DETECTION OF BEESWAX ADULTERATION WITH HYDROCARBONS USING GAS CHROMATOGRAPHY WITH

MASS DETECTOR (GC-MS)

DETECTION OF BEESWAX DETECTION OF BEESWAX ADULTERATION WITH HYDROCARBONS ADULTERATION WITH HYDROCARBONS USING GAS CHROMATOGRAPHY WITH USING GAS CHROMATOGRAPHY WITH

MASS DETECTOR (GCMASS DETECTOR (GC--MS)MS)

Ewa Waś, Helena Rybak-Chmielewska, Teresa Szczęsnae-mail: ewa.was@man.pulawy.pl

Research Institute of Pomology and Floriculture, Apiculture Division,Department of Bee Products,

24-100 Puławy, Kazimierska 2, Poland.

Ewa WaEwa Waśś, Helena Rybak, Helena Rybak--Chmielewska, Teresa SzczChmielewska, Teresa Szczęęsnasnaee--mail: mail: ewa.was@man.pulawy.plewa.was@man.pulawy.pl

Research Institute of Pomology and Floriculture, Apiculture DiviResearch Institute of Pomology and Floriculture, Apiculture Division,sion,Department of Bee Products,Department of Bee Products,

2424--100 Pu100 Pułławy, Kazimierskaawy, Kazimierska 2, Poland. 2, Poland.

Beeswax adulteration with hydrocarbonsBeeswax adulteration with hydrocarbons

For a few years, due to the lack of mandatory regulations concerning beeswax quality, this product has been increasingly adulterated with much cheaper hydrocarbons of alien origin. As aresult, the quality of the product is compromised which makes itunusable by the industries.

For a few years, due to the lack of mandatory regulations concerning beeswax quality, this product has been increasingly adulterated with much cheaper hydrocarbons of alien origin. As aresult, the quality of the product is compromised which makes itunusable by the industries.

Adulteration of beeswax with hydrocarbons has also a negative impact on the quality of comb foundation. When used on regular basis in bee farming adulterated beeswax interferes with normal brood rearing and development(Jimenez i in., 2005).

Adulteration of beeswax with hydrocarbons has also a negative impact on the quality of comb foundation. When used on regular basis in bee farming adulterated beeswax interferes with normal brood rearing and development(Jimenez i in., 2005).

Beeswax adulteration with hydrocarbonsBeeswax adulteration with hydrocarbons

According to the requirement laid down by PN-R-78890 „Wosk pszczeli” the content of hydrocarbons cannot exceed 16.5%.

According to the requirement laid down by PN-R-78890 „Wosk pszczeli” the content of hydrocarbons cannot exceed 16.5%.

Higher contents of those compounds indicate contamination of that product with alien hydrocarbons.

Higher contents of those compounds indicate contamination of that product with alien hydrocarbons.

Detection of beeswax adulteration with Detection of beeswax adulteration with hydrocarbonshydrocarbons

The analysis of the chemical composition of beeswax because of its special nature is exceptionally difficult even if state-of-the-art equipment is used.

The analysis of the chemical composition of beeswax because of its special nature is exceptionally difficult even if state-of-the-art equipment is used.

Until recently, in order to detect adulteration of beeswax with hydrocarbons column chromatography combined with weight analysis was used in Poland(PN-R-78890 „Wosk pszczeli”-„Beeswax”). The method allows only the determination of total hydrocarbons occurring in beeswax.

Until recently, in order to detect adulteration of beeswax with hydrocarbons column chromatography combined with weight analysis was used in Poland(PN-R-78890 „Wosk pszczeli”-„Beeswax”). The method allows only the determination of total hydrocarbons occurring in beeswax.

The aim of the studyThe aim of the study

The aim of the study was to describe hydrocarbons in natural beeswax.

Another objective was to develop a newmethod to detect beeswax adulteration with paraffin.

The aim of the study was to describe hydrocarbons in natural beeswax.

Another objective was to develop a newmethod to detect beeswax adulteration with paraffin.

Material and methods Material and methods

Hydrocarbon assays were made on a gas chromatograph with a Shimadzu mass detector (Gas Chromatograph Mass Spectrometer, GC-MS-QP 2010 Plus).

Hydrocarbon assays were made on a gas chromatograph with a Shimadzu mass detector (Gas Chromatograph Mass Spectrometer, GC-MS-QP 2010 Plus).

Material and methods Material and methods

• Hydrocarbon fraction was isolated on a silica column according to Polish Standard PN-R-78890.

• Hydrocarbons were separated on a non-polar ZB- 5HT 30m x 0.25mm x 0.25µm column manufactured by Phenomenex. Chromatography separation of hydrocarbons in temperature program was done with helium as a carrier gas.

• An electron source was used to ionize sample molecules in the gaseous phase. The temperature of the electron source was 2300C. EI mass spectra were recorded at a standard electron energy of 70eV.

• Based on the electron spectra of the NIST 05 library and on the retention indexes, identyfication of studied compounds was done.

• Hydrocarbon fraction was isolated on a silica column according to Polish Standard PN-R-78890.

• Hydrocarbons were separated on a non-polar ZB- 5HT 30m x 0.25mm x 0.25µm column manufactured by Phenomenex. Chromatography separation of hydrocarbons in temperature program was done with helium as a carrier gas.

• An electron source was used to ionize sample molecules in the gaseous phase. The temperature of the electron source was 2300C. EI mass spectra were recorded at a standard electron energy of 70eV.

• Based on the electron spectra of the NIST 05 library and on the retention indexes, identyfication of studied compounds was done.

Table 1.Table 1. The The homologous series of saturated hydrocarbons homologous series of saturated hydrocarbons with unramified carbon chains occurring in naturalwith unramified carbon chains occurring in natural beeswaxbeeswax

* Molecular ion not observed.

ChromatogramChromatogram of alkanesof alkanes in naturalin natural beeswaxbeeswax

20 25 30 35 40 45 50 55 60

0.25

0.50

0.75

1.00

1.25

1.50

(x10,000,000) TIC

C19C20* C21

C22*

C23

C24

C25

C26

C27

C28

C29

C30

C31

C32*

C33

C34*C35*

Abu

ndan

ce

Time [min]

Electron impact spectrum of alkanesElectron impact spectrum of alkanes

Hydrocarbons containing more than eight carbon atoms show very similar spectra and the identification of those compounds depends on the detection of the molecular ion peak.

Hydrocarbons containing more than eight carbon atoms show very similar spectra and the identification of those compounds depends on the detection of the molecular ion peak.

As a rule, a peak of the molecular ion of saturated hydrocarbons with a simple (unramified) chain is visible. However, its intensity decreases with length and ramification degree of the chain. In the spectra of simple-chain alkanes peak groups are observed at m/z values differing by 14 daltons (CH2).

As a rule, a peak of the molecular ion of saturated hydrocarbons with a simple (unramified) chain is visible. However, its intensity decreases with length and ramification degree of the chain. In the spectra of simple-chain alkanes peak groups are observed at m/z values differing by 14 daltons (CH2).

Electron impact spectrum of alkanesElectron impact spectrum of alkanes

50.0 75.0 100.0 125.0 150.0 175.0 200.0 225.0 250.0 275.0 300.0 325.0 350.0

0 .0

25.0

50.0

75.0

100.0 57

71

85

99113

127 155 169 183 197 211 239 253 267 352281 309 323

C25H52

Abu

nda

nce

The [CnH2n+1]+ ions are accompanied by ions of the [CnH2n]+. [CnH2n-1]+

and formulas which arise as a result of the primary fragmentation ions being eliminated.

m/z

Within each group, the peak of the highest intensity corresponding to a [CnH2n+1]+ fragment occurs at m/z= 14n+1 (m/z= 29, 43, 57, 71, 85, 99 etc.) and it arises from the breakdown of C-C bonds at different sites of the carbon chain.

Within each group, the peak of the highest intensity corresponding to a [CnH2n+1]+ fragment occurs at m/z= 14n+1 (m/z= 29, 43, 57, 71, 85, 99 etc.) and it arises from the breakdown of C-C bonds at different sites of the carbon chain.

Table 2. Alkenes and dienes Table 2. Alkenes and dienes occurring in naturaloccurring in natural beeswaxbeeswax

* :1, :2- the number of double bonds.** (1,2)- the number of identified isomeres.

ChromatogramChromatogram of alkenesof alkenes and dienes and dienes in naturalin natural beeswaxbeeswax

37.5 40.0 42.5 45.0 47.5 50.0 52.5 55.0

0.25

0.50

0.75

1.00

1.25

(x10,000,00)

TIC

C23:1 C25:1C27:1

C29:1

C29:1

C31:2

C31:1C31:1

C33:2

C33:1

C35:2

C35:1C35:1

Time [min]

Abu

ndan

ce

ChromatogramChromatogram of alkanesof alkanes inin commercial paraffincommercial paraffin

20.0 25.0 30.0 35.0 40.0 45.0 50.0 55.0 60.0

0.25

0.50

0.75

1.00

1.25 (x10,000,000)

TIC

C19

C20

C21

C22

C23

C24C25

C26 C27

C28

C29 C30

C31

C32

C33

C34*

C35*

Aba

unda

nce

Time [min]

ChromatogramChromatogram of alkanesof alkanes inin samples of paraffin samples of paraffin ––adulterated beeswax adulterated beeswax

20.0

25.0 30.0 35.0 40.0 45.0 50.0 55.0 60.0

0.25

0.50

0.75

1.00

1.25

1.50

(x10,000,000)TIC

C19

C20

C21C22

C23C24

C25 C26

C27

C28

C29

C30

C31

C32 C33

C34

C35*

C36*

Time [min]

Abu

ndan

ce

The comparison of alkanes in natural beeswax and The comparison of alkanes in natural beeswax and alkanes in commercial paraffinalkanes in commercial paraffin

20.0 25.0 30.0 35.0 40.0 45.0 50.0 55.0 60.0 65.0

0.25

0.50

0.75

1.00

1.25

(x10,000,000) TIC

C19

C20

C21C22

C23C24

C25 C26

C27

C28C29

C30

C31

C32 C33

C34

C35*

20.0 25.0 30.0 35.0 40.0 45.0 50.0 55.0 60.0

0.1 0.3

0.5

0.7

0.9 1.1

1.3 (x10,000,000)

TIC

C19C20*

C21C22*

C23

C24

C25

C26

C27

C28

C29

C30

C31

C32*

C33

C34*C35*

The comparison of alkanes in commercial paraffin The comparison of alkanes in commercial paraffin and alkanes in paraffin and alkanes in paraffin –– adulterated beeswaxadulterated beeswax

20.0 25.0 30.0 35.0 40.0 45.0 50.0 55.0 60.0 65.0

0.25

0.50

0.75

1.00

1.25

1.50

(x10,000,000)TIC

C19

C20

C21 C22C23C24

C25 C26

C27

C28

C29

C30

C31

C32 C33

C34

C35*

20.0 25.0 30.0 35.0 40.0 45.0 50.0 55.0 60.0

0.25

0.50

0.75

1.00

1.25 (x10,000,000) TIC

C19

C20

C21

C22

C23C24C25

C26C27 C28

C29 C30

C31

C32

C33

C34*

C35*

ConclussionsConclussions

ConclussionsConclussions

Thank You for attentionThank You for attentionThank You for attention