Extraction Technologies for Medicinal and Aromatic ... - Capacity4Dev

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11 PROCESS-SCALE HIGH PERFORMANCE LIQUID CHROMATOGRAPHY FOR MEDICINAL AND AROMATIC PLANTS Capacity factor (K ’ ), selectivity (α) and column effi ciency (N) are three fundamental parameters that infl uence the resolution of a chromatographic separation, as follows: R s = 1 4 ( α – 1 α ) √⎯N ( K ' 1 + K ') 11.2.3 Main Components of HPLC An HPLC system contains the following components: a) Reservoir. This is meant for the mobile solvents. Acetonitrile, methanol, heptane, isopropanol and cyclohexane are the organic modifi ers most commonly used. Trifl uoroacetic acid, heptafl uorobutyric acid, phosphoric acid and triethylamine phosphate are ion-pairing reagents for better chromatographic results. All tubing and fi ttings should be chemically inert. Solvent must be fi ltered through a 0.45-μm fi lter unit. b) Degasser. In analytical operations, the mobile phase should be free of air bubbles. For this purpose, a degasser is used. c) Pumps. These are devices that deliver the mobile solvent at a controlled flow rate to the separation system. HPLC uses reciprocating pumps: a pump with a single or multiple chambers, from which the mobile phase is displaced by reciprocating pistons or diaphragms. Binary gradients are created by the selected mixing of two solvents, on a single-headed two-pump system. Accurate gradient is maintained by microprocessor control. d) Injector/autosampler. This device introduces a liquid sample into the mobile phase or onto the chromatographic bed. An autosampler can perform repeated functions without operator attendance, and thus is a labor-saving device. e) Column. Silica and modifi ed silica columns are available for various applications. Examples are octyl (C 8 ), octadecyl (C 18 ), phenyl (C 6 H 5 ), and cyno (CN) columns. f) Guard column. This is used to protect the main column. g) Detectors. No universal detector is available for all molecules. However, according to the characteristic of the molecules investigated, various detectors are used (Table 1). h) Fraction collector. This device collects the fractions containing the molecules of interest during the chromatographic run. i) Records. A computer is used for chromatographic data acquisition. Table 1: Characteristics of various HPLC detectors Detector Application Advantages and limitations Electrochemical Responds to substances that are oxidizable or reducible Commercially available. Non-specifi c. High LOD 184
EXTRACTION TECHNOLOGIES FOR MEDICINAL AND AROMATIC PLANTS Detector Application Advantages and limitations Fluorescence Detects trace-level analytes such as afl atoxins, carbamates and polycyclic aromatic hydrocarbons Very specifi c. Low LOD. Not everything fl uoresces Infrared Works for all molecules Many solvents are infrared-active Mass spectrometry Photodiode array Analyte identifi cation Works for wavelengths 190-800 nm Ability to ionize analyte. Low LOD High LOD Refractive index Works for nearly all molecules Temperature sensitive. High LOD Scattering Uniform response Non-specifi c. LOD, 5 ng per 25 mL. Interference from solvent Ultraviolet and visible LOD, level of detection Works for molecules with chromophores and for complex samples 11.2.4 HPLC Classification Non-specifi c. All molecules that absorb UV and visible light can be detected. HPLC may be characterized depending on column diameter, which is the governing factor for fl ow rate from microscale to industrial scale chromatography. Column internal diameter (i.d.) defi nes the sample load and fl ow rates (Figure 1). Figure 1: Classifi cation of HPLC according to column diameter 185
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Extraction Technologies for Medicin
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CONTRIBUTORS Chapter 6 Aqueous Alco
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7 DISTILLATION TECHNOLOGY FOR ESSEN
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