DOI: 10.1515/JAS-2015-0018 J. APIC. SCI. Vol. 59 No.

2 2015
J. APIC. SCI. Vol. 59 No. 2 2015
Original Article

AN APPROACH FOR ROUTINE ANALYTICAL DETECTION

OF BEESWAX ADULTERATION USING FTIR-ATR SPECTROSCOPY
Lidija Svečnjak1*
Goran Baranović2
Marko Vinceković3
Saša Prđun1
Dragan Bubalo1
Ivana Tlak Gajger4
1
University of Zagreb Faculty of Agriculture, Department of Fisheries,
Apiculture and Special Zoology, 10000 Zagreb, Croatia
2
Rudjer Boskovic Institute, Division of Organic Chemistry and Biochemistry
(Laboratory of Molecular Spectroscopy), 10000 Zagreb, Croatia
3
University of Zagreb Faculty of Agriculture, Department of Chemistry,
10000 Zagreb, Croatia
4
University of Zagreb, Faculty of Veterinary Medicine,
Department of Biology and Pathology of Fish and Bees, 10000 Zagreb, Croatia
*corresponding author: [email protected]
Received 8 October 2014; accepted 10 July 2015

Abstract
Although beeswax adulteration represents one of the main beeswax quality issues, there
are still no internationally standardised analytical methods for routine quality control.
The objective of this study was to establish an analytical procedure suitable for routine
detection of beeswax adulteration using FTIR-ATR spectroscopy. For the purpose of this
study, reference IR spectra of virgin beeswax, paraffin, and their mixtures containing dif-
ferent proportions of paraffin (5 - 95%), were obtained. Mixtures were used for the estab-
lishment of calibration curves. To determine the prediction strength of IR spectral data
for the share of paraffin in mixtures, the Partial Least Squares Regression method was
used. The same procedure was conducted on beeswax-beef tallow mixtures. The model
was validated using comb foundation samples of an unknown chemical background which
had been collected from the international market (n = 56). Selected physico-chemical pa-
rameters were determined for comparison purposes. Results revealed a strong predictive
power (R2 = 0.999) of IR spectra for the paraffin and beef tallow share in beeswax. The
results also revealed that the majority of the analysed samples (89%) were adulterated
with paraffin; only 6 out of 56 (11%) samples were identified as virgin beeswax, 28% of
the samples exhibited a higher level of paraffin adulteration (>46% of paraffin), while
the majority of the analysed samples (50%) were found to be adulterated with 5 - 20% of
paraffin. These results indicate an urgent need for routine beeswax authenticity control.
In this study, we demonstrated that the analytical approach defining the standard curves
for particular adulteration levels in beeswax, based on chemometric modelling of specific
IR spectral region indicative for adulteration, enables reliable determination of the adul-
terant proportions in beeswax.

Keyword: adulterants share detection, beeswax adulteration, fingerprint region,

FTIR-ATR spectroscopy.

INTRODUCTION honey and brood are being capped (Breed et  al.,
1995; Fröhlich et al., 2000; D’Ettorre et al., 2006;
A specific lipid-based chemical composition of Buchwald et al., 2009). It is, therefore, important
beeswax plays an important role in a honeybee that the foundations on which the honeycombs will
colony as a source of nest-mate recognition cues and be built are genuine.
as construction material for honeycomb cells where

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Although beeswax adulteration has been one of methods were not performed. Also, a general inter-
the main beeswax quality issues for more than pretation of beeswax IR spectrum and investigation
a  decade, there are still no internationally stand- of molecular structure and dynamics of beeswax by
ardised analytical methods for routine beeswax IR and Raman spectroscopy were reported in several
authenticity control (Bogdanov, 2004a). Nowadays, spectroscopic studies (Birshtein and Tul‘chinskii,
more than 15 different natural (petroleum-derived, 1977; Edwards et al., 1996; Zimnicka and Hacura,
mineral, animal, and plant waxes) and artificial/ 2006; Muscat et. al., 2014).
industrial waxes can be used as beeswax adulter- There has been a recent report on the feasibility
ants (Bogdanov, 2009). Among them, the adultera- study of a single-reflection Attenuated Total Re-
tion with paraffin waxes represents the greatest flectance Fourier Transform Infrared Spectroscopy
problem due to its wide availability, low price, (FTIR-ATR) for detection of beeswax adulterants
and physico-chemical properties (a chemically inert, (Maia et al., 2013). This spectroscopic study revealed
white or colourless and odourless substance) that direct and reagent free method that provides good
altogether makes it “ideal” for adulteration. detection limits (0.5 - 5%) of four of the most
The analytical methods defining pure-beeswax- commonly employed beeswax adulterants: paraffin,
composition criteria based on 10 classical physico- microcrystalline wax, tallow, and stearic acid.
chemical parameters determined in accordance with Considering the reported advantages of this spec-
European Pharmacopoeia (Council of Europe, 2007) troscopic technique and its detection limits for
and the Methods of German Society for Fat Science beeswax adulterants, the objective of this study
(DGF-M-V-6, 1957), have been proposed for routine was to establish an analytical procedure suitable
beeswax authenticity testing (Bogdanov, 2004b; for routine detection of beeswax adulteration by
2009). However, as reported in the past and current paraffin and beef tallow using the FTIR-ATR spec-
literature, there are several factors affecting the troscopy. The existing methodology was modified
proposed analytical range values for pure beeswax in terms of faster spectra acquisition, different
and detection of its adulteration (Tulloch, 1973; data processing, and establishment of a calibration
Bernal et al., 2005; Jiménez et al., 2007; Maia and curve procedure. Also, the purpose of this work was
Nunes, 2013). Along with possible deviations in to validate the method by comparing the results of
proposed range values arising from the beeswax the performed analytical procedure with those of
origin (geographical and/or race-related) (Beverly commonly used physico-chemical analyses.
et al., 1995), a temperature treatment (an integral
part of the production of comb foundation) can cause MATERIAL AND METHODS
measurable changes of certain physico-chemical
values (primarily acid and ester value). Consequent- Samples
ly, particular beeswax samples can be displaced To obtain the reference infrared (IR) spectra of
beyond the limits proposed for pure beeswax. These pure beeswax, virgin beeswax samples (wild-built
findings undermine the use of these measurements combs) were collected directly from the beehives
as reliable analytical tools for detection of beeswax maintained at experimental apiaries in different
adulteration. geographical regions across Croatia in 2013 - 2014
Along with the mentioned classical analytical (n = 21). Furthermore, a total of 56 comb-foun-
methods, very sophisticated analytical tools have dation samples were collected from the beeswax
been employed to investigate detailed beeswax foundation manufacturers and/or specialised
chemical composition and/or to find more reliable beekeeping shops in 9 European countries (Austria,
analytical tools for detection of beeswax adultera- Bosnia and Herzegovina, Croatia, Germany, Hungary,
tion. The tools referred to are different techniques Italy, Macedonia, Serbia, Slovenia; n = 52) and in
of high-temperature gas chromatography - mass other countries (Russia, Argentina, China; n = 4).
spectrometry (Aichholz and Lorbeer, 1999, 2000; Four paraffin samples with different melting points
Jiménez et al., 2003; 2004; 2006; 2009; Serra were obtained from Kemig (paraffinum solidum, Ph.
Bonvehi and Orantes Bermejo, 2012; Maia and Eur. 7.8, Croatia) and from the beekeepers (paraffin
Nunes, 2013; Waś et al., 2014). These methods, used for hive waxing). Beef tallow was obtained
though, have not yet met the analytical eligibility for from the local butcher shop. Samples were analysed
routine beeswax control. The reason being, that the directly as obtained, without using purification or
establishment of calibration curves and validation any other chemical reagents. Samples were stored
by comparing the results with other commonly used in the dark at room temperature before analysis.

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Preparation of beeswax-paraffin and beeswax- using Origin version 8.1 (Origin Lab Corporation).
beef tallow mixtures for calibration (standard Given that the most characteristic spectral features
curves) of pure beeswax appear in the spectral region
Mixtures of virgin beeswax containing different pro- between 1800 - 900 cm-1, this particular region, a fin-
portions (5 - 95%) of paraffin wax were prepared. gerprint region, was used for further chemometric
This procedure was performed to obtain reference and statistical modelling. The use of the IR spectra
samples exhibiting physico-chemical features of prepared beeswax-paraffin mixtures was carried
and IR spectra specific for particular adulteration out to generate the calibration curves covering the
levels (5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95%). entire range (0 - 100%) of paraffin adulteration. The
These samples were used for the establishment overall fingerprint region was used for that purpose.
of calibration (standard) curves exhibiting a rela- The same procedure was performed on beeswax-
tionship between the measurement response and beef tallow mixtures.
the known concentrations of the analyte. Virgin Additionally, the IR spectra of microcrystalline wax,
beeswax (wild-built combs) collected from 7 hives, stearic acid, and carnauba wax were recorded. This
and Kemig paraffin wax (with a melting point of was done to create a reference spectra collection
56°C) were used for the mixture preparation (w/w). for comparative qualitative detection of their
Mixtures were homogenised by melting at 90°C for potential presence in analysed comb foundation
3 h and re-solidified by allowing the mixture to cool samples. Detection of other adulterants was inves-
to room temperature. The same analytical procedure tigated by comparing the analysed foundations to
was initially assayed for the virgin beeswax-beef the IR spectra in available spectral databases (such
tallow mixtures that were prepared in the same as shellac wax and wool lanoline in the FTIR-ATR
way in order to validate the procedure on another spectral database of the University of Tartu, Estonia,
adulterant type. Institute of Chemistry).
To investigate the possible influence of a high-
FTIR-ATR spectra acquisition and data temperature treatment on the spectral features
processing of pure beeswax, a virgin beeswax sample used in
The IR spectra of all sample types (virgin beeswax, this study for mixtures preparation, was subjected
paraffin, tallow, beeswax-paraffin, and beeswax-tal- to 125°C for 45 minutes (the most commonly used
low mixtures) were recorded using an ABB Bomem treatment in the comb foundation production). After
MB102 FTIR (fourier-transform infrared) spectrom- cooling to room temperature, the sample’s spectrum
eter with a DTGS (deuterated triglycine sulfate) was recorded and compared to the one obtained
detector and CsI (cesium iodide) optics. A  single- before the exposure to 125°C.
reflection high temperature heated diamond ATR
(Attenuated Total Reflectance) system (Golden Quality assessment of beeswax using the
Gate, Specac) and beam condensing optics made of physico-chemical parameters
ZnSe were used to acquire spectra. The depth of Selected physico-chemical analyses, i.e. determina-
the beam penetration into the sample was around tion of acid number, ester number, ester/acid ratio,
2 µm. Spectra were recorded in a mid-infrared region saponification number, and melting point, were
(4000 - 400 cm-1) in accordance with the method performed according to European Pharmacopeia
described by Maia et al. (2013), including a few Commission (Council of Europe, 2007) in all samples.
modifications regarding spectra acquisition and data The relationship between the paraffin adulteration
processing. Prior to the acquisition of the spectra, level and the obtained values of the physico-chemical
each sample was left on the ATR plate for 3 minutes parameters was, thus, assessed. The results of the
to allow melting, homogenisation, and stabilisation physico-chemical parameters have been assessed
of the sample. Approximately 0.05 g of the sample according to the quality criteria proposed by Inter-
was used to acquire the sample’s IR spectrum. Two national Honey Commission (Bogdanov, 2009).
replicate spectra of each sample were recorded
using the same aliquot, and 64 scans were collected Statistical analysis
for each spectrum. A new background spectrum Chemometric modelling and statistical analyses
was acquired before each sample. To exclude the were performed using the statistical packages
noisy parts of the spectra, only the spectral range MatLab 7.11 2010b (MathWorks Inc.) and Statistica
between 3700 and 600 cm-1 was taken into consid- 7  (StatSoft Inc.). A factor–based Partial Least
eration for further data analysis. Raw IR spectra of Squares Regression (PLSR) method was used to
the analysed samples were stored and pre-analysed determine the prediction strength of FTIR-ATR spec-

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troscopy for the paraffin and tallow adulteration level assessed on the mixtures using simple regression
using the sample set of prepared beeswax-paraffin models. The paraffin share prediction performances
and beeswax-tallow mixtures for calibration. The of IR spectral data and physico-chemical measurands
model was validated on a set of samples consisting were also compared.
of 56  comb foundations of unknown chemical
background collected from the market. Model cali- RESULTS
bration performance and accuracy were evaluated
using coefficient of determination (R2) and mean FTIR-ATR spectra of virgin beeswax, paraffin,
squared error (MSE) value. For comparison purposes, and tallow
the relationship between the values of physico- Results presented in Fig. 1a show the charac-
chemical parameters and the paraffin share was teristic FTIR-ATR spectrum of genuine beeswax

Fig. 1. (a) Characteristic FTIR-ATR spectrum of virgin beeswax with assignment of underlying
absorption bands;
(b) Distinctive spectral differences between virgin beeswax, paraffin, and tallow occurring in
the fingerprint region.

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and underlying molecular vibrations. Beeswax represent valuable and indicative spectroscopic data
represents a complex organic mixture of numerous for detection of beeswax adulteration. Contrary to
compounds. Yet, only the most dominating ones the lack of these particular signals in the fingerprint
are observable in a typical IR spectrum of genuine region of a paraffin spectrum, a characteristic IR
beeswax; mostly analyte signals belonging to hy- spectrum of beef tallow is dominated by the strong
drocarbons, esters and free fatty acids (Fig. 1a). A intensive absorption bends that correspond to the
typical FTIR-ATR spectrum of paraffin in comparison esters vibrations, occurring at 1745 and 1158
to characteristic virgin beeswax is presented in Fig. cm-1. These signals are considerably stronger and
1b. It is characterised by a simple molecular structure characterised by the absorption maxima shifts in
related to hydrocarbon absorption bands (at 2921, comparison to beeswax signals (Fig. 1b).
2852, 1464 and 719 cm-1). The main spectral dif-
ferences between pure beeswax and paraffin are Spectral features (FTIR-ATR) of beeswax
observed in the fingerprint region. The analyte mixtures with paraffin and tallow
signals related to the ester and free fatty acids As presented in Fig. 2a, IR spectra of prepared virgin
vibrations (at 1739, 1714 and 1172 cm-1) are not beeswax-paraffin mixtures containing different
present in the IR spectrum of paraffin, and therefore, proportions of paraffin (5 - 95%), revealed a clear

Fig. 2. (a) IR spectra of virgin beeswax, paraffin, and beeswax-paraffin mixtures containing different pro-
portions of paraffin with an emphasis on indicative spectral regions; (b) Spectral variations between
different virgin beeswax samples.
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 Svečnjak et al. Detection of beeswax adulteration by FTIR-ATR
linear decrease in the absorption intensities of the the most variable part of a virgin beeswax spectra
lipid components following the increasing percent- belongs to the absorption vibrations of alcohols and
ages of the paraffin. These effects were observed amines, observed in the spectral region between
in the spectral region between 1765 and 1700 cm-1 1130 and 970 cm-1. Furthermore, no spectral dif-
(esters and free fatty acids; absorption maximums ferences between samples of genuine beeswax
at 1739 cm-1 and 1714 cm-1, respectively) and before and after the temperature treatment (125°C,
1300 and 1130 cm-1 (esters; absorption maximum 45  minutes) were determined (results not shown).
at 1172 cm-1). As presented in Fig. 3, the results showed a similar
When comparing the spectral variations identified in detection accuracy for the tallow adulteration
different virgin beeswax samples (n = 21) (Fig.  2b) level in beeswax-beef tallow mixtures and linear
with absorption changes observed in beeswax- behaviour where IR absorption intensities (most
paraffin mixtures, it is obvious that the spectral prominent in the spectral ranges between 1745 -
variations determined in different beeswaxes do 1739 cm-1 and 1172 - 1158 cm-1) increased linearly
not intercept those crucial for detection of beeswax for the increasing percentages of the tallow added
adulteration with paraffin. As presented in Fig. 2b, to beeswax (Fig. 3a).

Fig. 3. (a) IR spectra of virgin beeswax, beef tallow, and beeswax-tallow mixtures (b) PLSR calibration plot of the real beef-
tallow share values versus FTIR-ATR predicted values.

Fig. 4. Prediction performance parameters of PLSR model constructed for determination of the
paraffin share in beeswax: (a) PLSR calibration plot of the real-paraffin share values versus
FTIR-ATR predicted values; (b) Effects of the first PLS latent component.

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Detection of adulterants in beeswax by samples exhibited a higher level of paraffin adulter-
FTIR-ATR spectroscopy ation (>46% of paraffin), while the majority of the
Partial least squares regression (PLSR) calibra- analysed samples (50%) were found to be adulterat-
tion and model performance parameters of the ed with 5 - 20% of paraffin. The red line represents
paraffin share in adulterated mixtures predicted the limit-area that distinguishes genuine beeswax
by IR spectral data are presented in Fig. 4a. The samples (below the red line) from adulterated ones.
results obtained by using the fingerprint region have The results have revealed paraffin (and/or microc-
revealed a strong predictive strength (R2  =  0.999) rystalline wax) as the only adulterants detected in
and good calibration accuracy (MSE = 2.173) of the studied comb foundations. No other adulterant
FTIR spectroscopy for detection of the paraffin traces (no spectral features indicating the presence
share in beeswax. An investigation of PLS latent of other adulterants) were found in the collected
components revealed that most of the variance in comb foundations, based on the qualitative
the spectral data set is explained by the first 6 latent inspection of their IR spectra and comparison to IR
components. Most of the information (99.84%) was spectra of the most common adulterants (personal
captured in eigenvalues compressed in the first PLS spectral collection and the literature). These obser-
component, which correspond to the alterations of vations confirm the prevalence of paraffin adultera-
esters (~1739 cm-1 and 1172 cm-1) and free fatty tion on the beeswax market.
acid (~1714 cm-1) absorption bands (Fig. 4b). Partial
least squares regression calibration and model per- Measurements of physico-chemical parameters
formance parameters of the tallow share predicted used for detection of beeswax adulteration
by IR spectra (Fig. 3b) also revealed good predictive Descriptive statistics for analysed physico-chemical
strength (R2 = 0.999) and calibration accuracy parameters for virgin beeswax and comb foundations
(MSE = 1.247). collected from the international market is presented
A constructed PLSR model was used to predict the in Table 2. The results revealed anomalous values
paraffin share in comb foundation sample set in for particular virgin beeswax samples in which the
which the samples were of an unknown chemical values were out of the range officially proposed for
background. The results of the distribution of pure beeswax. Higher acid number values (>22 mg
comb foundation samples according to the paraffin KOH/g) were determined for 8 samples. Values of sa-
proportion predicted by the IR spectra, are presented ponification and ester number outside the proposed
in Fig. 5. As summarised and categorised in Tab. 1, ranges were found for 5 virgin beeswax samples,
the majority of the analysed samples (89%) were reaching the maximum value of 134 and 112.2 mg
adulterated with paraffin. Only 6 out of 56 (11%) KOH/g, respectively.
samples were identified as virgin beeswax, 28% of

Fig. 5. PLSR prediction of the paraffin share in comb foundation samples (n = 56) using FTIR-ATR
spectra with the fingerprint region (1800-900 cm-1).

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The results showing the correlation between the the paraffin proportion proved to be more accurate
values of the analysed physico-chemical parameters (Fig. 4a) in comparison to acid number prediction
and paraffin share in beeswax-paraffin mixtures performance (Fig. 6a). Thus, it can be concluded that
are presented in Fig. 6. It is clear from the scatter such a  discrepancy is related to the acid number
plot presented in Figure 6a that as the paraffin anomalies.
share increases, the acid value tends to decrease
(negative correlation). This strong linear correlation DISCUSSION
between acid number and paraffin share indicates a
good predictive strength of acid number for paraffin Infrared (IR) spectroscopy permits acquisition of the
share (R2 = 0.989) in beeswax-paraffin mixtures. absorption spectra of compounds that are a  unique
However, the changes of other physico-chemical reflection of their molecular structure. An infrared
values as the paraffin share increases did not follow spectrum of a compound provides a unique “fin-
the linear pattern well. Ester number (R2 = 0.788) gerprint” which is readily distinguished from the
and saponification number (R2 = 0.883) showed absorption patterns of all other compounds and
a moderate linear correlation with the paraffin share, it is, therefore, a valuable tool for identification
as presented in Figure 6b and 6c, respectively. On and analysis of different substances in organic
the other hand, the ester/acid ratio (R2 = 0.611) specimens. The method and observations reported
and melting point (R2 = 0.564) exhibited almost no by Maia et al. (2013) provided a valuable foundation
linear correlation with significant deviations (Fig. 6d and guidelines for further research; establishment
and 6e). of calibration curves (covering the entire concentra-
The relationship between the prediction of the tion range of adulteration: 0 - 100%) and validation
paraffin share in comb foundation samples by of analytical procedure on unknown samples, which
IR spectral data and by acid number (as the was performed in this study. The entire fingerprint
only parameter showing consistent results with region (1800 - 900 cm-1) comprising the crucial
respect to paraffin share detection) is presented in absorbance intensity changes (at 1739, 1714 and
Figure 7. It is obvious that the results obtained by 1172 cm-1) was used in this study for chemomet-
two predictors are not congruent in paraffin share ric modelling. In comparison to the investigation
determination. The linear fit of IR spectral data for approach demonstrated in the feasibility study by
Table 1.
Distribution of analysed comb foundation samples (n = 56) by adulteration category
Adulteration category Paraffin share n of samples % of samples
High level >71% 12 21
Higher middle level 46 - 70% 4 7
Lower middle level 21 - 45% 6 11
Low level 5 - 20% 28 50
Genuine beeswax < 5% 6 11

Table 2.
Physico-chemical parameters determined in virgin beeswax and comb foundation from the international market
Sample Physico-chemical parameter n Min Max Mean SD CV
Melting point (°C) 17 60 65 62.74 1.64 0.03
beeswax

Acid number (mg KOH/g) 15 20.70 30.20 25.74 3.88 0.15

Virgin

Ester number (mg KOH/g) 11 31.10 112.20 74.46 23.13 0.31

Ester/acid ratio 11 1.18 5.14 3.01 1.11 0.37
Saponification number (mg KOH/g) 11 57.50 134.00 99.45 22.12 0.22
Melting point (°C) 55 48 64 57.23 3.97 0.07
foundations

Acid number (mg KOH/g) 55 0.86 25.80 16.04 8.05 0.50

Comb

Ester number (mg KOH/g) 53 1.40 92.50 48.89 26.39 0.54

Ester/acid ratio 53 0.46 4.95 2.92 0.94 0.32
Saponification number (mg KOH/g) 53 4.20 117.70 65.24 33.80 0.52

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Fig. 6. Simple regression scatter plots showing the relationship between the values of physico-chemical
measurands and the paraffin share in mixtures: (a) Acid number; (b) Ester number; (c) Saponifica-
tion number; (d) Ester/acid ratio; (e) Melting point.

Maia et al. (2013) that involved the development of could not be distinguished by FTIR-ATR. These
a quality index based on the calculation of chosen findings were reported by Maia et al. (2013) as well.
absorbance ratios (I1739 cm-1 / I2852 cm-1, I1714 cm-1 / I2852 It can be assumed that such effects appear due
cm-1
and I1739 cm-1 / I1714 cm-1), a different spectral data to similar spectral features arising from the same
processing approach was performed in this study. We petroleum-based origin (both waxes are made of
used this approach to make the analytical procedure long-chain alkane hydrocarbons; microcrystalline
simpler, faster, and applicable for routine purposes. wax is additionally characterised by very fine crys-
It was observed in this study that adulteration of talline structure). Given that the price of microcrys-
beeswax with paraffin and microcrystalline wax talline wax is almost the same as beeswax price, it

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Fig. 7. Comparison of the paraffin share prediction performance by FTIR-ATR

spectral data and acid number for comb foundation samples.
is unlikely from the economical point of view that different virgin beeswax samples in the spectral
beeswax is being adulterated with a substance of region indicative for paraffin detection. The samples
equal price to a larger degree. Thus, it can be assumed exhibiting less than a 5% share of paraffin were,
that paraffin prevails as a beeswax adulterant due therefore, identified as genuine beeswax. The mean
to its significantly lower price. As far as adultera- squared error (MSE) value (2.173%) calculated in
tion goes, though, it is irrelevant which substance is this study and the 5% detection limit determined
added to beeswax since both substances represent for paraffin in previous spectroscopic findings (Maia
contaminants that should not be present in beeswax. et al., 2013), were in this regard taken into account.
The spectral analysis of genuine beeswax samples The quality criteria for routine beeswax testing
revealed that no particular spectral differences proposed by the IHC (International Honey
appear when comparing white and yellow beeswax Commission), defined range values for 10  physico-
samples. Beeswax colour is mostly related to the chemical parameters. Values of saponification and
fat-soluble carotenoid pigments that originate from ester number outside the proposed range-values
pollen, but various undefined contaminants that are (87 - 102 mg KOH/g and 70 - 90 mg KOH/g, respec-
collected and absorbed in the beeswax over time tively) were determined in this study for 5 virgin
may also affect the colour (Winston, 1991; Berry beeswax samples, which resulted in anomalous
and Delaplane, 2001). These substances appear in minimum and maximum values. Additionally,
beeswax in traces, so one can assume that they higher acid number values (>22 mg KOH/g) were
provide little or no IR information. It could also be also observed for 8 samples. Nevertheless, the
that the signals of these substances are overlapped same samples did not show deviations in their IR
by more prominent absorption bands, and thus, do spectra; they exhibited spectrum characteristic for
not compromise chemical analyte signals favourable pure beeswax. The beeswax samples used in this
for adulteration detection. study were definitely authentic (collected directly
According to the prediction of the paraffin share in from the experimental beehives, containing no
comb foundation samples based on their IR spectral unnatural residues). Thus, the results suggest that
data, 6 samples were placed below the 5% detection the definition and identification of pure beeswax by
limit reported for paraffin. It can be assumed, that currently proposed physico-chemical values is not
the paraffin share that was less than 5% was ten- reliable and may lead to a wrong interpretation of
tatively detected in those samples due to minor a sample‘s authenticity. As expected, values of the
absorbance variations that might occur among proposed physico-chemical parameters obtained for

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comb foundation revealed even a higher degree of on a larger data set of both virgin beeswax and
inconsistencies and anomalous values. Similar results different beeswax adulterant types. To the best of
were reported by Bernal et al. (2005) who investigat- our knowledge, no method with a calibration set
ed the influence of the relative amounts (2  - 50%) covering the entire concentration range of paraffin
of adulterants (paraffin, stearic acid, animal tallow, and tallow adulteration in beeswax was employed
and carnauba wax) on the values of the commonly until now. It was a crucial step prior to the establish-
used physico-chemical parameters. The results have ment of a protocol that provides accurate determina-
revealed inconsistency and a frequent appearance tion of the share of adulterants in beeswax.
of anomalous values for the majority of the analysed It can be assumed, that adulterated beeswax
parameters. foundation can cause economic losses in beekeeping.
Furthermore, the reliability of the proposed It was reported that adulterated beeswax foundation
analytical values for pure beeswax characterisation may have an impact on brood rearing. The results can
and detection of adulteration is jeopardised by the be abnormalities and disorders of brood development,
temperature treatment >100°C which can cause and consequently, weakness and increased mortality
a  significant increase in ester value and decreases of honeybee colonies (Wallner, 2005). Frequent
in acid value (Tulloch, 1973; Bogdanov, 2009). reports on deformed or completely destroyed combs
Such effects inevitably occur in practice (comb by the Croatian beekeepers (personal communica-
foundation production process) given that beeswax tion) indicate the above described effects might
is commonly being subjected to different high tem- be possible. An investigation on this subject was
perature treatments (121 - 140°C) necessary to kill recently launched by Semkiw and Skubida (2013),
the spores of the heat-resistant Paenibacillus larvae investigating the comb construction and brood de-
larvae. velopment on beeswax foundation adulterated with
The results of this research revealed disturbing paraffin. The results have revealed that paraffin
findings; 89% of comb foundation samples collected used in their experiment had no negative impact on
from the market were found to be adulterated with the bee colonies. However, the authors emphasized
paraffin. We believe that this kind of outcome is the that it is impossible to exclude the possibility that
consequence of the larger-scale problem of a general paraffin of different chemical compositions and
deficit of beeswax and a “chronic” accumulation and purities may cause harmful effects on bees.
circulation of the paraffin in the comb foundation We believe that the fact that adulteration of any
production process. The lack of routine analytical product represents deliberate destruction of the
tools, and regulations controlling the beeswax origin product‘s quality aiming to achieve an illegal profit
or any kind of its monitoring, definitely contribute and deceive consumers / beekeepers, along with
to this issue. The chemical composition of beeswax indications on negative effects of adulterated foun-
is generally not being controlled routinely, and dations on honeybee colonies, should trigger the
therefore, allows continuous and successive contam- general initiative for routine monitoring of the au-
ination of beeswax by adulterants, primarily paraffin. thenticity of the beeswax comb foundations present
Similarly to the results obtained in this study, a high on the market.
percentage of comb foundation samples collected
from the Portuguese market (60%) were adulter- CONCLUSIONS
ated with paraffin/microcrystalline wax, as reported
by Maia et al. (2013). The disconcerting situation on In this study we demonstrated that an analytical
the comb foundation market, indicated by current approach defining the standard curves for particular
and previous studies, implies an urgent need for adulteration levels (5 - 95%) based on chemomet-
routine beeswax authenticity control. ric modelling of specific spectral data set indicative
The objective and results presented in this study fit for beeswax adulteration (fingerprint region), allows
the current objective of the IHC; to work out standards the same contamination levels to be determined for
for other bee products besides honey, including the unknown samples (comb foundations).
development of better and new analysis methods. The PLS regression model performance parameters
The FTIR-ATR analytical procedure and adultera- showed the strong predictive power of FTIR-ATR
tion-level categorisation, established in this study spectroscopy for predicting the share of paraffin
for distinguishing pure from adulterated beeswax, and tallow in beeswax (R2 = 0.999). The predictive
can be used as a guideline to further upgrade of power indicates the analytical reliability of this
this method. But verification should be performed method for routine quality control. Moreover, good

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 Svečnjak et al. Detection of beeswax adulteration by FTIR-ATR
calibration performance and detection accuracy for Bernal J. L., Jiménez J. J., del Nozal M. J., Toribio L., Martín
the paraffin and tallow adulteration levels initially M. T. (2005) Physico-chemical parameters for the char-
investigated in this study, strongly suggest the pos- acterization of pure beeswax and detection of adultera-
sibility of developing the same analytical protocol tions. European Journal of Lipid Science and Technology
for other commonly used beeswax adulterants. On 107(3): 158-166. DOI: 10.1002/ejlt.200401105
the other hand, unsatisfactory regression perform-
ance parameters obtained for the correlation inves- Berry A. J., Delaplane S. K. (2001) Effects of comb age on
tigation between the values of the studied physico- honey bee colony growth and brood survivorship. Jour-
chemical parameters and paraffin share in mixtures, nal of Apicultural Research 40(1): 3-8. DOI: 10.1002/
confirmed the unsuitability of existing physico- ejlt.200401105
chemical analytical tools for reliable detection of
adulterants in beeswax (with the exception of acid Beverly M. B., Kay P. T., Voorhees K. J. (1995) Principal
number). component analysis of the pyrolysis mass spectra from
Besides providing results that are accurate, African, Africanized hybrid and European beeswax. Jour-
consistent, comparable, and within acceptable limits nal of Analytical and Applied Pyrolysis 34(2): 251-263.
of precision, the advantages of the demonstrated DOI: 10.1016/0165-2370(95)00891-H
method over commonly used methods are that it is
fast, reliable, cheap, and easy-to-use. Also, FTIR-ATR Birshtein V. Y, Tul‘chinskii V. M. (1977) Determination of
measurements require no sample preparation (rea- beeswax and some impurities by IR spectroscopy. Chem-
gent-free method) and only a small amount of the istry of Natural Compounds 13(2): 232-235.
sample (~0.05g). These factors further support the
applicability of the proposed FTIR-ATR analytical Bogdanov S. (2004a) Beeswax: quality issues today. Bee
procedure for routine beeswax analysis and quality World 85(3): 46-50.
control.
Bogdanov S. (2004b) Quality and standards of pollen
ACNOWLEDGMENTS and beeswax. Apiacta 38: 334-341.

This work has been supported in part by the Croatian Bogdanov S. (2009) Beeswax: Production, properties,
Science Foundation under the project NaPro-Flav composition and control. Beeswax book. Bee Product
(8547). The research was also supported by the Science. 17 pp.
Paying Agency for Agriculture, Fisheries and Rural
Development and VIP Project No. 2013-12-62. The Breed M. D., Garry M. F., Pearce A. N., Hibbard B. E., Bjostad
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also thank the Croatian Beekeepers Association and bee nestmate recognition. Animal Behaviour 50(2): 489-
Mr. Predrag Manger for the sampling support. 496. DOI: 10.1006/anbe.1995.0263

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