Extraction Technologies for Medicinal and Aromatic ... - Capacity4Dev
Extraction Technologies for Medicinal and Aromatic ... - Capacity4Dev Extraction Technologies for Medicinal and Aromatic ... - Capacity4Dev
14 QUALITY CONTROL OF MEDICINAL AND AROMATIC PLANTS AND THEIR EXTRACTED PRODUCTS BY HPLC AND HIGH PERFORMANCE THIN LAYER CHROMATOGRAPHY Figure 2: Calibration curve of DPH-1 The TLC-based method of analysis and fi ngerprint development is quick and reliable, and can be used conveniently in different laboratories. Similarly, it is possible to apply this technique to other plant drugs to develop fi ngerprint profi les and also to estimate the percentage of marker substances in the crude drugs or in fi nished products. 14.5 High Performance Liquid Chromatography In a period of less than 50 years, HPLC has become the most widely used analytical tool in most laboratories of the world. The technique has received great attention for innovations leading to its overall development, regarding both consumables and equipment. HPLC separations are achieved using any of the fi ve basic chromatographic modes: liquid-solid (adsorption), liquid-liquid (partition), bonded-phase (partition), ion exchange, and size exclusion chromatography. The selected mode depends on the nature and properties of the analyte. Bonded-phase chromatography, in which a stationary phase of organosilanes of varying carbon lengths is chemically bonded to silanol groups, is the most commonly used mode of separation. In liquid-liquid chromatography, the solid support (usually silica or kieselguhr) is mechanically coated with a fi lm of high boiling point organic liquid, unlike bonded-phase chromatography where non-polar hydrocarbon chains are chemically bonded to hydroxyls of the silica support. Liquid-liquid chromatography, by virtue of its mechanism, is more susceptible to changes by interaction with mobile phase than bonded-phase chromatography. A typical HPLC operation includes pumping of mobile phase at moderately high pressure through a narrow-bore column containing adsorbent. The separation of the mixture takes place in the column and separated components are detected by employing a suitable detector. As the mobile phase is being pumped at high pressures, a system is required to inject the mixture into the system without dropping the pressure and disturbing the fl ow characteristics, i.e. rate and pressure. To accomplish these requirements, an HPLC system requires a pump to push the mobile phase against high pressure, an injector to insert a solution of standard substance or test 254
EXTRACTION TECHNOLOGIES FOR MEDICINAL AND AROMATIC PLANTS mixture, a column to effect separation, a detector to reveal the presence of analyte in the eluate, and a suitable data processing unit. 14.5.1 Pumps The pump is considered a heart of the HPLC system, as all depends on the composition of the mobile phase and its fl ow rate accuracy. The pump gives a pulse-free fl ow of mobile phase; the expected variations in fl ow rate are less than 1.0%. Online mixing of solvents is preferred to manual mixing. However, compositions containing less than 10% of a particular solvent are better prepared by manual mixing. The composition of the mobile phase is either constant during the analysis (isocratic mode) or it is changed (gradient mode). The type and design of modern pumps allow low pressure mixing of up to four solvents; else, different pumps, one for each solvent, are required for gradient operation and the solvents are mixed at high pressure. A typical analytical procedure uses a fl ow rate of about 1 ml/ min and operating pressure between 1000 and 2000 psi. Higher fl ow rates generating higher pressure should always be used with justifi cation, as they decrease column life besides requiring frequent servicing of the pump. The fl ow accuracy of the pumps is critical for analysis. The constancy of retention time of the last eluted peak is a measure of long-term fl ow accuracy of the pumps, whereas short-term fl ow accuracy is checked from the average peak areas of each component and their standard deviations. The mobile phase must be free of dissolved gases to ensure an accurate fl ow and to minimize noise due to bubbles. Vacuum fi ltration, sonication and helium gas purging are methods for degassing. 14.5.2 Injector The injector allows a predetermined volume of test solution to be introduced into the fl ow channel of the system, without disturbing the fl ow kinetics. Typically, fi xed volume injections are preferred over variable volume injections. When using fi xed volume loops, it is advisable to fl ush higher volumes of the sample through the loop to ensure complete fi lling of the loop with the sample solution. The mobile phase close to the inner walls of the loop can only be assured to have pushed out after injecting volumes larger than the loop volume, e.g. injecting 20 μl test solution into a 20-μl loop cannot assure accurate injection; if the quantity of the test material is not a problem, fl ush the loop with over 100 μl test solution. Only the appropriate needle (compatible with the injection port) should be employed for the purpose of making an injection. It is important to select a syringe of appropriate size when giving variable injections; a thumb rule for any analytical technique is not to use the volumetric apparatus if less than 20% of its total volume is being used. Thus, a syringe of 25 μl should not be used to measure or inject volumes less than 5 μl. 255
- Page 208 and 209: 12 FLASH CHROMATOGRAPHY AND LOW PRE
- Page 210 and 211: 12 FLASH CHROMATOGRAPHY AND LOW PRE
- Page 212 and 213: 12 FLASH CHROMATOGRAPHY AND LOW PRE
- Page 214 and 215: 13 COUNTER-CURRENT CHROMATOGRAPHY C
- Page 216 and 217: 13 COUNTER-CURRENT CHROMATOGRAPHY K
- Page 218 and 219: 13 COUNTER-CURRENT CHROMATOGRAPHY 1
- Page 220 and 221: 13 COUNTER-CURRENT CHROMATOGRAPHY a
- Page 222 and 223: 13 COUNTER-CURRENT CHROMATOGRAPHY 1
- Page 224 and 225: 13 COUNTER-CURRENT CHROMATOGRAPHY W
- Page 226 and 227: 13 COUNTER-CURRENT CHROMATOGRAPHY S
- Page 228 and 229: 13 COUNTER-CURRENT CHROMATOGRAPHY 1
- Page 230 and 231: 13 COUNTER-CURRENT CHROMATOGRAPHY
- Page 232 and 233: 13 COUNTER-CURRENT CHROMATOGRAPHY E
- Page 234 and 235: 13 COUNTER-CURRENT CHROMATOGRAPHY 1
- Page 236 and 237: 13 COUNTER-CURRENT CHROMATOGRAPHY F
- Page 238 and 239: 13 COUNTER-CURRENT CHROMATOGRAPHY 1
- Page 240 and 241: 13 COUNTER-CURRENT CHROMATOGRAPHY H
- Page 243 and 244: EXTRACTION TECHNOLOGIES FOR MEDICIN
- Page 245 and 246: EXTRACTION TECHNOLOGIES FOR MEDICIN
- Page 247 and 248: EXTRACTION TECHNOLOGIES FOR MEDICIN
- Page 249 and 250: EXTRACTION TECHNOLOGIES FOR MEDICIN
- Page 251 and 252: EXTRACTION TECHNOLOGIES FOR MEDICIN
- Page 253 and 254: EXTRACTION TECHNOLOGIES FOR MEDICIN
- Page 255 and 256: EXTRACTION TECHNOLOGIES FOR MEDICIN
- Page 257: EXTRACTION TECHNOLOGIES FOR MEDICIN
- Page 261 and 262: EXTRACTION TECHNOLOGIES FOR MEDICIN
- Page 263 and 264: EXTRACTION TECHNOLOGIES FOR MEDICIN
14 QUALITY CONTROL OF MEDICINAL AND AROMATIC PLANTS AND THEIR EXTRACTED PRODUCTS<br />
BY HPLC AND HIGH PERFORMANCE THIN LAYER CHROMATOGRAPHY<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
Figure 2: Calibration curve of DPH-1<br />
The TLC-based method of analysis <strong>and</strong> fi ngerprint development<br />
is quick <strong>and</strong> reliable, <strong>and</strong> can be used conveniently in different laboratories.<br />
Similarly, it is possible to apply this technique to other plant drugs to<br />
develop fi ngerprint profi les <strong>and</strong> also to estimate the percentage of marker<br />
substances in the crude drugs or in fi nished products.<br />
14.5 High Per<strong>for</strong>mance Liquid Chromatography<br />
In a period of less than 50 years, HPLC has become the most<br />
widely used analytical tool in most laboratories of the world. The technique<br />
has received great attention <strong>for</strong> innovations leading to its overall development,<br />
regarding both consumables <strong>and</strong> equipment. HPLC separations are<br />
achieved using any of the fi ve basic chromatographic modes: liquid-solid<br />
(adsorption), liquid-liquid (partition), bonded-phase (partition), ion exchange,<br />
<strong>and</strong> size exclusion chromatography. The selected mode depends on the nature<br />
<strong>and</strong> properties of the analyte. Bonded-phase chromatography, in which<br />
a stationary phase of organosilanes of varying carbon lengths is chemically<br />
bonded to silanol groups, is the most commonly used mode of separation.<br />
In liquid-liquid chromatography, the solid support (usually silica or<br />
kieselguhr) is mechanically coated with a fi lm of high boiling point organic<br />
liquid, unlike bonded-phase chromatography where non-polar hydrocarbon<br />
chains are chemically bonded to hydroxyls of the silica support. Liquid-liquid<br />
chromatography, by virtue of its mechanism, is more susceptible to changes<br />
by interaction with mobile phase than bonded-phase chromatography.<br />
A typical HPLC operation includes pumping of mobile phase at<br />
moderately high pressure through a narrow-bore column containing adsorbent.<br />
The separation of the mixture takes place in the column <strong>and</strong> separated<br />
components are detected by employing a suitable detector. As the mobile<br />
phase is being pumped at high pressures, a system is required to inject the<br />
mixture into the system without dropping the pressure <strong>and</strong> disturbing the<br />
fl ow characteristics, i.e. rate <strong>and</strong> pressure. To accomplish these requirements,<br />
an HPLC system requires a pump to push the mobile phase against<br />
high pressure, an injector to insert a solution of st<strong>and</strong>ard substance or test<br />
254
Change language