physico-chemical parameters for the characterization of pure beeswax and detection of adulterations

José Luis BernalJuan José JiménezMaría Jesús del NozalLaura ToribioMaría Teresa Martín

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

Physico-chemical parameters for thecharacterization of pure beeswax and detectionof adulterations

Pure beeswaxes produced in different climatic regions of Spain have been character-ized by the determination of nine physico-chemical parameters: density, acid, saponi-fication, ester, ratio number, iodine, peroxide, melting point and ash content values.The official methods of analysis to determine density and saponification values in fatsand oils were not suitable for beeswaxes; alternative methods are proposed in thiswork for both parameters. After verifying the precision of the methods and the valueguidelines, the usefulness of the physico-chemical parameters to detect adulterationswith paraffin, stearic acid, carnauba wax and tallow was tested: adulteration percent-ages of 5%, or higher, were commonly detected. Marketed foundation beeswaxsheets, rejected or badly accepted by the bees, were also analyzed to discuss theirquality; 25 out of 27 beeswax sheets show anomalous values for at least oneparameter.

Keywords: Beeswax, quality, physico-chemical parameter analysis, adulteration.

1 Introduction

Beeswax is a natural fatty product. Nowadays, with aworld production of around 60 000 metric tons, beeswaxhas multiple uses in cosmetics, pharmacy, food and otherindustrial activities, apart from being used by beekeepers.These ones, either to make a better use of the floweringsor due to the lack of suitable means to extract the bees-wax from the combs, turn to the purchase of beeswaxfoundations sheets from industries devoted to the recy-cling of the beeswax from old combs. The quality of thefoundation beeswax sheets is one of the main concernsof apiarists and a determinant factor in the beehivedevelopment.

Some physico-chemical parameters used to analyze oilsand fats and described in the official methods of analysisfrom many pharmacopoeias and countries [1–6] arecommonly applied to evaluate the beeswax quality anddiscern possible adulterations. Melting point, acid, sapo-nification, ester and ratio number values are the mostcommonly determined parameters, whereas the peroxidevalue and iodine absorption number have also been con-sidered in a few works [7–10]. The proposed value rangesfor the parameters in pure beeswax differ from one coun-try to another [2, 5, 6]. These differences could be related

to the point of origin of the beeswax because the envi-ronmental and geographical factors play a significant rolein the bee adaptation and, as a result, in beeswax com-position [7–9].

The relatively high cost of beeswax in comparison withother vegetal or industrial waxes and lower-price prod-ucts, such as microcrystalline wax or paraffin, tallow,stearine and stearic acid, promotes its mixture with thesementioned fatty products, which is also favored by a lackof regulations [11]. The presence of these products canaffect the values of some physico-chemical parameters[8, 10–14]. Thus, several authors have discriminated theadulterated beeswaxes according to the anomalousvalues of some analytical parameters, for instance themelting point [9], the saponification value [7] or the iodinevalue [7, 12].

In this work, the feasibility and precision of the commonanalytical methods for oils and fats applied to the bees-wax have been studied, and data about the physico-chemical properties of beeswax from Apis mellifera yiel-ded in Spanish beehives are supplied, differentiatingwhite from yellow beeswaxes. In some previous manu-scripts, pure beeswaxes were not distinguished fromfoundation beeswaxes, mixing data of all of them [7, 13,15].

The measurement of physico-chemical parameters hasbeen accepted for a long time as appropriate to establishthe quality of beeswax, but it has not been verified if theirmeasurement is really efficient for this purpose. Thus, the

Correspondence: Juan José Jiménez, Department of AnalyticalChemistry, Faculty of Sciences, University of Valladolid, Prado dela Magdalena s/n, 47005-Valladolid, Spain. Phone: 134 983423262, Fax: 134 983 423013, e-mail: [email protected]

158 DOI 10.1002/ejlt.200401105 Eur. J. Lipid Sci. Technol. 107 (2005) 158–166

2005 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.ejlst.de

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Eur. J. Lipid Sci. Technol. 107 (2005) 158–166 Pure beeswax and detection of adulterations 159

influence of some probable adulterants (paraffins withdifferent melting points, stearic acid, animal tallow andcarnauba wax) and their relative amounts in the value ofphysico-chemical parameters often used to characterizethe beeswaxes is tested in this work. With these experi-ments, we try to find the cause of the rejection or badacceptation by the bees of many batches of foundationbeeswax sheets. Thus, the procedures are also applied to52 beeswax sheets, from which 25 were well accepted bythe bees and 27 were removed from the combs owing torejection observed in the beehives. The quality of thefoundation beeswax sheets is discussed according to thedeviations found in the values of the analytical parame-ters measured in the adulterant-beeswax mixtures.

2 Materials and methods

2.1 Materials and reagents

Beeswax used to study the analytical methods and threeparaffins with different melting points (54–56 7C, 58–60 7C, 68–74 7C) were obtained from Fluka (Buchs,Switzerland). Stearic acid (95% purity) and carnauba wax(refined number 1, yellow) were provided by Sigma-Aldrich (St. Louis, MO), and cow tallow was purchased inthe local market. Analysis-grade acetic acid, hydrochloricacid, sodium hydroxide, potassium iodide, sodium thio-sulfate, potassium iodate, iodine monobromide, 1% phe-nolphthalein solution in ethanol and a saturated starchsolution were obtained from Panreac (Barcelona, Spain)and Merck (Darmstadt, Germany). Analytical-grademethanol and chloroform were supplied by Lab-scan(Dublin, Ireland).

2.2 Methods for the determination ofphysico-chemical parameters

Official methods of analysis of fats and oils were used atfirst to determine the physico-chemical parameters [1].These procedures proved to be appropriate, with minimummodifications, to analyze beeswax samples, except for twoparameters: density and saponification value. Anothermethod, by Hager, was used for density [16], while it wasnecessary to make some experiments to optimize an alter-native method for the saponification value.

2.2.1 Density

A calibrated pycnometer was weighed empty first, andthen full of water. Both determinations were made at25 7C and in triplicate, to calculate the mass mean values.Then, an amount of 10.0 6 0.1 mg beeswax was placed

into a beaker containing water. As beeswax is less densethan water, the first one floats on the surface of the liquid.Next, small volumes of methanol were added from a bur-ette until beeswax was suspended into the liquid. At thatmoment, the density of the liquid matched up with that ofthe beeswax sample, so the beeswax density (in g/mL) ata temperature of 20 7C could be determined with thepycnometer by means of the expression:

density ¼ 0:9982ðm1 � m2Þ þ A� �

ðm3 � m2Þ þ A

where A = 0.0012 (m1–m2), with m1 being the mass, in g,of the pycnometer containing the hydroalcoholic mixture;m2 the mass, in g, of the empty and completely drypycnometer; and m3 the mass, in g, of the pycnometercontaining water. Three different portions of beeswaxsample were weighed to calculate the correspondingdensity values and an average for each sample.

2.2.2 Saponification value

A beeswax amount of 0.300 6 0.001 g was weighed andplaced into a 10-mL glass vial. Then, 4 mL of a 4 M NaOHaqueoussolution was added to the vial. Thevial was closedand put into an oven to carry out the saponification at100 7C for 1 h. After that, the aqueous phase was pouredinto a beaker, and the vial was washed with about 5 mLwater, which was also poured into the beaker. The solution,still hot, was titrated with a 0.5 M HCl solution, using phe-nolphthalein as indicator and constant manual shaking. Ablank assay was also conducted. The determinations weremade in triplicate. The saponification value, expressed asmg KOH/g, was calculated as follows:

saponification value ¼ 56:1MðV � V0Þ

w

where V is the volume, in mL, of HCl solution required bythe blank; V’ is the volume, in mL, of HCl solution requiredfor the sample; M is the molarity of the HCl solution; and wis the mass, in g, of the beeswax sample.

2.2.3 Acid value

A beeswax amount of 1.00 6 0.01 g was dissolved in50 mL chloroform with the help of an ultrasonic bath.Then, two drops of phenolphthalein were added and thesolution was titrated with 0.05 M NaOH in methanol withcontinuous manual shaking until indicator turning. A blankwas also titrated to correct solvent acidity. The acid value(in mg KOH/g) was calculated by the formula:

acid value ¼ 56:1MðV � V0Þ

w


160 J. L. Bernal et al. Eur. J. Lipid Sci. Technol. 107 (2005) 158–166

where V is the volume, in mL, of NaOH solution in metha-nol required by the sample; V’ is the volume, in mL, ofNaOH solution required for the blank; M is the molarity ofthe NaOH solution; and w is the mass, in g, of the bees-wax sample.

The NaOH solution was previously normalized against anaqueous solution of 0.05 M potassium acid phthalate withphenolphthalein as indicator. The analyses were alsodone in triplicate.

2.2.4 Ester value and ratio number

The ester value was determined by subtracting the acidvalue from the saponification value, and the ratio numberwas calculated as the ester value divided by the acidvalue.

2.2.5 Melting point

This index was determined following the “capillary tubemethod” [17]. Melted beeswax was introduced in a 10 cmlong 6 2 mm internal diameter thin-wall hollow capillarytube, until reaching a height of about 1 cm. Once beeswaxsolidified, the tube was kept at room temperature forabout 24 h. After that, the capillary tube, containing thebeeswax, was introduced into a bath of water that wasslowly warmed at 1–2 7C/min; the temperature waschecked with a thermometer whose bulb had to be asclose as possible to the beeswax column introduced inthe capillary tube. The melting temperature was that atwhich the beeswax was completely molten: the beeswaxliquid was entirely transparent without turbidity. The ana-lyses were done in triplicate.

2.2.6 Iodine absorption number

This parameter was determined by the method of Hanus[1, 3]. An amount of 0.300 6 0.005 g beeswax was dis-solved in 10 mL chloroform. Then, 5 mL, exactly meas-ured, of Hanus reagent (solution prepared dissolving 2 gof IBr in 100 mL acetic acid) was added to the beeswaxplaced in a beaker. The mixture was softly shaken for 30 sand then kept in darkness and at room temperature for 1 hto complete the addition of I2 to the double bonds. Afterthat, 5 mL of an 8% KI aqueous solution was added, and itwas titrated with a 0.1 M Na2S2O3 solution, with constantshaking and using starch solution as an indicator. Theindicator was added when the titration was close to thefinal point. As in the above-mentioned parameters, ablank was also conducted under the same conditions tocorrect the possible influence of the reagents. The iodine

absorption number (in g I/100 g of sample) was calculatedby the following expression:

Iodine value ¼ 1:269ðV � V0Þ

w

where V is the volume, in mL, of 0.1 M Na2S2O3 solutionrequired by the sample; V’ is the volume, in mL, of 0.1 MNa2S2O3 solution required for the blank; and w is themass, in g, of the beeswax sample. The Na2S2O3 wasnormalized with a 0.05 M KIO3 solution to which 10 mL2 M HCl and 10 mL KI were also added. Three iodinevalues were measured for each sample to calculate anaverage.

2.2.7 Peroxide value

This parameter was determined by the official method ofanalysis for fats and oils [5]. A beeswax amount of1.000 6 0.005 g was dissolved in 10 mL chloroform withthe help of an ultrasonic water bath. Then, 10 mL of a 2 MHCl solution and 5 mL of an 8% KI solution were added.The mixture was gently shaken and kept in darkness andat room temperature for 5 min. Then, the solution wastitrated with 0.00005 M Na2S2O3 (normalized with KIO3),using a starch solution to observe the final point. A blankwas performed under the same conditions. Three por-tions of sample were measured to calculate the meanvalue. The peroxide value (milliequivalents of oxygen/kg)was calculated as:

Peroxide value ¼ 1000ðV � V0ÞM

w

where V is the volume of Na2S2O3, in mL, spent in thetitration of the sample; V’ is the volume of Na2S2O3, in mL,used to titrate the blank; M is the molarity of the Na2S2O3

solution; and w is the mass, in g, of the beeswax sample.

2.2.8 Ash content

The official method for fats and oils has been adapted todetermine this parameter [5]. To tare the porcelain cap-sules, these were kept in the muffle at 600 7C for 30 minand were then cooled within a dessicator until constantweight. An amount of 2.000 6 0.001 g was placed in thecapsules, heated prudently until inflammation point in themuffle port and finally heated at 600 7C in the muffle for1.5 h. After that, the capsules with the ashes were cooledin a dessicator until constant weight. The ash percentagewas determined as follows:

Ash % ¼ 100W1�W2

w



where W1 is the mass, in g, of the porcelain capsule withthe ash content; W2 is the mass, in g, of the empty cap-sule; and w is the mass, in g, of the beeswax sample.Sample analyses were done in triplicate.

2.3 Samples of beeswax analyzed

Beeswax samples from different Spanish regions (Anda-lucía, Aragón, Canarias, Castilla La Mancha, Castilla yLeón, Comunidad Valenciana and Extremadura) havebeen studied. Nine of them were white beeswaxes,13 were yellow, and 52 samples were sheets of marketedfoundation beeswax. White beeswax came directly frombee scales and was invariably white, whereas the color ofyellow beeswax was yellow-brown, owing to its mixturewith soluble pigments from the pollen lipidic fraction thatbees keep in the beehive. As regards the foundationsheets, 27 had been removed from the beehives as aconsequence of the acceptation problems observed, and25 were perfectly used by the bees for their constructions.

All marketed beeswaxes were received as stampedsheets and did not require a treatment previous to analy-sis. White and yellow beeswaxes were provided by localapiarists and were mixed with rests of the beehive, so itwas necessary to perform a previous clean-up step. Tothis end, beeswax was added to a beaker with boilingwater, in proportion 100 g beeswax/liter, and boiling waskept up for 45 min. Then, the mixture was cooled at roomtemperature for about 12 h; the beeswax purified bymelting (less dense) solidified over the water, and theimpurities placed at the bottom of the solidified beeswaxwere removed with a scraper.

The melting of the beeswax was repeated at least twomore times with fresh water portions to achieve an aque-ous phase clear and transparent without any impuritiesplaced at the bottom of the beeswax. Samples were keptat room temperature and darkness until their analysis.

2.4 Preparation of the adulterant-beeswaxmixtures

The mixtures were carried out by weighing knownamounts of Fluka beeswax and one of the followingadulterants: paraffin with melting point between 54 and56 7C, paraffin with melting point between 58 and 60 7C,paraffin with melting point between 68 and 74 7C, stearicacid, tallow or carnauba wax. The percentage of adul-teration varied from 2 to 50%.

To achieve a good mixture and homogeneity in the sam-pling, the beeswax-adulterant mixture was heated in analuminum vessel up to the total melting of the mixture,

which required a temperature of about 70 7C and a timeclose to 5 min. After that, the mixtures were allowed tocool at room temperature for 24 h and kept at room tem-perature and darkness until their analysis.

2.5 Statistical analysis

Correlation and multiple variance analysis (MANOVA)were made with Statistica, release 4.3, for Windows soft-ware package (StatSoft Inc.). Each type of beeswax(white, yellow or foundation) was considered as an inde-pendent variable. Variance analyses comparing types ofbeeswax in pairs were also done.

3 Results

3.1 Precision of the methods of analysis

The often used analytical methods to determine the acid,iodine, peroxide, ash content and melting point valuesproved to be appropriate and provided a good precision,as can be seen in Tab. 1.

The determination of density, as the official methodsindicate [1, 4], is based on the weight of a pycnometer fullof molten beeswax. However, and according to ourexperience, when the pycnometer cools off, air bubblesare formed inside the mass of beeswax placed in thepycnometer on account of the beeswax contraction whenit solidifies. Thus, the measurement was not repetitive anddid not supply the real value of density; measured den-sities were about 0.83 g/mL. For this reason, the methodof Hager was applied for this parameter.

Tab. 1. Precision of the methods applied for the deter-mination of physico-chemical parameters (n = 5). Experi-ments were made with beeswax obtained from Fluka,except for peroxide value and ash content, for which afoundation beeswax sheet was used.

Parameter Mean 6 ConfidenceInterval{

Density [g/mL] 0.957 6 0.018Melting point [7C] 65.40 6 0.40Acid value [mg KOH/g] 18.41 6 0.23Saponification value [mg KOH/g] 92.0 6 3.5Ester value [mg KOH/g] 73.5 6 1.1Ratio number 3.99 6 0.11Iodine absorption number [g I/100g] 12.51 6 0.39Peroxide value [meq O/kg] 0.0051 6 0.0002Ash content [%] 0.00246 6 0.00003

{ Confidence level 99.5%.



As regards the saponification value, the official methodwas initially assayed. This method consists of a hydrolysiswith KOH in ethanol, followed by a titration; however, inour experimentation, the beeswax could never be hydro-lyzed. In fact, this method is recommended for fattysamples whose wax content is less than 5%. The hydro-lysis with ethanolic KOH under strongest conditions wasalso not satisfactory. For this reason, an alternativemethod was considered to establish the saponificationvalue in beeswax samples.

The repetitivity obtained by the proposed method for thesaponification value can be seen in Tab. 1. This latter to-gether with the peroxide value are the least precisedeterminations. These precision experiments were car-ried out with Fluka beeswax, except for the peroxidevalue and ash content for which a foundation beeswaxsheet was used. On the other hand, it is worth mentioningthat in those titrations that involved the use of phe-nolphthalein in the presence of beeswax, it was better toadd the indicator at the end of the titration, to diminish itsadsorption and to clearly observe the final point.

3.2 Physico-chemical parameters of purebeeswax

Tab. 2 shows the mean values and ranges obtained forthe analyzed parameters in white and yellow beeswaxes.As can be inferred from the results obtained both for per-oxide value and for ash content, the white pure bees-waxes contain neither mineral residues nor oxidizingsubstances, whereas there are very small amounts ofboth types of compounds in some yellow pure bees-waxes.

The mean value of the different parameters is, indeed,within the intervals commonly accepted by the literature[2, 5–8, 13–15, 18–20]. On the other hand, acid values andiodine absorption numbers are slightly higher in yellowbeeswaxes than in white beeswaxes, whereas saponifi-cation, ester, and ratio number values are somewhathigher in white beeswaxes. There were no significant dif-ferences between white and yellow beeswaxes ( p,0.05),although for iodine number, the p level was very close tothe imposed significance limit: p = 0.0584. In relation tothe maximum value of the saponification index, excep-tionally high values were found in three samples, two ofwhite beeswax and one of yellow beeswax, all of themabove the values stated in the pharmacopeias [2, 5, 6]; inthese samples, the saponification values were 110.5,147.1 and 137.6 mg KOH/g, respectively. As a con-sequence, their ester values were 92.2, 128.9 and115.7 mg KOH/g, respectively, and the ratio numberswere 5.04, 7.08 and 5.30, respectively. A satisfactoryexplanation for these results has not been reached.

The results suggest that the content in unsaturated andacidic compounds in pure beeswaxes increases withtime, with a consequent decrease in the ester compoundamounts that were initially secreted by the bees. This factis probably a consequence of the bee inputs from theoutside of the beehive.

According to the results, guide value ranges in purebeeswax have been proposed for each studied parame-ter, without considering the strange saponification, esterand ratio values obtained in three samples (Tab. 2). Themaximum guide values for saponification index, ester andratio numbers in pure beeswaxes often cited in the litera-ture are also described. The maximum guide value for

Tab. 2. Mean values and ranges of the physico-chemical parameters measured in white and yellow beeswaxes. Guidevalue ranges proposed for the physico-chemical parameters in pure beeswax.

Parameter White beeswax Yellow beeswax Guide value for purebeeswax. Proposed ranges

Mean Range Mean Range Commonvalues in theliterature

Own data

Acid value [mg KOH/g] 18.5 17.1–20.4 19.3 17.6–21.9 17.0–24.0 17.1–21.9Saponification value [mg KOH/g] 99.3 82.8–92.5 96.3 83.0–92.4 83.0–103.0 82.8–92.5Ester value [mg KOH/g] 80.9 65.7–74.7 76.9 62.7–74.8 66.0–82.0 62.7–74.8Ratio number 4.39 3.52–4.20 3.97 3.09–4.25 3.00–4.30 3.09–4.25Iodine absorption number [g I/100g] 8.9 7.6–10.6 10.2 8.4–13.1 Not defined 7.6–13.1Peroxide value [meq O/kg] 0.00 – 0.01 0.00–0.01 Not defined ,0.01Density [g/mL] 0.936 0.920–0.947 0.934 0.921–0.957 0.960 0.920–0.957Melting point [ 7C] 64.8 64.5–65.0 65.1 64.0–66.0 61.0–66.0 64.0–66.0Ash content [%] 0.000 – 0.010 0.000–0.055 Not defined ,0.055



acidity proposed in this work is about 2 mg KOH/g, lowerthan that pointed out in the pharmacopeias, and theminimum guide value proposed for the melting point is3 7C higher. The guide value range proposed now fordensity is higher than the range commonly considered.

3.3 Analysis of beeswax-adulterant mixtures

The variation of each parameter, when the adulterantpercentage increased up to 50% in beeswax, is summa-rized in Tab. 3. Data are the means of three experiments.Almost all the parameters vary linearly with the percent-

age of adulterant. The density decreased when the adul-terant amount increased, on account of the lower densityof the assayed adulterants in comparison with that ofbeeswax, except for carnauba wax, the density of whichis higher than that of beeswax. For paraffins and tallow,the decrease followed a polynomic trend. The mixtureswith stearic acid showed a different behavior; the densityof the mixture decreased for low stearic acid percentagesand increased for high ones. This fact could be explainedtaking into account that the volumes are not additives andthat the mixture of these two compounds was not com-pletely homogeneous, as it was observed during theexperimentation.

Tab. 3. Variation of the physico-chemical parameters for increasing amounts of some adulterants. Linear fitting: parametervalue = (slope)(percentage) 1 intercept. Polynomic fitting{: parameter value = (second-degree coefficient)(percenta-ge)2 1 (first-degree coefficient)(percentage) 1 intercept.

Added adulterant Paraffin Paraffin Paraffin Stearic acid Tallow Carnaubawax

54–56 7C 58–60 7C 68–74 7C

DensityIntercept 0.954{ 0.954{ 0.955{ nd 0.954 0.957First-degree coefficient/slope 20.0017{ 20.0013{ 20.0015{ nd 20.0009{ 0.0008Second-degree coefficient* 0.000021{ 0.000010{ 0.000011{ nd 0.000008{ ndr2 0.994{ 0.992{ 0.997{ nd 0.994{ 0.991

Acid valueIntercept 18.43 18.68 18.81 17.76 19.16 18.98Slope 20.183 20.208 20.214 1.899 20.191 20.113r2 0.98 0.995 0.98 0.9990 0.994 0.998

Saponification valueIntercept 80.76 81.26 81.37 81.04 87.05 ndSlope 21.342 21.337 21.329 10.765 1.050 ndr2 0.990 0.992 0.992 0.998 0.98 nd

Ester valueIntercept 62.13 62.38 62.26 62.25 68.08 63.76Slope 21.153 21.123 21.108 9.042 1.237 0.113r2 0.990 0.991 0.98 0.998 0.98 0.996

Ratio numberIntercept 3.51 3.49 3.50 3.50 3.11 3.28Slope 20.053 20.047 20.048 0.058 0.169 0.037r2 0.98 0.98 0.98 0.94 0.990 0.98

Melting temperatureIntercept 64.19 64.35 64.73 64.50 64.40 64.52Slope 20.105 20.109 0.057 0.050 20.089 0.498r2 0.993 0.993 0.98 1.000 0.994 0.994

Iodine valueIntercept 9.05 9.25 9.05 9.12 9.01 9.37Slope 20.090 20.098 20.090 20.092 0.299 0.009r2 0.995 0.994 0.995 0.993 0.994 0.93

nd – no data{ Polynomic fitting



The acid value of the mixtures decreased linearly forincreasing percentages of the adulterants, except stearicacid. For this latter, a notable increase of the acid value,also linear, was observed when the adulteration percent-age increased. As regards the saponification value, itdecreased when the beeswax was mixed with paraffins,whereas it increased in the adulterations with tallow andstearic acid; the variation was more marked in the pres-ence of stearic acid. In the mixtures with carnauba wax,the saponification value was kept virtually constant. Thevariation of the ester value and ratio number was similarto that of the saponification and acid values, from whichthey are calculated, for most of the adulterants. Tallowand stearic acid were the products that most affected theratio number and ester value, respectively.

As was expected, the melting point was the only parameterfor which the evolution of the three paraffin-beeswax mix-tures was not similar. The melting point decreased whenthe beeswax was mixed with increasing amounts of tallowand the two paraffins with lower melting point (54–56 7Cand 58–60 7C ranges), whereas it increased in the mixtureswith increasing amounts of stearic acid, carnauba wax andthe paraffin with the highest melting point (68–74 7C). Themost remarkable variation was observed when carnaubawax was added to the beeswax. For iodine value, itdecreased when the proportion of the adulterantsincreased, except for tallow, which notably increased theindex. In the mixtures with carnauba wax, the iodine valueincreased somewhat. Finally, it is interesting to remark thata soft heating – as it was done in this work – did not changethe value of the assayed parameters of pure beeswax, as itwas verified in this work. The variation was within the pre-cision range of the analytical methods.

Tab. 4 shows the minimum adulteration percentages thatcan be detected by the measurement of each analyticalparameter and for each type of fatty compound added. To

this purpose, the fittings between the concentration ofeach compound and the adulterant percentage wereused. Then, the adulteration percentage that can bedetected was estimated from the guide-value range pro-posed for pure beeswax. If the parameter value decreasedfor increasing percentages, the lowest value of the rangewas introduced into the fitting equation to calculate thepercentage. When the parameter value increased with theadulteration percentage, the highest value for pure bees-wax was taken as reference.

To carry out the estimations, two criteria related to theguide values in pure beeswaxes have been considered:on the one hand, the value ranges used as reference inthe literature for pure beeswaxes [5–7, 16, 17, 21]; on theother, the more restrictive guide value ranges observed inthis work, which made a clear distinction between purebeeswaxes and marketed beeswaxes, since the lattercould be pure or not.

As a rule of thumb, the measurement of the stated phy-sico-chemical parameters enables the detection ofadulterated beeswaxes if the amount of adulterant isroughly higher than 2 or 5%. The adulteration with stearicacid is easily identified because a small percentage of thisproduct in the mixture substantially modifies all the ana-lytical parameters, and mainly the acid value. To detectparaffins, the determination of the ester value is prefer-able, while for the carnauba wax, the most suitable pa-rameter is the melting point. Some authors use the melt-ing point as the only parameter to discriminate betweenadulterated and pure beeswaxes [10]; however, our datasuggest that the exclusive determination of only one pa-rameter, whichever it is, is not reliable to this aim. Themeasurement of several parameters is advisable todetect the adulteration of the beeswaxes on account ofthe different natures of the possible adulterants.

Tab. 4. Minimum adulteration percentages detected by the measurement of analytical parameters according to the valueranges commonly proposed in the pharmacopoeias and official methods of analysis and to the more restrictive rangesobtained in the analysis of the pure beeswaxes (in brackets).

Paraffin Paraffin Paraffin Stearic acid Tallow Carnaubawax

54–56 7C 58–60 7C 68–74 7C

Density 10 (40) 15 (40) 15 (50) 3 (–) 20 (–) 15 (3)Melting temperature 30 (5) 30 (5) 50 (3) 30 (5) 40 (10) 5 (5)Acid value 10 (10) 10 (10) 10 (10) 2 (2) 10 (10) 20 (15)Saponification value 10 (5) 10 (5) 10 (5) 3 (3) 15 (6) – (–)Ester value 5 (5) 5 (5) 5 (5) 5 (2) 10 (6) – (–)Ratio number 10 (10) 10 (10) 10 (10) 15 (12) 10 (7) 40 (25)Iodine value (15) (15) (15) (15) (15) (–)

– : Not useful to detect the adulteration.



For iodine value, there is not a defined and commonlyaccepted range, and the values found in beeswaxes arevery different: from 3 to more than 30 g I/100 g [7, 16, 17,21]. Thus, we have only considered our own guide value.As deduced from this guideline, beeswax with an iodinevalue below 8.0 g I/100 g should be considered as suspi-cious of adulteration.

Finally, if the guide values from the analysis of beeswaxes,about whose purity there is not doubt, are adopted asvalid, the detectable adulteration percentages (in brack-ets; Tab. 4) could change substantially in comparison withthose obtained from the literature data. So, by the firstprocedure, the saponification value and mainly the melt-ing point allow the determination of lower adulterationpercentages, whereas the density determination is onlyvalid to detect higher percentages.

3.4 Quality of the marketed foundationbeeswax sheets

Foundation beeswax sheets supplied by Spanish bee-keepers were received and analyzed in the laboratory. Asit was expected, the parameter values of the beeswaxsheets accepted by the bees were within the guide valueranges, while most of the 27 sheets that belonged to badlyaccepted or rejected lots by the bees had extreme valuesof iodine absorption number, ratio number, melting point,acid, saponification and ester values. They were higher orlower than in the pure beeswax samples, which indicateda possible adulteration. Furthermore, the highest peroxidevalues and ash contents have been found, curiously, inthese marketed sheets, probably owing to the use ofsome chemical products during their processing.

Tab. 5 shows the parameters whose values were unac-ceptable after analyzing the 27 rejected sheets. Of the27 samples, 25 (93%) seem to be adulterated with highenough amounts of strange products to be discriminatedby the measurement of their physico-chemical parame-ters. Acid, saponification, ester and ratio values were outof the proposed guidelines in many samples; furthermore,in some cases, all of the three latter parameters hadanomalous values in the same sample. This fact couldindicate the addition of fatty substances, when the pa-rameter values are above the guide values, or a mixturewith paraffins, if the parameter values are below. On theother hand, a low iodine value (related to the number ofunsaturations) could also be associated with a likelypresence of paraffins.

It is worth to remark that these 25 foundation sampleshave at least one incorrect parameter, but this fact is notenough to state that the anomalous parameters are the

Tab. 5. Anomalous physico-chemical parameters in therejected or badly accepted foundation beeswax sheets.

Sample Incorrect parameter

1 Acid, ash2 Saponification, ester, ratio, peroxide, ash3 Acid, melting temperature4 Saponification, ester, ratio5 Acid, saponification, ester, ratio, peroxide,

melting temperature6 Peroxide7 Ester, ratio8 Ester, ratio, peroxide9 Peroxide

10 Acid, saponification, ester, meltingtemperature

11 Ester, ratio, peroxide, ash12 Ester, ratio, ash13 Ester, ratio14 Ester, ratio15 Saponification, ester, ratio, ash16 Saponification, ester, ratio17 Acid, saponificaction, ester, ratio, iodine,

density, melting temperature, ash18 Ratio, iodine19 Acid, saponification, ester, ratio, iodine,

melting temperature20 Iodine21 Iodine22 Iodine, ash23 Saponification, ester, ratio, iodine24 Saponification, ester, ratio, iodine, ash25 Saponification, ester, ratio, iodine, ash

cause of the rejection, which could be attributed to thepresence of other products not detected by the determi-nation of physico-chemical parameters. The existence oftwo rejected beeswax samples whose parameters arewithin normality corroborates this statement.

Obviously, to identify the possible adulterant added, it isnecessary to turn to the information provided by instru-mental techniques, mainly chromatographic and spec-troscopic ones [22, 23]. We are working on this aspectnow.

After an ANOVA considering the pure beeswaxes (whiteand yellow ones together) and the rejected foundationbeeswax sheets as sample groups, significant differenceswere found for melting point, peroxide value, iodineabsorption number and ash content (p = 0.0140,p = 0.0450, p = 0.0038 and p = 0.0074, respectively). Thestatistical results seem to confirm that foundation bees-waxes have a higher mineral composition than purebeeswaxes and that the melting points and the acidvalues are different. By comparing yellow and rejected



foundation beeswaxes, it was observed that acid valuesand melting points were significantly different (p = 0.0380and p = 0.0294, respectively). Moreover, a correlationstudy revealed that the acid and iodine values were posi-tively correlated in foundation beeswaxes (r2 = 0.77,p ,0.05). This suggests that many adulterants mixed withthe beeswaxes must contain compounds with acidgroups and double bonds in their structures.

It is commonly expected that the foundation beeswaxsheets be made from old pure beeswaxes, which arestamped after purifying them by successive melting pro-cesses. However, the data obtained in this work suggestthat the recycled beeswax is mixed not only with otherfatty products but also with mineral and even oxidizingcompounds before stamping.

4 Conclusions

The official methods of analysis for oils and fats are notsuitable for the determination of density and saponifica-tion index in beeswax. Alternative and more reliablemethods are proposed.

The guide value ranges of some physico-chemical pa-rameters have been verified for pure beeswaxes. The ashcontent and peroxide value are parameters that shouldalso be regularly evaluated to control the quality of foun-dation beeswax sheets.

Adulteration percentages higher than 2 or 5% can bedetected by the determination of physico-chemical pa-rameters, although we suggest that the exclusive meas-urement of only one parameter cannot be used to detectthe adulteration of beeswax. The acid value, iodineabsorption number, peroxide value, melting point and ashcontent are the best parameters to discriminate betweenpure and adulterated beeswaxes.

The measurement of physico-chemical parameters doesnot allow the discrimination of all rejected or badlyaccepted foundation beeswax samples.

Acknowledgments

We are grateful to the Spanish Instituto Nacional deInvestigaciones Agrarias for financing this work (projectsAPI99-003 and API01-005) and to the Centro ApícolaRegional (Marchamalo, Guadalajara), Apiarist associa-tions and individual Apiarists for supplying beeswaxsamples.

References

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[Received: November 25, 2004; accepted: January 3, 2005]


physico-chemical parameters for the characterization of pure beeswax and detection of adulterations

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