The Scientific Bulletin of VALAHIA University – MATERIALS and MECHANICS – Nr. 7 (year 10) 2012 39 SPECTRAL IDENTIFICATION AND HPLC QUANTIFICATION OF BENZOIC ACID FROM NATURAL JUICES Rusăndica Stoica 1,2 , Sanda-Maria Doncea 1,2 , Virgil Bădescu 1 , Raluca Senin 1 , Rodica-Mariana Ion 1,2 1 National Research and Development Institute for Chemistry and Petrochemistry - ICECHIM, Bucharest, Romania 2 Valahia University of Targoviste, Romania E-mail: irusandica@yahoo.com Abstract. Benzoic acid from four natural juices was investigated in this paper. The identification of benzoic acid was achieved with FTIR using ATR technique. A high-performance liquid chromatography (HPLC) with photodiode array detection (PDA) method was developed for determination of benzoic acid. The highest concentration of benzoic acid was found in orange juice in the range of 71 mg·L -1 . Precision, linearity, sensitivity (limit of detection and limit of quantitation) and uncertainty were established. The obtained results suggested that ATR and HPLC are suitable methods for the identification and quantification of benzoic acid in natural juices. Keywords: Benzoic acid, FTIR, HPLC, natural juices 1. INTRODUCTION Phenolics are an important constituent of fruit quality because of their contribution to the taste, colour and nutritional properties of fruit [1]. Plant phenolics include phenolics acids (hydroxybenzoic acids and hydroxycinnamic acids), flavonoids, tannins and the less common stilbenes and lignans [2]. Benzoic acid (C 6 H 5 –COOH), the simplest aromatic carboxylic acid, is present naturally either free or bound as benzoic acid esters and can be found in many natural products [3]. Benzoic acid is extensively used as preserving agent most suitable for foods, fruit juices, and soft drinks. It is generally effective in controlling mould and inhibiting yeast growth and also in preventing a wide range of bacterial aggressions. Although this prevents or delays nutritional losses due to microbiological, enzymatic, or chemical changes of foods during its shelf life, it is harmful at higher than permitted safety levels [4]. In humans, the acute toxicity of benzoic acid is low, this substance is known to cause non-immunological contact reactions (pseudo allergy). Cases of urticaria, asthma, rhinitis, or anaphylactic shock have been reported following oral, dermal, or inhalation exposure to benzoic acid and sodium benzoate. The symptoms appear shortly after exposure and disappear within a few hours, even at low doses [5]. Analytical methods for the determination of benzoic acid include spectrophotometric methods [6], which need extensive extraction procedures and are not very specific; gas chromatographic (GC) methods [7], which are more sensitive and specific but need lengthy sample preparation and derivatization prior to determination; and high-performance liquid chromatography (HPLC)[8], which has a high specificity and minimum sample preparation and does not require derivatization. 2. MATERIALS AND METHODS Chemicals and materials All reagents: acetic acid (CH 3 COOH) glacial, methanol (CH 3 OH), for HPLC, ammonium acetate (CH 3 COONH 4 ), 0.01 mol/L solution, potassium hexacyanoferrate (II) trihydrate, K 4 [Fe(CN) 6 ]·3H 2 O, zinc sulfate heptahydrate, (ZnSO 4 ·7H 2 O), 300 g/L solution used were of analytical grade. Benzoic acid was purchased from Sigma-Aldrich. Four commercial juices (two orange juices, one multivitamin juice and one berry juice) were purchased from retail stores. ATR analysis Spectra were recorded on a Perkin Elmer Spectrum GX device type, the field 4000-600 cm -1 with 32 scans and a resolution of 4 cm -1 . Each standard and each sample were recorded three times and was a very good reproducibility of the spectra. Standard spectrum for benzoic acid was compared with spectra library of the instrument and a very good fitting was obtained. Attenuated total reflectance, ATR, is an infrared spectroscopy analytical technique most used in recent years, according to the literature [9]. ATR allows analyze samples without any previous preparation and high speed of analysis. The lack of sample preparation for analysis and penetration depths to reach an all too small incidence of the sample, a few μm, placed on a diamond crystal, which has a high refractive index, are the main advantages of this analytical technique. Penetration depth is proportional to the incident beam wavelength and therefore increase the penetration depth is observed at wavelengths greater, so the smaller wave numbers. The intensity of radiation absorbed depends on the quantity of sample comes in contact with the diamond surface and the number of contact points that radiation has the sample. By increasing the number of reflections by pressing the sample, IR absorption intensity increases and thus the spectrum intensity. Liquid or fine powder