,5-tri-tert-

butylbenzene

7.47792 1478373


MOAH 14.85 260150071 184.85 27.73

46

Figure 36: Ag/Polymer column MOSH H 100 mg/mL

Table 24: GC-FID Ag/Polymer column MOSH H 100 mg/mL


(mg/mL)

undecane 3.88225 1507808

bicyclohexyl 6.16608 1570748 1 0.150

tridecane 6.28392 1736569

5-α-Cholestane 15.5457 852628

MOSH 6.94292 525145915 334.33 50.15

47

Figure 37: Ag/Polymer column MOAH I H 100 mg/mL

Table 25: GC-FID Ag/Polymer column MOAH I H 100 mg/mL


(mg/mL)

5-α-cholestane 15.5452 35706

MOAH 14.6123 22798466 2.24

48

Figure 38: Ag/Polymer column MOAH II H 100 mg/mL

Table 26: GC-FID Ag/Polymer column MOAH II H 100 mg/mL


(mg/mL)


1-

methylnaphthalene

6.19358 1432082

biphenyl 6.87025 1523321 1 0.150

1,3,5-tri-tert-

butylbenzene

7.47558 1649191


MOAH 14.1016 245459578 161.13 24.17

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Analysis of mineral oil hydrocarbons in consumer products using … · 2020-03-13 · Aromatic Hydrocarbons (MOAH), with regards to their toxicity, an individual analysis is needed