This research aims at separation of polyphenols from Jordanian olive mill wastewater which have possible applications in pharmaceutical industry. The phenolic compounds were isolated using silica column chromatography based on using different solvents after extracting the acidified solution with n-hexane and ethyl acetate. The structural elucidation of the separated compounds was achieved using 1H-NMR, 13C-NMR and mass spectrometry. The concentrations of these compounds were determined by GC-MS after derivatization with N, O-bis(trimethylsilyl)trifluoroacetamide (BSTFA). The concentrations of the main isolated phenolic compounds in the Jordanian olive mill wastewater were ferulic acid (93.6 mg/L),
Olive mill wastewater (OMWW) is a dark red-to-black-colored, mildly acidic liquid of high conductivity, obtained from mechanical olive processing during olive oil production [
The treatment of OMWW is extremely difficult due to its large volume and the high concentration of organic matter. The major factor of the environmental problems imposed by the OMWW is the high concentration of polyphenols. These compounds are difficult to decompose [
The olive fruit is very rich in phenolic compounds, but only 2% of the total phenolic content of the olive fruit passes in the oil phase, while the remaining amount is lost in the OMWW (approx. 53%) and in the pomace (approx. 45%) [
In general, polyphenols are thought to deliver health benefits by several mechanisms, including: (1) direct free radical quenching, (2) protection and regeneration of other dietary antioxidants, (3) chelation of metal ions [
One of the most abundant polyphenol present in OMWW and very interesting from the nutritional point of view is hydroxytyrosol, which has been widely studied demonstrating its antioxidant and health-beneficial properties as well as its good bioavailability: hydroxytyrosol scavenges free radicals during the oxidation process [
Hydroxytyrosol is not commercially available in large amounts as food additive; it is an expensive costly for scientific/experimental purposes [
Various studies were conducted to isolate phenolic compounds from OMWW by using liquid-liquid extraction (LLE). A large number of solvents were tried, but it has been shown that ethyl acetate exhibits a higher extraction power compared to other solvents, such as methyl isobutyl ketone, methyl ethyl ketone, and diethyl ether [
The used chromatographic methods for the determination of the various phenolic compounds in OMWW are high-performance liquid chromatography (HPLC) [
The aims of the present study are (1) to separate polyphenols from Jordanian OMWW, (2) to identify the isolated phenolic compounds by nuclear magnetic resonance spectrometry (NMR) and high resolution mass spectrometry (HRMS), and (3) to identify and quantify the isolated phenols by GC-MS after derivatization with BSTFA and to determine the limit of detection and the limit of quantification of each isolated compound.
m-Methoxy-acetophenone (internal standard-I.S.) and N, O-bis (trimethylsilyl) trifluoroacetamide (BSTFA) used for derivatization were purchased from Across Chemical (USA).
The following solvents of HPLC-grade and GC-grade were purchased from Riedel-de Haën (Germany): n-Hexane, ethyl acetate, methanol, chloroform, dichloromethane, ammonia, benzene, and pyridine.
The used silica gel was Silica Gel 60 (for column chromatography) and Silica Gel G/UV254 (for thin layer chromatography) which were purchased from Riedel-de Haën (Germany). The type of plates used for separation was silica gel glass precoated TLC plates SILG/25 with a thickness of 0.5 mm, purchased from Riedel-de Haën (Germany).
The olive mill wastewater sample of 5 L volume was collected from the olive mill in Al-Balqa area, 20 km north of the capital Amman, and stored in aspirators at low temperature until required for experimental use. The storage of OMWW at low temperature is necessary because of the time-variable composition.
Scheme for the column and TLC separation of the phenolic compounds.
A sample of OMWW was filtrated, in order to get rid of any solid materials, followed by acidification to pH 2 with 2 M HCl. Four liters of the acidified sample was extracted three times with 150 × 4 mL n-hexane in order to remove the lipid fraction. The aqueous layer was extracted with 100 mL ethyl acetate (seven times) in order to collect the phenolic compounds. The pooled ethyl acetate extracts were dried over anhydrous sodium sulfate and evaporated using a rotary evaporator at 75°C to obtain 4.68 g of the crude extract sample.
The crude sample (4.68 g) was dissolved in chloroform/methanol solvent mixture, and 5 g of silica gel was added to the mixture. The solvent was evaporated in the fume hood, and then the mixture was loaded on a silica gel column (155 g silica type: MN Silica Gel 60, 3.5 cm in diameter), which was packed in chloroform. The column was eluted with chloroform, and then the polarity was gradually increased using methanol. The content of each collected fraction was evaporated and followed by TLC. Fractions of similar compositions were collected together to give a total of three pooled fractionated groups [A (I)–A (III)]. Group A (I) contained one compound that was eluted in a system of 100% chloroform. Group A (II) contained five compounds which were eluted in a system of 5% methanol: 95% chloroform. Group A (III) contained one compound that was eluted in a system of 10% methanol: 90% chloroform.
The crude sample from group A (II) was dissolved in chloroform/methanol solvent mixture, and 4 g of silica gel was added to the mixture. The solvent was evaporated in the fume hood, and then the mixture was loaded on a silica gel column (150 g silica type: MN Silica Gel G/UV254, 3.5 cm in diameter), which was packed in benzene. The column was eluted with benzene, and then the polarity was gradually increased using ethyl acetate. The collected fractions were evaporated and separated by TLC. Fractions of similar compositions were pooled together to give a total of three collective groups [B (I)–B (III)]. Group B (I) contained one compound that was eluted in a system of 20% ethyl acetate: 80% benzene. Group B (II) contained three compounds which were eluted in a system of 25% ethyl acetate: 75% benzene. Group B (III) contained one compound that was eluted in a system of 40% ethyl acetate: 60% benzene. Group B (II) was separated on silica gel plates using a system of 30% methanol: 70% dichloromethane in presence of ammonia vapor.
Each of the isolated compounds was purified by either preparative TLC or fractional crystallization.
Derivatization causes a nonvolatile sample to become volatile, or it improves the detectability upon derivatization. Furthermore, the derivatives may also be more thermally stable. N,O-bis (trimethylsilyl) trifluoroacetamide (BSTFA) is mostly used as derivatizing agent and it has a high reactivity. The derivatization of phenolic compounds for GC-MS analysis was performed according to the procedure described by Zafra et al. [
A 0.1 g of crude sample was dissolved in ethyl acetate under sonication and the volume was completed to the mark in 100 mL volumetric flask with the same solvent. 12
The isolated pure phenolic compounds as shown in Section
The limit of detection (LOD) for each standard phenolic compound was calculated from a chromatogram of a diluted standard mixture solution, on the basis of signal-to-noise (S/N) ratio of 3.
The limit of quantification (LOQ) for each standard phenolic compound was calculated from a chromatogram of a diluted standard mixture solution, on the basis of signal-to-noise (S/N) ratio of 10.
Constituents were detected as spots in each fraction after separation from the column using short UV radiation lamp at wavelength of 254 nm.
1H and 13C-NMR spectra were recorded at 300 and 75.5 MHz on a BRUKER DPX spectrometer, using TMS as an internal standard in order to elucidate the structure of the isolated compounds.
High-resolution mass spectrometry (BRUKER APEX IV) was used in order to identify the correct molecular ion for each compound. The ionization method used was electrospray ionization (ESI) with a resolution of 180000 (at
The gas chromatographic analysis was performed using an Agilent 6890 Series II. A gas chromatograph fitted with an autosampler injector. A capillary column HP-5 fused silica column (30 m × 0.25 mm, film thickness 0.25
The chemical structures of the seven pure isolated, characterized, and studied phenolic compounds are reported in Figure
Structures of the isolated phenolic compounds 1–7.
This compound belongs to group A (I) which was isolated from silica gel column of the crude sample as a white powder using the solvent (100% chloroform).
The mass spectrum showed the molecular ion at
The 1H-NMR (CDCl3) spectrum exhibited signals for a monosubstituted benzene ring as a multiplet at
The 13C-NMR (CDCl3) spectrum showed signals for eight carbon atoms. The signal at
The DEPT (135) (CDCl3) spectrum showed only five signals in the positive direction at
This compound belongs to group A (II) and group B (I) who was isolated from silica gel column of the crude sample as a white crystalline powder using the solvent ethyl acetate/benzene (20 : 80).
The mass spectrum showed the molecular ion at
The 1H-NMR (CD3OD) spectrum showed the presence of two doublets at
The 13C-NMR (CD3OD) spectrum showed the presence of six signals at (
The DEPT (135) (CD3OD) spectrum showed only four signals, two of them appeared in the positive direction at
This compound belongs to group A (II) and group B (II) which was isolated from the crude sample by TLC on silica gel plates as a pale yellow powder using the solvent methanol/dichloromethane/ammonia (30 : 70 : vapor). The lowest band
The mass spectrum showed the molecular ion at
The 1H-NMR (CD3OD) spectrum revealed the presence of two doublets at
The 13C-NMR (CD3OD) spectrum showed the presence of eight carbons in the spectrum in which three were methine carbons at 114.8, 112.4, and 124.0 ppm assigned to C-2, C-5 and C-6, respectively. The four quaternary carbons at 121.7, 147.3, 151.3, and 168.8 ppm were assigned to C-1, C-3, C-4 and C-7, respectively. The methoxyl carbon atom showed a signal at
The DEPT (135) (CD3OD) spectrum showed only four signals in the positive direction at
This compound belongs to group A (II) and group B (III) which was isolated from silica gel column of the crude sample as a yellowish-brown powder using the solvent ethyl acetate/benzene (40 : 60).
The mass spectrum showed the molecular ion at
The 1H-NMR (CD3OD) spectrum revealed the presence of two doublets at
The 13C-NMR (CD3OD) spectrum showed the presence of eight signals in which three were methine carbons at 115.2, 115.9, and 120.2 ppm assigned to C-2, C-5 and C-6, respectively. The three quaternary carbons at 130.6, 144.7, and 143.2 ppm were assigned to carbon signals at positions C-1, C-3, and C-4, respectively. Finally, the chemical shift signals at
The DEPT (135) (CD3OD) spectrum showed only five signals, three of which appeared in the positive direction at
This compound belongs to group A (II) and group B (II) which was isolated from the crude sample by TLC on silica gel plates as a white powder using the solvent methanol/dichloromethane/ammonia (30 : 70 : vapor). The middle band
The mass spectrum showed the molecular ion at
The 1H-NMR (CD3OD) spectrum showed the presence of two aromatic protons signals resonating as two doublets at
The 13C-NMR (CD3OD) spectrum showed seven signals for nine carbon atoms. The signal at
The DEPT (135) (CD3OD) spectrum showed only four signals in the positive direction at
This compound belongs to group A (II) and group B (II) which was isolated from the crude sample by TLC on silica gel plates as a white powder using the solvent methanol/dichloromethane/ammonia (30 : 70 : vapor). The third highest band
The mass spectrum showed the molecular ion at
The 1H-NMR (CD3OD) spectrum showed the presence of one methoxyl group signal at
The 13C-NMR (CD3OD) spectrum showed signals for ten carbon atoms. The signal at
The DEPT (135) (CD3OD) spectrum showed only six signals in the positive direction at
This compound belongs to group A (III) which was isolated from silica gel column of the crude sample as a yellow powder using the solvent methanol/chloroform (10 : 90).
The mass spectrum showed the molecular ion at
The 1H-NMR (CD3OD) spectrum revealed the presence of two doublets at
The 13C-NMR (CD3OD) spectrum showed the presence of nine carbons in the spectrum in which five were methine carbons (three aromatic carbons and two aliphatic carbons) at 113.5, 112.1, 119.9, 144.1, and 112.5 ppm assigned to C-2, C-5, C-6, C-7, and C-8, respectively. The four quaternary carbons at 124.8, 143.8, 146.5, and 168.1 ppm were assigned to C-1, C-3, C-4, and C-9, respectively.
The DEPT (135) (CD3OD) spectrum showed only five signals in the positive direction at
The concentrations of the phenolic compounds identified in the crude sample of OMWW were determined quantitatively by preparing a solution of crude the sample with 0.5 mg/L IS and then the compounds were quantified by comparing the relative peak area (RPA) for each compound in the chromatogram of the diluted crude sample with the RPA of the same compound in the chromatogram of the standard solution.
The GC-MS chromatogram of the prepared diluted sample is shown in Figure
GC-MS chromatogram of the prepared crude sample with 0.5 mg/L IS.
Table
GC-MS parameters of silylated derivatives of the phenolic compounds 1–7.
Compound | Retention time (min) | Identified ions ( | LOD (ppb) | LOQ (ppb) |
---|---|---|---|---|
1 | 11.37 | 220, 205, 131, 103 | 2.93 | 9.78 |
2 | 11.62 | 282, 267, 179, 73 | 1.23 | 4.09 |
3 | 13.34 | 312, 297, 282, 267, 253, 223 | 1.54 | 5.15 |
4 | 13.38 | 370, 267, 179, 73 | 0.33 | 1.09 |
5 | 14.74 | 308, 293, 219, 73 | 3.74 | 12.45 |
6 | 15.92 | 338, 323, 308, 249, 73 | 1.19 | 3.98 |
7 | 16.30 | 396, 381, 219, 191, 73 | 2.82 | 9.40 |
Calibration curves were constructed by a linear regression of the peak area ratio of the individual phenolic standard to the IS, versus the concentration of each phenolic standard in the solution mixture. As a result, the calibration curves were linear for all phenolic compounds with regression coefficient of
The results of the analysis of the extract from OMWW are reported in Table
Concentration of identified phenolic compounds in OMWW.
Compound | Concentration of OMWW (mg/L) |
---|---|
105.3 | |
Tyrosol | 210.6 |
Vanillic acid | 128.7 |
Hydroxytyrosol | 315.9 |
117.0 | |
Ferulic acid | 93.6 |
Caffeic acid | 140.4 |
In conclusion, separation and analysis of the phenolic compounds extracted from Jordanian OMWW was achieved using a new procedure. Hydroxytyrosol and Tyrosol are the predominant products in Jordanian OMWW with relatively high concentrations of 315.9 mg/L and 210.6 mg/L, respectively. GC-MS has appeared to be a simple and sensitive analytical tool for the determination of phenolic compounds in olive mill wastewater.
We recommend studying the following parameters and their effect on the constituents obtained and their respective concentrations: (a) effect of method of olive oil pressing, (b) type of olive trees, (c) age of olive trees, (d) area where olive trees are grown, and (e) storage of wastewater.
The authors wish to thank the Higher Council for Science and Technology/National Center for Biotechnology for funding this research.