Supercritical Carbon Dioxide - American Chemical Society

Chapter 10

Downloaded by NORTH CAROLINA STATE UNIV on November 5, 2013 | http://pubs.acs.org Publication Date: August 31, 2003 | doi: 10.1021/bk-2003-0860.ch010

Hot Water Extraction Followed by Solid-Phase Microextraction of Active Ingredients in Rosemary: An Organic Solvent-Free Analytical Technique Youxin G a n and Yu Yang

*

Department of Chemistry, East Carolina University, Greenville, NC 27858

An organic solvent-free technique was developed in this study to determine the concentrations of the active ingredients (pinene, camphene, limonene, camphor, citronellol, and carvacrol) of rosemary in the water phase after cooking at 100 °C. The water extract obtained from the hot water extraction was then used as the sample for solid-phase microextraction (SPME) followed by GC analysis. Polydimethylsiloxane fiber was employed in SPME. SPME was optimized by evaluating the carryover effect and effects of water volume and sorption time on SPME efficiency. The concentrations of six active components in rosemary in the water phase after cooking were determined using this green analytical technique. Up to 15% of the active ingredients in rosemary were found in the water phase after a 15-min hot water extraction at 100 °C. This organic solvent-free coupling technique could have a good potential in pharmaceutical and food analysis.

© 2003 American Chemical Society

In Supercritical Carbon Dioxide; Gopalan, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2003.

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146 INTRODUCTION Tracing the history of food and pharmaceuticals, plants have served human beings for thousands o f years. Chinese people have a long history o f preparing and using herbs for the treatment o f all kinds o f diseases. Traditional Chinese medicines are prepared with dried herbs, which are cooked with boiling water in order to extract die active ingredients into the soup. Patients take the soup according to the prescription. This cooking process is actually a so-called hot water extraction. In order to make better application of medicinal herbs, people extract the active ingredients from the raw plante to make pills, tablets, juice, and injecting solutions. Extraction methods applied to natural products generally include hot water extraction, steam distillation, organic solvent extraction, supercritical fluid extraction, and microwave-assisted extraction [1-6]. Rosemary (Rosemarinus officinalis) is one o f the widely used natural products in everyday life. It is generally used as a spice for cooking, but it is also an important herb. The leaves and tops o f rosemary can be used as herbs for tonic, diaphoretic, stomachic, and antirheumatic purposes [6]. The major active ingredients in rosemary have been studied and identified [7,8], and they include pinene, camphene, limonene, camphor, citronellol and carvacrol. The concentrations o f these active ingredients in raw rosemary are well understood, but the fraction o f the active ingredients i n the water phase after hot water extraction (cooking) process is less studied. The water phase is normally treated by solid phase extraction (SPE) or liquid-liquid extraction for quantitation. Therefore, organic solvents are involved i n the analysis of the active ingredients in the water phase. Solid-phase microextraction (SPME) uses a very small amount o f organic phase coated on the needle o f a specially designed syringe to extract analytes from water samples or the headspace [9-11]. The S P M E process involves two steps: partitioning o f analytes between the coating and the sample matrix, followed by desorption of the concentrated extracts into an analytical instrument. In the first step, die coated fiber is exposed to the sample or its headspace, which causes the target analytes to partition from die sample matrix into the coating. The fiber bearing extracted analytes is then transferred to an instrument for desorption, whereupon separation and quantification o f extracts can take place. Since S P M E can only be applied to gas or liquid samples, hot (or subcritical) water extraction can convert solid samples into water samples so that S P M E can be used for solid matrices. Therefore, the coupling o f S P M E with subcritical water extraction has also been studied [12-15]. The goal o f this research was to develop an organic solvent-free technique to determine the concentrations of the active ingredients of rosemary in the water phase after the hot water extraction at 100 °C. Hot water extraction was used to simulate the cooking process, and the water extracts from the hot water extraction were then used as samples for solid-phase microextraction followed


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by G C analysis. Therefore, no organic solvents were involved in the entire extraction/analysis process. Please note that the emphasis of this technique is not to exhaustively extract the active ingredients from herbs but to determine how much of them can be found in the water phase after cooking, the traditional way of preparing Chinese medicinal herbs. EXPERIMENTAL Samples, Chemicals, and Reagents The rosemary used i l l this project was directly purchased from a local grocery store. The raw rosemary was grounded using ceramic mortar and pestle. Methylene chloride and acetone used for solvent extraction were of analytical grade and purchased from Fisher Scientific (Pittsburgh, PA). Pinene, camphene, limonene, camphor, citronella, and carvacrol were obtained from Aldrich (Milwaukee, WI). Hot Water Extraction ofRosemary Approximately 0.5 g of grounded rosemary was weighed into a 20-mL glass vial as a single sample for hot water extraction at 100 °C. Approximately 8 m L of distilled water was added into the rosemary vial. The loaded vial was put on a hot plate to perform the hot water extraction. The vial was loosely sealed with a rubber cap. The water extraction time was 15 min. After the hot water extraction at 100 °C, the glass vial was removed from the hot plate and cooled down to room temperature. Clear aqueous solution was obtained by filtration using small filter paper. This water sample was then ready for solid-phase microextraction. Solid-Phase Microextraction Before applying solid-phase microextraction to the water extracts obtained from hot water extraction o f rosemary, optimization of experiments were conducted to determine die optimum working conditions for S P M E , including water volume, S P M E time, and carry over effect. Polydimethylsiloxane fiber (PDMS, 100 μπι, Supelco, Bellefonte, PA) was used for performing solid-phase microextraction. Three different extraction volumes (2, 4, and 8 mL) were tested with S P M E extraction time set to 15 min. The solid-phase microextraction time studied was 15, 30, and 60 min while the water volume was set to 4 mL. In order to study the fiber carryover effect in G C analysis, the total thermal desorption time (at 250 °C) was set to 10 min. Two sets of experiments were performed: 1-min thermal desorption followed by 9-min desorption, and 5-min desorption followed by 5-min desorption. The water extractant of rosemary was separated from the rosemary residue. The P D M S fiber was dipped into the glass vial containing the water extracts.


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SPME was performed for IS min. In order to determine the concentration of the target analytes in the water phase, calibration curves were prepared using the same SPME procedure. All SPME extractions were performed at room temperature while the water samples were stirred during the SPME extraction process. Sonication Extraction ofRosemary In order to determine the total concentration of the target analytes in rosemary, solvent extraction of rosemary was also performed using a Fisher FSS sonication bath. A solvent mixture of methylene chloride and acetone (50:50, v%) was chosen for solvent extraction. For the determination of the total concentration of analytes in rosemary, approximately 0.3-0.4 g of grounded rosemary was weighed into a glass vial, and 3 mL of methylene chloride and acetone mixture was then added into the vial. The solution was thoroughly mixed before setting into a water bath for sonication. Sonication extractions were performed for 12 hours. After the sonication extraction, die upper layer of the clear solution was injected into GC for analysis. Gas Chromatographic Analysis A Hewlett-Packard (Wilmington, DE) 6890 GC was used in this project. A HP-35MS capillary column (30 m χ 0.25 mm id, 0.25 μπι film thickness) was used for separation, while a flame ionization detector (FID) was employed for detection. Both injector and FID temperatures were held at 250 °C. The initial oven temperature was 40 °C. After 5-min holding time, the oven temperature was increased at 8 °C/min to afinaltemperature of 300 °C, with 15-min holding time. Splitless injection was employed for SPME injections while split mode was used for most solvent injections. RESULTS AND DISCUSSION SPME Carryover Effect Unlike any other traditional GC injection methods (e.g., solvent injection using a traditional syringe), carryover effect plays an important role in SPME/GC analysis. If the thermal desorption time is not properly chosen, the compounds remaining on the SPME fiber lead to poor accuracy and precision in SPME/GC analysis. Two experiments were performed. First, the SPME fiber was thermally desorbed in the hot GC injector (250 °C) for 1 min, then GC analysis was performed. After the GC analysis for the 1-min desorption, the fiber was replaced in the GC injector for another 9-min desorption then GC analysis was again performed. Another experiment was done in a similar way but with 5min desoprtion/GC analysis followed by a second 5-min desorption/analysis. The total thermal desorption time was 10 min for both experiments. As shown


149 in Table 1, the 5-min desorption yielded higher percentage of desorption, therefore, 5-min desorption was used as desorption time for the remainder o f this work. Table 1. Influence of Thermal Desorption Time on Carryover Effect of S P M E Fiber a


Compound

%Desorbed(%RSD ) 1 min

5 min

Pinene

99.34

(

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