Eur Food Res Technol (2001) 213:372–376 DOI 10.

1007/s002170100373

O R I G I N A L PA P E R

Anton Kaufmann · Bianca Ryser · Bea Suter

HPLC with evaporative light scattering detection for the determination of polar compounds in used frying oils

Received: 1 March 2001 / Published online: 15 August 2001 © Springer-Verlag 2001

Abstract An HPLC method for the determination of polar compounds in used frying oils/fats is described. It is based on normal phase liquid chromatography, i.e., the same separation mode as the reference method. The use of a mass-sensitive evaporative light scattering detector (ELSD) permits the quantification of the polar and non-polar compounds under two symmetrical, baselineresolved peaks. A polarity gradient is used for the elution of the polar oil components. The results correlate well with the official reference method (r2>0.97). Simplicity, speed, and low consumption of organic solvent are the main advantages. Keywords Polar compounds · Frying oils · Frying fats · HPLC

fined conditioning and elution process [4]. This method has been approved by international organizations like IUPAC and AOAC as the official standard for the quality control of frying fats. It is considered to be time-consuming, labor-intensive [5], and reliability is not fully satisfactory. A recent inter-laboratory test with 35 participating laboratories produced coefficients of variation (CV) of 17.2% (3.6% after the removal of eight outlying laboratories) [6]. Alternative methods As an alternative, a number of chemical quick tests are available [7]. Acceptable correlations have been reported between the polar compounds and the dielectric constant as measured by the Food Oil Sensor (FOS) [5, 8]. NIR in combination with chemometric calibration techniques is reported to permit very fast measurements [9]. A “micro method” described the potentials of miniaturization of the reference method [10]. The imitation of the reference method by other methodology is difficult, because of the vague definition of polar compounds. Most quick chemical tests as well as the dielectric constant measurements are supposed to determine the concentration of polar moieties like carboxyl groups. However, aging of frying oils also produces oligomers, i.e., triglycerides linked by C-O-C or C-C bonds. These as well as epoxides are retained on the silica column together with the fraction of polar compounds, as shown by Aitzetmüller [11]. As a consequence, the determination of such a heterogeneous group of compounds by means other than normal phase liquid chromatography is rather difficult. There were attempts to imitate the reference method by reversed phase liquid chromatography (RPLC) and refractometry index (RI) detection [12]. We found it difficult to obtain a clear-cut separation between the polar and non-polar fraction and noted a different selectivity. In RPLC the dimeric triglycerides are probably eluted within the non-polar fraction.

Introduction
Frying oils/fats are used as a heat transfer medium for the preparation of a variety of food products. Heating of such oils in the presence of oxygen, water, and food causes a gradual thermal degradation of the triglycerides. A large number of artifacts [1, 2, 3] have been reported. Reference method for polar compounds In order to monitor the quality of frying oils, a gravimetric column chromatography method was developed by Guhr and Waibel [4]. The measured polar compounds are not a clearly defined group of substances, but are probably best described as all substances which are retained in a silica column having been subjected to a deA. Kaufmann (✉) · B. Ryser · B. Suter Official Food Control Authority of the Canton of Zurich (Kantonales Labor Zürich), P.O. Box, CH-8030 Zürich, Switzerland e-mail: anton.kaufmann@klzh.ch Tel.: 0041-12525654, Fax: 0041-12624753

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Normal phase liquid chromatography The most promising approach to substitute the reference method by an automated analysis deviates as little as possible from the original principle. Aitzetmüller proposed frontal elution liquid chromatography [11] using a moving wire detector which is no longer commercially available. He not only reported one of the earliest attempts to automate liquid chromatography (in 1973) but also gave a still valid discussion about the unique character of the normal phase liquid chromatography on silica gels. Chromatography on silica is based on both partitioning and adsorption. A water-free silica surface contains active sites and primarily separates by adsorption [13]. If the activity of silica is reduced by water or ethanol, chromatography occurs by partitioning. As a consequence, the control of the water content of silica is essential. Silica adsorbs water from the eluent or desorbs water into a dry eluent until a steady state is reached. These changes cause drifting retention times [13]. If a gradient is employed, a steady-state situation might never be reached. The concept of isohydric solvents was introduced [15, 16], permitting polarity gradients without affecting the water concentration of the stationary phase. However, isohydric eluents require the careful adjustment of the water concentration of the two eluents. Mostly a dry and a saturated solvent are mixed. Detection technique How can gravimetric quantification be replaced by a chromatographic detection? UV response cannot be used as it strongly differs for the compounds of interest. RI detectors approach mass-sensitive measurement more closely, but can only be employed for isocratic runs. Hence the peak of the polar compounds (requiring gradient elution) cannot be quantified. ELSD represents another mass-sensitive type of detector. Its main features are excellent gradient capability, ease of operation, and ruggedness. However, because of the different underlying light scattering mechanisms, ELSDs do not produce linear response [14]. This paper presents an HPLC method which mimics the reference method for the determination of the polar compounds [4] as closely as possible. The main advantages are speed, low labor-requirement, low solvent consumption, and improved repeatability. The validation of the method as well as the comparison with the reference method for polar compounds and the method measuring the dielectric constant (FOS) will be presented in a second paper [17].

autosampler (Gynkotek Gina 50), a column thermostat (Gynkotek STH 585), and a light scattering detector (Polymer Laboratories PL-ELS 1000). The column flow rate was 2.0 ml/min, while the column temperature was held at 10 °C. Injection volume was 20 µl. The gradient program consisted of 0.0–0.5 min 0% B, 0.5–1.9 min 0–100%B, 1.9–2.4 min 100% B, 2.4–2.9 min 100–0%B, 2.9-x min 0% B. The conditioning time (x) required depends on the dead volume of the HPLC pump and might vary between 4 and 9 min. The dead volume was determined by injecting the reference oil while maintaining 100% eluent B. The conditioning time was increased stepwise until the first peak of the reference oil (non-polar compounds) eluted between k′ 0.6 and 1.0. Three consecutive injections were made for any evaluated conditioning time in order to ensure that stable performance 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 evaporation temperature to 90 °C. A nitrogen gas flow of 1 l/min was used. Chemicals and materials. Hexane 96% (multisolvent, HPLC grade, Scharlau, Barcelona), ethanol, abs., HPLC quality, Mächler AG, Reinach, Switzerland), bidistilled water, diethyl ether (Ph.H.V. Siegfried, Zofingen, Switzerland). A reference oil (25–30% polar compounds) was used as a standard. A 30×4.6 mm i.d., 10 µm particle size cyano Nucleosil HPLC column, (Macherey Nagel) was used for the separation. A peek tubing (3.0 m×0.5 mm i.d.) was installed between the column and the detector. Solutions. The extraction solvent consisted of hexane:diethyl ether, 10:3 (by volume), the mobile phase A of hexane, and mobile phase B of hexane:ethanol:water, 50:50:1 (by volume). Both eluents were stored in brown bottles. Standards. Stock solution: 0.625±0.02 g reference oil warmed to 40–50 °C was weighed in a graduated flask. The latter was filled with extraction solvent to 50 ml. Reference oil was produced by prolonged heating of a frying fat in an open, air-exposed vessel until FOS measurement indicated values corresponding to 25–30% polar compounds. The exact concentration of polar compounds was determined by the reference method (six independent determinations, each with an individually deactivated silica batch performed by three different operators). Six standards solutions (A, B, C, D, E, F) were prepared by transferring 1, 3, 6, 8, 10, and 12 ml stock solution into 50-ml graduated flasks and filling up to the mark with hexane. In a refrigerator solutions are stable for more than three months. Preparation of samples. Warmed oil (15 drops, 40–50 °C) were dissolved in 20 ml extraction solvent. Then 0.4 ml of this solution was diluted in the HPLC sample vial with 1.2 ml hexane and the vial closed with a PTFE only or a Viton seal. Calibration. Peak areas of the polar and non-polar compounds are calibrated by a quadratic, non-zero offset calibration curve. The calibration included the concentration of the polar and non polar compounds. For example, if 0.625 g reference oil of 30.4% polar compound is used, standard A contains 0.174 g/l non-polar and 0.076 g/l polar compounds. The percentage of polar compounds in a sample was calculated as polar compounds (%)=100×amount polar/(amount non-polar+amount polar).

Results Materials and methods
Instruments. The HPLC instrument consisted of a gradient pump (Gynkotek P 580A NDG/Agilent HP1100 quaternary), with an

Figure 1 shows a chromatogram of a heavily used (top) and a fresh frying oil. Two well separated peaks were obtained in less than 2.5 min.

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Fig. 1 Chromatograms of a heavily used (top) and a fresh frying oil

Selection of the stationary and mobile phase Separations on underivatized silica HPLC columns were strongly affected by the water concentration in the eluent. A cyano-modified column used under normal phase chromatography conditions was preferred. Such phases are equilibrated after a rather short period of time [13]. The reference method uses a mixture of petroleum ether and diethyl ether as mobile phase. Because of the higher point (to prevent cavitation) hexane and ethyl alcohol were preferred. A column temperature of 10 °C improved the peak shape of the peak containing the nonpolar compounds. Conditioning/deactivation of the stationary phase The selectivity of the column used by the reference method strongly depends on the water content of the silica [10]. The cyano HPLC column used showed a similar behavior. After a gradient run it was deactivated and had to be conditioned by hexane to restore the required activity. Figure 2 shows the effect of varying the duration of reconditioning on chromatography of a used frying oil. The top chromatogram in Fig. 2 was obtained by injection into 100% mobile phase B. The retention time of the single peak (polar and apolar compounds) reflects the dead volume of the system. The sample was injected into a column of drifting activity rather than stabilizing (equilibrating) the column at a given activity. This speeds up the analysis and provides increasing activity during chromatography of the non-polar compounds, causing an accentuated separation between the non-polar and the polar compounds. Removal of the ethanol during conditioning increases column activity, i.e., prolonged conditioning results in a more retentive column. At the same time, more material of intermediate polarity is shifted from the non-polar to the polar fraction. Selectivity of the separation must therefore be adjusted to that of the reference method by reconditioning such that the same amount of polar material is found. Low dead

Fig. 2 Chromatograms of the same oil sample by identical gradient 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 6 min column conditioning before starting the next injection/run. The LC pump used for this experiment was the Gynkotek P 580A

Table 1 Detector response for different substances: relative peak area for equal amounts of different analytes Analyte Sunflower oil Tristearate 1,3-Dipalmitate 1,2-Dipalmitate Monopalmitate Epoxylates soy oil Palmitinic acid Absolute peak area 105 114 89 107 106 93 86

volume pumps like the tested Agilent 1100 dry the column in such a short time that it is feasible to reach a stable equilibrium within a reasonable time. If such a pump is available, proper column activity can also be obtained by adding ethanol to the mobile phase A. Ethanol (0.1%) in the mobile phase A permitted the elution of the non-polar compounds within the specified k′ range. Oils analyzed by these two different approaches (conditioning vs equilibration) did not produce significantly different results. Ten frying oils from local restaurants were analyzed with the reference method and by HPLC. HPLC analysis was performed three times, each run with a different conditioning time. Figure 3 shows the correlation between the classical polar compound and the HPLC method. Correlation was best at k′ 0.5–1.0. The k′ value and the peak width depends on the dead volume of the HPLC pump mixing system. Hence reconditioning time has to be adjusted 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 technique Quantification required that the various compounds comprised in the two peaks produce almost identical detector responses. Table 1 shows relative peak areas for an equal

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aration power of a short column with coarse particle diameter was still excessive, peak broadening was deliberately introduced by inserting a 3 m×0.5 mm i.d. tubing between the column and the detector. The strong peakbroadening introduced by this feature rendered negligible the rather slight peak-broadening caused by partial separation of analytes within the non-polar peak. There is no corresponding problem for the peak of the polar compounds because of the steep gradient. Ruggedness of the method A number of tests were performed in order to assess the robustness of the method. The water content of the hexane (eluent A) and the composition of eluent B were varied, with hardly any effect on the analytical results. However, commercial hexane contains “polar compounds” which are concentrated on the column and eluted as a peak with the same retention time as the polar compounds of the oil. Although this is accounted for by the calibration curve (intercept), such a peak should be kept as small as possible. Presence of this contamination did not seem to be related to the advertised quality or price of the hexane. As photoinduced reactions might be involved, eluents were be stored in brown eluent bottles. Repeated injection from the same sample vial caused septa material (silicone or rubber) to be partially dissolved, which also generated a peak eluted at the retention time of the polar compounds to be analyzed. Hence PTFE only or Viton seals were used.

Fig. 3 The dead volume of an HPLC pump affects the conditioning time. Hence this time has to be adjusted for every pump. High correlation is observed if the k′ value of the peak containing the non-polar compounds is around 0.6–1

Discussion
The advantage of the proposed technique is the tight control of a number of variance-generating factors (column packing, flow rate and column temperature). Further, a set of standards compensates for possibly deviating conditions, while the reference method relies on the assumption that all the non-polar compounds elute quantitatively with the defined 150 ml eluent. The adjustable duration of conditioning permits a fine tuning of the method. The retention time of the non-polar compounds is a quality relevant information, since it indicates the correctness of the column deactivation for each chromatogram. Much HPLC software permits the direct calculation of the polar compounds, hence producing, together with the chromatogram, easily interpretable reports. However, the main benefits of the HPLC method over the classical method are the simplicity and speed as well as the significant reduction of labor and organic solvent cost.

amount of given analyte. Responses vary relatively little. Furthermore, it can be expected that different types of oils undergo similar thermal and hydrolytic decomposition, such that the differences in detector response can be assumed to have no significant effect on the analysis. There were suspicions that triglycerides could be detected at varying sensitivities, depending on whether they pass the detector cell as droplets of liquid or solid particles. Even a strong reduction of the ELSD temperature did not significantly affect peak areas. This was observed for oils as well as pure, crystallization-prone tristearate. Therefore, the response of an ELSD is a good approximation of the gravimetric reference method. ELSDs produce an exponential, slightly sigmoid calibration curve [14]. The non-linearity of the detector is responsible for a rather insidious effect. The non-polar compounds, primarily intact triglycerides, are slightly separated, producing an asymmetric peak, whose shape depends on the composition of the oil or fat. Peaks are, therefore, of a varying area/height ratio. Integration of such peaks is biased by a systematic error if the detector shows non-linear response. For this reason the separation of the non-polar analytes was suppressed. Since the sep-

References
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