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10.1007%2Fs002170100373

10.1007%2Fs002170100373

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Abstract
An HPLC method for the determination of po-lar compounds in used frying oils/fats is described. It isbased on normal phase liquid chromatography, i.e., thesame separation mode as the reference method. The useof a mass-sensitive evaporative light scattering detector(ELSD) permits the quantification of the polar andnon-polar compounds under two symmetrical, baseline-resolved peaks. A polarity gradient is used for the elu-tion of the polar oil components. The results correlatewell with the official reference method (r
2
>0.97). Sim-plicity, speed, and low consumption of organic solventare the main advantages.
Keywords
Polar compounds · Frying oils · Frying fats ·HPLC
Introduction
Frying oils/fats are used as a heat transfer medium forthe preparation of a variety of food products. Heating of such oils in the presence of oxygen, water, and foodcauses a gradual thermal degradation of the triglyce-rides. A large number of artifacts [1, 2, 3] have been re-ported.Reference method for polar compoundsIn order to monitor the quality of frying oils, a gravimet-ric column chromatography method was developed byGuhr and Waibel [4]. The measured polar compoundsare not a clearly defined group of substances, but areprobably best described as all substances which are re-tained in a silica column having been subjected to a de-fined conditioning and elution process [4]. This methodhas been approved by international organizations likeIUPAC and AOAC as the official standard for the qualitycontrol of frying fats. It is considered to be time-con-suming, labor-intensive [5], and reliability is not fullysatisfactory. A recent inter-laboratory test with 35 partic-ipating laboratories produced coefficients of variation(CV) of 17.2% (3.6% after the removal of eight outlyinglaboratories) [6].Alternative methodsAs an alternative, a number of chemical quick tests areavailable [7]. Acceptable correlations have been reportedbetween the polar compounds and the dielectric constantas measured by the Food Oil Sensor (FOS) [5, 8]. NIR incombination with chemometric calibration techniques isreported to permit very fast measurements [9]. A “micromethod” described the potentials of miniaturization of the reference method [10].The imitation of the reference method by other meth-odology is difficult, because of the vague definition of polar compounds. Most quick chemical tests as well asthe dielectric constant measurements are supposed to de-termine the concentration of polar moieties like carboxylgroups. However, aging of frying oils also producesoligomers, i.e., triglycerides linked by C-O-C or C-Cbonds. These as well as epoxides are retained on thesilica column together with the fraction of polar com-pounds, as shown by Aitzetmüller [11]. As a conse-quence, the determination of such a heterogeneous groupof compounds by means other than normal phase liquidchromatography is rather difficult.There were attempts to imitate the reference methodby reversed phase liquid chromatography (RPLC) andrefractometry index (RI) detection [12]. We found it dif-ficult to obtain a clear-cut separation between the polarand non-polar fraction and noted a different selectivity.In RPLC the dimeric triglycerides are probably elutedwithin the non-polar fraction.
A. Kaufmann (
) · B. Ryser · B. SuterOfficial Food Control Authority of the Canton of Zurich(Kantonales Labor Zürich), P.O. Box,CH-8030 Zürich, Switzerlande-mail: anton.kaufmann@klzh.chTel.: 0041-12525654, Fax: 0041-12624753Eur Food Res Technol (2001) 213:372–376DOI 10.1007/s002170100373
ORIGINAL PAPER
Anton Kaufmann · Bianca Ryser · Bea Suter
HPLC with evaporative light scattering detection for the determinationof polar compounds in used frying oils
Received: 1 March 2001 / Published online: 15 August 2001©Springer-Verlag 2001
 
373
Normal phase liquid chromatographyThe most promising approach to substitute the referencemethod by an automated analysis deviates as little aspossible from the original principle. Aitzetmüller pro-posed frontal elution liquid chromatography [11] using amoving wire detector which is no longer commerciallyavailable. He not only reported one of the earliest at-tempts to automate liquid chromatography (in 1973) butalso gave a still valid discussion about the unique char-acter of the normal phase liquid chromatography on sili-ca gels.Chromatography on silica is based on both partition-ing and adsorption. A water-free silica surface containsactive sites and primarily separates by adsorption [13].If the activity of silica is reduced by water or ethanol,chromatography occurs by partitioning. As a conse-quence, the control of the water content of silica is es-sential. Silica adsorbs water from the eluent or desorbswater into a dry eluent until a steady state is reached.These changes cause drifting retention times [13]. If agradient is employed, a steady-state situation mightnever be reached. The concept of isohydric solvents wasintroduced [15, 16], permitting polarity gradients with-out affecting the water concentration of the stationaryphase. However, isohydric eluents require the careful ad- justment of the water concentration of the two eluents.Mostly a dry and a saturated solvent are mixed.Detection techniqueHow can gravimetric quantification be replaced by achromatographic detection? UV response cannot be usedas it strongly differs for the compounds of interest. RIdetectors approach mass-sensitive measurement moreclosely, but can only be employed for isocratic runs.Hence the peak of the polar compounds (requiring gradi-ent elution) cannot be quantified. ELSD represents an-other mass-sensitive type of detector. Its main featuresare excellent gradient capability, ease of operation, andruggedness. However, because of the different underly-ing light scattering mechanisms, ELSDs do not producelinear response [14].This paper presents an HPLC method which mimicsthe reference method for the determination of the polarcompounds [4] as closely as possible. The main advanta-ges are speed, low labor-requirement, low solvent con-sumption, and improved repeatability. The validation of the method as well as the comparison with the referencemethod for polar compounds and the method measuringthe dielectric constant (FOS) will be presented in asecond paper [17].
Materials and methods
 Instruments
. The HPLC instrument consisted of a gradient pump(Gynkotek P 580A NDG/Agilent HP1100 quaternary), with anautosampler (Gynkotek Gina 50), a column thermostat (Gynkotek STH 585), and a light scattering detector (Polymer LaboratoriesPL-ELS 1000).The column flow rate was 2.0ml/min, while the column temper-ature was held at 10°C. Injection volume was 20µl. The gradientprogram consisted of 0.0–0.5min 0% B, 0.5–1.9min 0–100%B,1.9–2.4min 100% B, 2.4–2.9min 100–0%B, 2.9-x min 0% B. Theconditioning time (x) required depends on the dead volume of theHPLC pump and might vary between 4 and 9min. The dead vol-ume was determined by injecting the reference oil while maintain-ing 100% eluent B. The conditioning time was increased stepwiseuntil the first peak of the reference oil (non-polar compounds) elut-ed between k 
0.6 and 1.0. Three consecutive injections were madefor any evaluated conditioning time in order to ensure that stableperformance was reached. A longer conditioning prolongs the k 
value of the first eluting peak while the k 
value of the second peak (polar compounds) remains constant.The ELSD spray temperature was set to 40°C and the evapo-ration temperature to 90°C. A nitrogen gas flow of 1l/min wasused.
Chemicals and materials
. Hexane 96% (multisolvent, HPLCgrade, Scharlau, Barcelona), ethanol, abs., HPLC quality, MächlerAG, Reinach, Switzerland), bidistilled water, diethyl ether (Ph.H.V.Siegfried, Zofingen, Switzerland). A reference oil (25–30% polarcompounds) was used as a standard.A 30
×
4.6mm i.d., 10µm particle size cyano Nucleosil HPLCcolumn, (Macherey Nagel) was used for the separation. A peek tubing (3.0m
×
0.5mm i.d.) was installed between the column andthe detector.
Solutions
. The extraction solvent consisted of hexane:diethylether, 10:3 (by volume), the mobile phase A of hexane, and mo-bile phase B of hexane:ethanol:water, 50:50:1 (by volume). Botheluents were stored in brown bottles.
Standards
. Stock solution: 0.625±0.02g reference oil warmed to40–50°C was weighed in a graduated flask. The latter was filledwith extraction solvent to 50ml. Reference oil was produced byprolonged heating of a frying fat in an open, air-exposed vesseluntil FOS measurement indicated values corresponding to 25–30%polar compounds. The exact concentration of polar compoundswas determined by the reference method (six independent determi-nations, each with an individually deactivated silica batch per-formed by three different operators).Six standards solutions (A, B, C, D, E, F) were prepared bytransferring 1, 3, 6, 8, 10, and 12ml stock solution into 50-mlgraduated flasks and filling up to the mark with hexane. In a re-frigerator solutions are stable for more than three months.
Preparation of samples
. Warmed oil (15 drops, 40–50°C) weredissolved in 20ml extraction solvent. Then 0.4ml of this solutionwas diluted in the HPLC sample vial with 1.2ml hexane and thevial closed with a PTFE only or a Viton seal.
Calibration
. Peak areas of the polar and non-polar compounds arecalibrated by a quadratic, non-zero offset calibration curve. Thecalibration included the concentration of the polar and non polarcompounds. For example, if 0.625g reference oil of 30.4% polarcompound is used, standard A contains 0.174g/l non-polar and0.076g/l polar compounds. The percentage of polar compounds ina sample was calculated as polar compounds (%)=100
×
amountpolar/(amount non-polar+amount polar).
Results
Figure1 shows a chromatogram of a heavily used (top)and a fresh frying oil. Two well separated peaks wereobtained in less than 2.5min.
 
374
Selection of the stationary and mobile phaseSeparations on underivatized silica HPLC columns werestrongly affected by the water concentration in the elu-ent. A cyano-modified column used under normal phasechromatography conditions was preferred. Such phasesare equilibrated after a rather short period of time [13].The reference method uses a mixture of petroleumether and diethyl ether as mobile phase. Because of thehigher point (to prevent cavitation) hexane and ethyl al-cohol were preferred. A column temperature of 10°Cimproved the peak shape of the peak containing the non-polar compounds.Conditioning/deactivation of the stationary phaseThe selectivity of the column used by the referencemethod strongly depends on the water content of thesilica [10]. The cyano HPLC column used showed a sim-ilar behavior. After a gradient run it was deactivated andhad to be conditioned by hexane to restore the requiredactivity. Figure2 shows the effect of varying the dura-tion of reconditioning on chromatography of a used fry-ing oil. The top chromatogram in Fig.2 was obtained byinjection into 100% mobile phase B. The retention timeof the single peak (polar and apolar compounds) reflectsthe dead volume of the system.The sample was injected into a column of drifting ac-tivity rather than stabilizing (equilibrating) the column at agiven activity. This speeds up the analysis and providesincreasing activity during chromatography of the non-po-lar compounds, causing an accentuated separation be-tween the non-polar and the polar compounds. Removalof the ethanol during conditioning increases column activ-ity, i.e., prolonged conditioning results in a more retentivecolumn. At the same time, more material of intermediatepolarity is shifted from the non-polar to the polar fraction.Selectivity of the separation must therefore be adjusted tothat of the reference method by reconditioning such thatthe same amount of polar material is found. Low deadvolume pumps like the tested Agilent 1100 dry the columnin such a short time that it is feasible to reach a stableequilibrium within a reasonable time. If such a pump isavailable, proper column activity can also be obtained byadding ethanol to the mobile phase A. Ethanol (0.1%) inthe mobile phase A permitted the elution of the non-polarcompounds within the specified k 
range. Oils analyzed bythese two different approaches (conditioning vs equilibra-tion) did not produce significantly different results.Ten frying oils from local restaurants were analyzedwith the reference method and by HPLC. HPLC analysiswas performed three times, each run with a different con-ditioning time. Figure3 shows the correlation betweenthe classical polar compound and the HPLC method. Cor-relation was best at k 
0.5–1.0. The k 
value and the peak width depends on the dead volume of the HPLC pumpmixing system. Hence reconditioning time has to be ad- justed for a given pump in order to permit the elution of the non-polar peak at a k 
value around 0.6–1.Detection and calibration techniqueQuantification required that the various compounds com-prised in the two peaks produce almost identical detectorresponses. Table1 shows relative peak areas for an equal
Fig.1
Chromatograms of a heavily used (
top
) and a fresh fryingoil
Fig.2
Chromatograms of the same oil sample by identical gradi-ent ramps but variations of the conditioning time. The trace at the
top
shows a chromatogram produced when injecting oil into 100%mobile phase B, hence reflecting the dead volume of the system.The following traces use 4, 5, and 6min column conditioning be-fore starting the next injection/run. The LC pump used for this ex-periment was the Gynkotek P 580A
Table1
Detector response for different substances: relative peak area for equal amounts of different analytesAnalyteAbsolute peak areaSunflower oil105Tristearate1141,3-Dipalmitate891,2-Dipalmitate107Monopalmitate106Epoxylates soy oil93Palmitinic acid86

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