Detection of beeswax adulterations using concentration guide-values

Juan José JiménezJosé Luis BernalMaría Jesús del NozalLaura ToribioJosé Bernal

Department of AnalyticalChemistry,Faculty of Sciences,University of Valladolid,Valladolid, Spain

Detection of beeswax adulterations usingconcentration guide-values

The concentrations of 102 chemical compounds (saturated and unsaturated hydro-carbons, palmitates, total and free acids, total hydroxyacids, total and free alcohols,acidic monoesters and monoesterified 1,2,3-propanetriols) have been determined byGC/FID on white and yellow comb beeswax of Apis mellifera from different regions ofSpain. Guide-value ranges are proposed for its characterization and to discriminateadulterated foundation beeswax sheets. The concentrations of many compoundsresulted to be statistically different for white and yellow beeswaxes, while the obser-vation of concentrations out of normal in some marketed foundation beeswax sheetssuggested their adulteration. However, the measurement of anomalous concentrationsin foundation beeswax sheets did not imply necessarily their rejection by the bees.

Keywords: Beeswax, organic compounds, quality, adulteration.

682 DOI 10.1002/ejlt.200600308 Eur. J. Lipid Sci. Technol. 109 (2007) 682–690

1 Introduction

Beeswax is a natural product with multiple applications;among them, it is interesting to emphasize its use asfoundation beeswax sheets to facilitate the work of thebees in the hive. Sometimes, beekeepers observe arejection of these sheets on the part of the bees and, inconsequence, there is an economical loss because thesheets must be substituted by new ones; and perhaps,most importantly, the beehive yield decreases. Trying toclarify the causes of the rejection, we have supposed thatin the course of their manufacture, new compounds areadded or the initially present ones are altered. For thisreason, we have intended to assess the composition ofpure beeswax and the possible composition differenceswith respect to those sheets that are rejected in the bee-hives. To this aim, an appropriate number of pure bees-wax samples have been selected to estimate the con-centration ranges of some organic compounds and totest the concentration values obtained in the problematicsheets.

The characterization of the quantitative composition ofbeeswax has usually been carried out in weight percent-ages for each organic compound family after isolatingthem by using adsorption column or thin-layer chroma-tography techniques. Thus, it has been stated that itcontains hydrocarbons (14%), monoesters (35%; pre-dominantly palmitates), free acids (13%), free alcohols

(1%), total hydroxyacids (11%), total acids (30%), totalalcohols (31%) and diols (3%), which are their total con-centrations measured after beeswax hydrolysis [1–7].

Another possibility to characterize the organic composi-tion of beeswax involves the determination of specificcompounds by gas chromatography (GC). The amount ofeach compound can be expressed in peak area, relativepercentage (normalization method) or in weight percent-age against an internal standard [1, 3–5, 8–11]. Withregard to this latter option, eicosane, methyl eicosanoate,octadecyl eicosanoate, p-dioctylbenzene and octadecyloctadecanoate have been used as a reference to deter-mine the compound concentration. Each one of them hasbeen used to quantify a specific family of compounds [5,8].

As it has been previously stated, many batches of foun-dation beeswax sheets are rejected or badly accepted bythe honeybees, and the addition of those strange prod-ucts is commonly thought to be the cause of this issue. Inthis respect, the measurement of physicochemical pa-rameters is usually carried out to discriminate adulteratedbeeswax, but the finding of anomalous values in theanalysis of certain samples has not resulted as useful toexplain the rejection of some foundation sheets observedin the beehives [12].

In this work, the concentrations of 102, free or total,compounds from beeswax of Apis mellifera are obtainedby GC/flame ionization detection (FID), to achieve a bettercharacterization of pure beeswax and to propose con-centration guide-values in weight percentage – referringto an internal standard: octadecyl octadecanoate. Thestudied compounds belong to different chemical families:

Correspondence: Juan José Jiménez, Department of AnalyticalChemistry, Faculty of Sciences, University of Valladolid, Prado dela Magdalena s/n, 47005 Valladolid, Spain. Phone: 134983423262, Fax: 134 983423013, e-mail: [email protected]

© 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.ejlst.com

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Eur. J. Lipid Sci. Technol. 109 (2007) 682–690 Detection of beeswax adulterations 683

alkanes, alkenes, palmitates of long-chain alcohols, freeacids and alcohols, total acids, alcohols and hydroxy-acids, and monoesterifed 1,2,3-propanetriols. The com-pounds are also determined in rejected and non-rejectedmarketed foundation beeswax sheets, to compare theircomposition and discuss their quality on the basis of theproposed guide-values. Furthermore, a distinction be-tween white and yellow comb beeswaxes is made toknow if there are differences in composition, a subjectthat has only been tackled by the analysis of physico-chemical parameters [12].

2 Experimental

2.1 Material and reagents

Residue analysis-grade chloroform, n-hexane, acetoni-trile and methanol were supplied by Labscan (Dublin, Ire-land). Octadecyl octadecanoate (99% minimum purity)from Sigma Aldrich (St. Louis, MO, USA) was used as aninternal standard. Some hydrocarbon, fatty acid methylester and fatty alcohol acetate standards were alsoobtained from Sigma Aldrich.

Boron trifluoride in methanol (20%), acetic anhydride,pyridine and potassium carbonate were obtained fromMerck (Darmstadt, Germany), and an analytical balanceMettler AE240 was provided by Mettler Toledo (Toledo,OH, USA). Ultrapure water was obtained from a Milli-Ro 6plus apparatus (Millipore, Mildford, MA, USA). All gaseswere supplied by Carburos Metalicos (Barcelona, Spain).A stove and mechanical shakers were obtained fromSelecta (Barcelona, Spain). Finally, an RE-111 rotaryevaporator was provided by Büchi (Plawil, Switzerland)and a 5810R centrifuge was purchased from Eppendorf(Hamburg, Germany).

2.2 Beeswax sampling and purification

Honeycomb beeswaxes from Apis mellifera collectedfrom beehives of different provinces of Spain were ana-lyzed. Sixteen beekeepers from several Spanish regionsgently collected 22 honeycombs and placed them inboiling water to melt the comb beeswax. Then, most ofthe comb debris was separated from the beeswax bysedimentation, and beeswax samples were sent to thelaboratory. The beeswax samples received in the labora-tory still contained rests of the beehive and required afurther purification. To this end, beeswaxes were purifiedby melting. Thus, beeswax was added to a beaker withboiling water, in proportion 100 g beeswax/L, and heatedfor 20 min. Then, the mixture was cooled at room tem-perature, the beeswax (less dense) solidified over the

water, and the impurities placed at the bottom of the soli-dified beeswax were removed with a scraper. Then, thepurified beeswax was collected and subjected to thesame treatment with fresh water twice more.

Comb beeswaxes were classified into two groupsaccording to their color: white (n = 9) or yellow (n = 12)ones. White beeswax came directly from the bee scalesbecause beekeepers had placed an empty frame in abeehive from their apiaries.

Beekeepers also sent us 52 foundation beeswax samplesobtained from different Spanish manufacturers. Of the52 foundation beeswax sheets, 27 had been rejected orbadly accepted in the beehives. We thought that thisproportion of problematical sheets does not reflect thereal occurrence. These samples were directly analyzed,without any purification step. All samples were kept atroom temperature and darkness until their analysis.

2.3 Sample preparation

The sample preparation methods used in this work hadbeen tested with the aim of characterizing the beeswax ina previous manuscript, in which they were shown in detail[13]. These methods are briefly described now. Hydro-carbons and palmitates were directly analyzed by high-temperature GC after dissolving beeswax in chloroform at1000 mg/L and adding octadecyl octadecanoate as aninternal standard at a concentration of 2 wt-% to quantifythe analytes.

The total content of acids was estimated by simultaneoushydrolysis and methylation with BF3 in methanol. Thus,beeswax dissolved in 4 mL chloroform at 1000 mg/L wasadded to 2 mL methanol and 2 mL BF3 in methanol at20%. Then, the solution was heated at 90 7C for 1 h and2 mL water was added to remove the excess of reagent.Finally, the chloroform phase was collected and a volumeof 10 mL octadecyl octadecanote was added to obtain aconcentration of 2 wt-%.

The total concentrations of alcohols and hydroxyacidswere determined after acetylating the chloroform solutionthat contained the methylated acids and released alco-hols. To this end, beeswax was subjected to the above-described transesterification, and a 2-mL chloroform ali-quot was mixed with 0.5 mL acetic anhydride and 50 mLpyridine. The solution was heated at 90 7C for 2 h, andthen a 1 M potassium carbonate solution was added toremove the excess of anhydride. After separating thephases by centrifugation, the organic layer was collectedand injected into the gas chromatograph.


684 J. J. Jiménez et al. Eur. J. Lipid Sci. Technol. 109 (2007) 682–690

A volume of 2 mL beeswax solution in chloroform(1000 mg/L) was acetylated as mentioned above, todetermine the concentrations of free alcohols by GC.

The free acids were extracted from beeswax, dissolved in4 mL n-hexane (always 1000 mg/L), with three portions of10 mL acetonitrile by mechanical shaking for 10 min. Thethree acetonitrile portions were combined and evapo-rated to dryness in a rotary evaporator at 35 7C and theresidue was dissolved in 4 mL chloroform. Then, theacids were methylated with BF3/methanol according tothe above-mentioned procedure and the internal stand-ard was also added to the derivatized extract.

Chloroform aliquots of 2 mL containing the methylatedfree acids were used to determine the content of mono-esterified propanetriols. To this purpose, the aliquots wereacetylated with acetic anhydride in the presence of pyr-idine as described in the alcohol analysis.

2.4 Determination by GC with FID

Analyses were made in a Hewlett-Packard 4890 high-temperature gas chromatograph (Little Falls, Wilmington,DE, USA) equipped with a flame ionization detector andan HP 7673A automatic sampler. A 25 m60.32 mm high-temperature column coated with 0.1 mm 5% phe-nylmethyl polysiloxane from SGE (Ringwood, Australia)was used in the determination of hydrocarbons, palmi-tates and free alcohols, in combination with the followingoven temperature program: initial temperature 50 7C, heldfor 2 min, a 50 7C/min ramp to 180 7C, then a 3.8 7C/minramp to 300 7C, and finally a 6 7C/min ramp to 420 7C,held for 5 min. The temperature of the detector was440 7C.

For the total content of acids and alcohols, a30 m60.32 mm column coated with 0.25 mm 5% phe-nylmethylpolysiloxane from Agilent Technologies (PaloAlto, CA, USA) was used, while a 25 m60.32 mm columnfrom Phenomenex (Torrance, CA, USA) coated with0.1 mm of the same stationary phase was used for the freeacid and monoesterified propanetriol determinations. Inthese cases, the oven temperature program was as fol-lows: initial temperature 50 7C, held for 2 min, then a 5 7C/min ramp to 310 7C, held for 15 min. The detector tem-perature was 325 7C.

The carrier gas (He) flow rate was 1 mL/min, measured at50 7C. Helium (30 mL/min), hydrogen (35 mL/min) andsynthetic air (350 mL/min) were used as auxiliary gasesfor the flame ionization detector. A 2-mL sample wasalways injected in splitless mode at an injection porttemperature of 250 7C. The purge valve was turned on at1.50 min after the injection.

The analytes were identified by the comparison of samplechromatograms obtained in GC-FID with those obtainedin GC-MS [14, 15], and by comparison of chromato-graphic peak retention times in samples with thoseobtained by injecting analytical standards for some com-pounds.

3 Results and discussion

3.1 Hydrocarbon and palmitate content inbeeswax

Tab. 1 resumes the concentration data obtained in theanalysis of hydrocarbons and palmitates in pure bees-wax; it also shows the concentration range of each com-pound in pure beeswaxes (white and yellow ones togeth-er) expressed in percent (wt-%) and related to the addedinternal standard. These ranges have been establishedtaking into account the minimum and maximum values foreach compound found in the analysis of those bees-waxes; they are proposed as guide-values to distinguishbetween pure and adulterated beeswaxes.

The hydrocarbon and palmitate concentrations of most ofthe 25 foundation beeswax sheets accepted in the bee-hives were within the proposed guide-value ranges.However, most of the 27 problematic foundation bees-waxes had anomalous concentrations, out of the guide-value ranges.

Fig. 1a, b shows mean concentrations of hydrocarbons inwhite, yellow and problematic foundation beeswaxes.The abbreviations of the corresponding compounds canbe found in the tables. As regards the saturated aliphatichydrocarbons with an odd number of carbon atoms, H27

was the most abundant hydrocarbon followed by H29, H31

and H25 in decreasing order. It was also observed thatodd-chain aliphatic hydrocarbons were less abundant inwhite beeswax in comparison with yellow beeswax –except for H27 – and, moreover, that problematic founda-tion beeswax sheets had the highest concentrations. Thehydrocarbons with an even carbon number were minorcompounds in white and yellow beeswaxes, with theconcentrations being higher in the last ones, too; thecontents of these hydrocarbons in the problematic foun-dation beeswaxes were notably higher in relation to thepure ones.

An ANOVA confirmed that there were significant differ-ences (p ,0.001) in the concentrations of H23, H24, H25,H26, H28, H29, H30, H31, H32, H33 and H35 among the threebeeswax types. When only the white and yellow bees-waxes were considered, there were also differences in theconcentrations of H22, H23, H24, H25, H29, H31, H32, H33 and



Tab. 1. Guide-values of concentration (in wt-%, relatedto the internal standard) proposed for hydrocarbons andpalmitates esterified with long-chain alcohols in purebeeswax.

Abbrev-iation

Compound Concentrationguide-values[wt-%]

H17 Heptadecane 0.03–0.22H19 Nonadecane 0.08–0.64H20 Eicosane 0.03–0.13H21 Heneicosene 0.19–0.61H22 Docosane 0.02–0.13H23:1 Tricosene 0.03–0.18H23 Tricosane 0.69–1.33H24 Tetracosane 0.04–0.18H25:1 Pentacosene 0.05–0.18H25 Pentacosane 1.26–1.78H26 Hexacosane 0.18–0.35H27:1a Heptacosene 0.05–0.12H27:1b Heptacosene isomer 0.09–0.21H27 Heptacosane 2.55–3.20H28 Octacosane 0.14–0.37H29:1 Nonacosene 0.06–0.52H29 Nonacosane 1.87–2.68H30:1a Triacontene 0.02–0.07H30:1b Triacontene isomer 0.02–0.05H30 Triacontane 0.11–0.31H31:2 Hentriacontadiene 0.01–0.08H31:1a Hentriacontene 0.61–0.89H31:1b Hentriacontene isomer 1.05–1.58H31 Hentriacontane 1.62–2.45H32:1 Dotriacontene 0.06–0.12H32 Dotriacontane 0.04–0.14H33:2 Tritiacontadiene 0.01–0.08H33:1a Tritriacontene 1.19–1.82H33:1b Tritriacontene isomer 0.06–0.38H33 Tritriacontane 0.34–0.72H35:1a Pentatriacontene 0.03–0.16H35:1b Pentatriacontene isomer 0.02–0.08H35 Pentatriacontane 0.01–0.09C16–22 Docosyl hexadecacanoate 0.17–0.31C16–24 Tetracosyl hexadecanoate 3.12–3.74C16–26 Hexacosyl hexadecanoate 2.10–2.85C16–28 Octacosyl hexadecanoate 2.02–2.62C16–30 Triacontyl hexadecanoate 2.40–3.52C16–32 Dotriacontyl hexadecanoate 1.55–2.62C16–34 Tetratriacontyl hexadecanoate 0.54–1.21C16–36 Hexatriacontyl hexadecanoate 0.07–0.26

H35, although the significance level was lower (p ,0.05).Except for the two isomers of hentriacontene (H31:1a andH31:1b) and the H33:1a isomer, the monounsaturated olefinsand the two dienes identified were minor compounds inpure beeswax, with mean concentrations lower than0.25%. The variation of the mean concentrations of theunsaturated hydrocarbons according to the beeswax

Fig. 1. Mean concentrations in white, yellow and prob-lematic foundation beeswaxes. (a) Saturated aliphatichydrocarbons with odd carbon atom number, (b) satu-rated aliphatic hydrocarbons with even carbon atomnumber, (c) palmitates.

type did not seem to follow a pattern. The mean con-centrations of H30:1a, H30:1b, H33:1b, H35:1a and H35:1b inproblematic foundation sheets were at least 30% higherthan in pure beeswaxes. On the other hand, the con-centrations of H27:1a, H27:1b, H30:1a, H30:1b, H31:1a, H32:1,H33:2, H35:1 and H35:1b were significantly different (p ,0.05)when problematic foundation beeswaxes were con-fronted with yellow pure ones.

The mean concentrations of the palmitates are shown inFig. 1c. The palmitate mean concentrations – except forC16–22 – were higher in yellow beeswaxes than in white



ones. In general terms, the concentrations of the palmi-tates in pure beeswax decreased in the following order:C16–30 . C16–24 . C16–26 . C16–28 . C16–32 . C16–34 . C16–22

. C16–36.

3.2 Acid content in beeswax

Tab. 2 shows the proposed guide-value ranges for thecontent of total and free acids, hydroxyacids and acidicmonoesters in pure beeswax. As regards the total satu-rated acids, 16:0 was the most abundant acid, asexpected, with a concentration of about 17% in purebeeswax, followed by the acids 24:0 (5%) and 28:0 (2%).The ratio between the acids 16:0 and 24:0 was about 3 : 1(see Fig. 2a); this ratio differs from the data obtained inother works [2, 3] in which a differentiation between pureand foundation beeswaxes was not made. An analysis ofvariance revealed that only the concentrations of 18:0 and22:0 were significantly different (p ,0.01) among the threetypes of beeswax: white, yellow and problematic foun-dation ones. Furthermore, the contents of 18:0, 22:0 and24:0 were significantly different between white and yellowbeeswaxes.

In relation to the unsaturated acids, 18:1 was more abun-dant than 18:2, and the mean concentrations found in yel-low beeswaxes were higher than those in white bees-waxes. Moreover, the concentrations of 18:1 and 18:2 werehigher in the problematic foundation beeswax sheets. Theconcentrations of 18:1 and 18:2 were significantly different(p ,0.05) for the white and yellow beeswaxes, while onlythe 18:2 concentration was significantly different (p ,0.05)between yellow and foundation beeswaxes.

Most of the acids detected in beeswax had an even car-bon atom number; however, a free acid with odd carbonatom number (15:0) was observed. Fig. 2b shows themean concentrations for the free acids. The most abun-dant acid was 24:0 with a concentration close to 2% inpure beeswax, followed by 16:0, a reverse concentrationorder to that observed in the content of total acids. Theamounts of free acids in yellow beeswaxes were clearlyhigher than in white beeswaxes. The problematic foun-dation beeswaxes had substantially higher concentra-tions of 16:0 and 18:1 with respect to the yellow purebeeswaxes, while the concentrations in the well-accept-ed foundation sheets were within the normal ranges formost of the samples as occurred for the other beeswaxcompounds. The concentrations of free 16:0, 18:0, 18:1,18:2, 20:0, 26:0 and 36:0 were statistically different(p ,0.05) when the three types of samples were includedin the ANOVA, while the concentration of free 15:0 wasthe only significantly different when the yellow beeswaxeswere confronted with the problematic foundation ones.

Tab. 2. Guide-values of concentration (in wt-%, relatedto the internal standard) proposed for total and free acids,hydroxyacids and acidic monoesters in pure beeswax.

Abbreviation Compound Concentrationguide-values[wt-%]

Total acids14:0 Tetradecanoic acid 0.07–0.1616:0 Hexadecanoic acid 15.03–19.2318:2 9,12-Octadecadienoic acid 0.02–0.4318:1 9-Octadecenoic acid 1.42–4.8618:0 Octadecanoic acid 0.43–0.8720:0 Eicosanoic acid 0.08–0.1622:0 Docosanoic acid 0.45–0.8524:0 Tetracosanoic acid 4.03–6.0226:0 Hexacosanoic acid 1.52–2.2028:0 Octacosanoic acid 1.62–2.4730:0 Triacontanoic acid 1.58–2.3032:0 Dotriacontanoic acid 1.43–2.6634:0 Tetratriacontaonic acid 1.16–2.0236:0 Hexatriacontanoic acid 0.11–0.26

Free acids14:0 Tetradecanoic acid 0.01–0.0615:0 Pentadecanoic acid 0.005–0.0216:0 Hexadecanoic acid 0.15–1.3618:2 9,12-Octadecadienoic acid 0.004–0.4118:1 9-Octadecenoic acid 0.03–1.4718:0 Octadecanoic acid 0.05–0.8520:0 Eicosanoic acid 0.01–0.0622:0 Docosanoic acid 0.17–0.5524:0 Tetracosanoic acid 1.16–3.8526:0 Hexacosanoic acid 0.23–0.9228:0 Octacosanoic acid 0.16–0.5730:0 Triacontanoic acid 0.09–0.2832:0 Dotriacontanoic acid 0.05–0.1434:0 Tetratriacontaonic acid 0.03–0.1336:0 Hexatriacontanoic acid 0.002–0.01

Hydroxyacids14OH-C16 14-Hydroxyhexadecanoic acid 0.62–1.2815OH-C16 15-Hydroxyhexadecanoic acid 3.13–5.9716OH-C16 16-Hydroxyhexadecanoic acid 0.04–0.0716OH-C18 16-Hydroxyoctadecanoic acid 0.12–0.2217OH-C18 17-Hydroxyoctadecanoic acid 0.23–0.4319OH-C20 19-Hydroxyeicosanoic acid 0.12–0.2221OH-C22 21-Hydroxydocosanoic acid 0.03–0.0923OH-C24 23-Hydroxytetracosanoic acid 0.23–0.50

Acidic monoesters14OH-16/C16 (1-ethyl-13-carboxytridecanoyl)

hexadecanoate0.40–0.60

15OH-16/C16 (1-methyl-14-carboxytetradecanoyl)hexadecanoate

2.33–3.47

15OH-16/C18:1 (1-methyl-14-carboxytetradecanoyl)9-octadecenoate

0.29–0.74

– Unknown 0.14–0.22



Fig. 2. Mean concentrations in white, yellow and prob-lematic foundation beeswaxes. (a) Total acids, (b) freeacids, (c) hydroxyacids.

The concentrations of hydroxyacids in problematicfoundation beeswax were lower than those in purebeeswax, as it can be seen in Fig. 2c where the meanvalues are shown. In fact, after performing an ANOVA,the concentrations of the hydroxyacids in yellow bees-wax were significantly higher (p ,0.05) with respect tothe concentrations measured in the foundations ones.The concentration of 23OH-C24 was also significantlydifferent (p ,0.05) in the comparison of white beeswaxagainst the yellow one. As regards the acidic mono-esters, their concentrations were relatively lower in theproblematic foundation beeswax and higher in the whiteone.

3.3 Alcohol content in beeswax

Tab. 3 lists the guide-values of concentration (in wt-%,and related to octadecyl octadecanoate) proposed forthe total and free alcohols detected in pure beeswax,according to the minimum and maximum concentrationsobtained for each compound in the white and yellowbeeswax analysis. The mean values of many of thesecompounds can be seen in Fig. 3. Regarding the totalalcohols, the saturated ones were predominant andwhite beeswaxes had concentrations a little higher thanyellow ones, except for C34OH. The problematic bees-wax foundation sheets had higher mean amounts of theunsaturated alcohols and C32OH than the pure bees-waxes. The concentration of C34:1OH was significantlydifferent (p ,0.05) between yellow and foundation bees-waxes.

Tab. 3. Guide-values of concentration (in wt-%, relatedto the internal standard) proposed for total and freealcohols and monoesterified propanetriols in pure bees-wax.

Abbreviation Compound Concentrationguide-values[wt-%]

Total alcoholsC22OH 1-Docosanol 0.06–0.10C24OH 1-Tetracosanol 4.94–7.27C26OH 1-Hexacosanol 3.94–5.89C28OH 1-Octacosanol 4.94–7.59C30OH 1-Triacontanol 10.45–15.44C32:1OH x-Dotriaconten-1-ol 0.29–1.17C32OH 1-Dotriacontanol 8.98–13.72C34:1OH x-Tetratriaconten-1-ol 0.09–0.38C34OH 1-Tetratriacontanol 0.82–2.22

Free alcoholsC24OH 1-Tetracosanol 0.03–0.11C26OH 1-Hexacosanol 0.03–0.09C28OH 1-Octacosanol 0.06–0.16C30OH 1-Triacontanol 0.20–0.52C32OH 1-Dotriacontanol 0.23–0.48C34OH 1-Tetratriacontanol 0.04–0.08

PropanetriolsC14/1-triol 2,3-Bis(hydroxy)propyl

tetradecanoate0.005–0.025

C16/2-triol 1,3-Bis(hydroxy)propylhexadecanoate

0.10–0.42

C16/1-triol 2,3-Bis(hydroxy)propylhexadecanoate

0.64–2.83

C18/2-triol 1,3-Bis(hydroxy)propyloctadecanoate

0.16–0.48

C18/1-triol 2,3-Bis(hydroxy)propyloctadecanoate

0.84–2.70



Fig. 3. Mean concentration in white,yellow and problematic foundationbeeswaxes. (a) Total alcohols, (b) freealcohols.

The free alcohols C30OH and C32OH were predominant.The concentration ratios between these two alcohols dif-fer according to the author [2, 3, 6, 9, 16]. In general, themean concentration of free alcohols was higher in prob-lematic foundation beeswaxes, mainly for alcohols with alow number of carbon atoms. The concentrations of freeC24OH, C26OH and C28OH were significantly higher(p ,0.01) in yellow than in white beeswax, while forC30OH, C32OH and C34OH their concentrations werehigher in white than in yellow beeswax. The concentra-tions of C24OH, C26OH, C34OH, C30OH and C32OH weresignificantly different (at p ,0.01 level for the three firstones, and p ,0.05 level for the other two compounds)between white and yellow beeswaxes. The total alcoholsC30OH, C32OH and C34OH were positively correlated withthe corresponding free alcohols (p ,0.05).

Fig. 4 shows the mean concentrations of four mono-esterified propanetriols for white, yellow and problematicfoundations. As it can be observed, C16/1-triol and C18/1-triol are the most abundant, with concentrations related to

the internal standard higher than 1 wt-%. For these twocompounds, the concentrations in yellow beeswaxeswere remarkably higher than those for white beeswaxes.

3.4 Foundation beeswax quality

From the 27 problematic samples, there were 25 forwhich the concentrations of many compounds wereclearly out of the value ranges, which suggests their mix-ture with other products. The beeswax compounds thatsurpassed the guide-value ranges coincide basically withthose whose mean values were commented in the abovesection. From those, the most frequent deviations havebeen found for saturated hydrocarbons with even andodd numbers of carbon atoms, olefins, free and totalalcohols and palmitates.

The number of compounds whose concentration wasincorrect in the problematic sheets was about 25 orhigher. In two samples, a small number of minor com-



Fig. 4. Mean concentration of monoesterified1,2,3-propanetriols in white, yellow and prob-lematic foundation beeswaxes.

pounds had anomalous concentrations; these sampleswere not unequivocally classified as adulterated, for tworeasons: (a) the delimitation of the guide-values for theminor compounds was subjected to a higher error and(b) the number of incorrect compounds was low. How-ever, it is necessary to point out that these two sampleswere adulterated according to the measurement of phy-sicochemical parameters; moreover, two samples whosephysicochemical parameters were within the normalranges now turn out to be adulterated as determined byGC [12].

As it was previously stated, the concentrations measuredin the bee-accepted foundation beeswax sheets werewithin the respective guide-values for most of the studiedcompounds and for most of the 25 samples: Only slightdeviations in the concentrations of a few minor com-pounds were found. Nevertheless, it must be remarkedthat eight samples had important concentration devia-tions for major compounds, mainly hydrocarbons. Tab. 4shows the concentration ranges found in rejected andnon-rejected beeswax sheets for some compounds.Consequently, and as happened for the measurement ofclassical parameters [12], the determination of anom-alous concentrations for some beeswax compounds isnot enough to state that the foundation beeswax will berejected by the bees. On the other hand, the compoundconcentrations in the accepted foundation sheets werecloser to those of yellow beeswax than to those of whiteones.

As the even-chain hydrocarbon concentrations seemedto be very useful to detect adulterations in the majority ofbeeswax foundation sheets, some even/odd hydro-carbon concentration ratios were calculated for adjacenthydrocarbons. These could be applied particularly todiscriminate pure beeswaxes from those adulterated withparaffin. For pure beeswax, and as a general rule, theconcentration ratios H24/H23, H24/H25, H26/H25, H26/H27,

Tab. 4. Minimum and maximum concentrations (in wt-%,related to the internal standard) found in the analysis ofsome compounds in accepted and problematic founda-tion beeswax sheets.

Compound Problematic sheets Acceptedsheets

H23 1.05–1.90 0.74–1.79H25 1.65–2.45 1.30–2.26H26 0.24–2.26 0.38–2.12H27 2.68–3.76 2.60–3.69H28 0.29–1.58 0.39–1.40H29 2.00–2.98 1.95–2.77H31:1a 0.41–0.79 0.60–0.90H33:1a 1.03–2.19 1.22–1.81C16–24 2.34–3.90 2.63–3.68C16–26 1.97–3.12 2.01–2.88C16–34 0.27–1.37 0.44–1.21Total 16:0 13.47–23.87 13.95–19.34Total 18:1 2.99–5.43 1.58–4.94Total 24:0 3.71–6.84 3.96–6.76Total 28:0 1.50–2.89 1.54–2.44Free 18:1 0.30–1.00 0.42–1.53Free 18:0 0.11–0.20 0.21–0.67Free 24:0 1.51–3.88 1.30–3.62Free 28:0 0.15–0.85 0.25–0.4915OH-C16 2.64–6.76 2.88–5.6616OH-C18 0.11–0.22 0.15–0.2021OH-C22 0.02–0.14 0.05–0.1014OH-16/C16 0.34–0.73 0.38–0.60Total C22OH 0.05–0.10 0.05–0.08Total C28OH 4.46–8.85 4.74–7.44Total C32:1OH 0.44–1.56 0.67–1.09Free C26OH 0.05–0.23 0.06–0.13Free C32OH 0.11–0.51 0.19–0.46C16/2-triol 0.07–0.34 0.11–0.39C18/1-triol 0.27–2.00 0.60–2.55

H28/H27, H28/H29, H30/H29 and H30/H31 did not exceed thevalue of 0.15. Except for five foundation beeswaxes, the



other marketed beeswaxes exceeded the proposedmaximum values for almost all the ratios.

A correlation statistical analysis done with foundationbeeswax sheets revealed that the concentrations of manysaturated hydrocarbons with even number of carbonatoms were negatively correlated with those of severalpalmitates of fatty alcohols (p ,0.05); this fact could beattributed to the expected decrease in the concentrationsof palmitates and the increase in the hydrocarbon con-centrations when paraffin is mixed with pure beeswax.

4 Conclusions

The concentration ranges of many compounds in purebeeswax have been proposed as guide-values to differ-entiate adulterated beeswaxes. It has been observed thatmost of the problematic foundation beeswaxes have highconcentrations of even-chain hydrocarbons, free alcoholsand short-chain free acids; meanwhile, the concentra-tions of 1,2,3-propanetriols and hydroxyacids decrease.

The rejection of beeswax sheets by the bees could not beassociated exclusively to the increase or decrease of theconcentrations of the studied chemical compounds; thesame happened when the values of some physicochem-ical parameters were evaluated to this aim.

The concentration ratios between hydrocarbons witheven and odd carbon atom number are useful to dis-criminate the beeswaxes mixed with paraffins. The max-imum value of some ratios for saturated hydrocarbonswith adjacent carbon atom number in pure beeswax isproposed.

Significant differences in the content of some hydro-carbons, acidic and alcoholic compounds in white andyellow beeswaxes have been established for the firsttime. So, yellow beeswax contains higher amounts ofsaturated hydrocarbons, palmitates, free acids and somehydroxyacids. In contrast, yellow beeswax has loweramounts of some total alcohols.

Acknowledgments

The authors thank the Spanish Instituto Nacional deInvestigaciones Agrarias (project API01-005) and InstitutoTecnológico Agrario from Junta de Castilla y León (projectVA12-2005) for providing funds for this investigation.

References

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[Received: December 22, 2006; accepted: April 13, 2007]


Detection of beeswax adulterations using concentration guide-values

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