Extraction Technologies for Medicinal and Aromatic ... - Capacity4Dev
Extraction Technologies for Medicinal and Aromatic ... - Capacity4Dev Extraction Technologies for Medicinal and Aromatic ... - Capacity4Dev
13 COUNTER-CURRENT CHROMATOGRAPHY 13.5.1.3 Preparation of Sample Solution • The sample for HSCCC-CPC may be dissolved directly in the stationary phase or in a mixture of the two phases. • The recommended sample volume is less than 5% of the total column capacity. • Introduction of a larger sample volume into the column will reduce peak resolution of the analytes, especially for those having small K values. • Ideally the analyte is injected in a small volume of the stationary phase to preserve the sharpness of the early elution peak. 13.5.1.4 Separation Column • We must understand the head–tail orientation of the separation column. • A lower (heavier) mobile phase should be introduced through the head toward the tail, and an upper (lighter) mobile phase in the opposite direction. • This is extremely important because the elution of either phase in the wrong direction results in an almost complete loss of the stationary phase from the column. 13.5.1.5 Choice of the Mobile Phase • In HSCCC-CPC, either phase can be used as the mobile phase provided that the K value of the analyte is in a proper range. • If one has a choice, the lower phase may be used as the mobile phase, because the system provides more stable retention of the stationary phase and one can avoid trapping air bubbles in the fl ow cell of the detector by introducing the effl uent from the lower end of the cell. • On the other hand, when the upper organic mobile phase (excluding the chloroform system) is used as the mobile phase, it will facilitate the evaporation of solvent from the collected fractions. 13.5.1.6 Flow Rate of the Mobile Phase • The fl ow rate of the mobile phase determines the separation time, the amount of stationary phase retained in the column, and therefore the peak resolution. • A lower fl ow rate usually gives higher retention level of the stationary phase and improves the peak resolution, although it requires a longer separation time. • The typical fl ow rates for the commercial multilayer coil are as follows: i) 5–6 ml/min for a preparative column with 2.6 mm i.d. PTFE tubing (600–800 rpm) (up to 1 g sample load); 224
EXTRACTION TECHNOLOGIES FOR MEDICINAL AND AROMATIC PLANTS ii) 2–3 ml/min for a semi preparative column with 1.6 mm i.d. PTFE tubing (800–1000 rpm) (up to 500 mg sample load); iii) 1 ml/min for an analytical column with 0.85–1.0 mm i.d. PTFE tubing (1000–1200 rpm) (up to 50 mg sample load). • These fl ow rates should be modifi ed according to the settling time of the two-phase solvent system as well as other factors. When the settling time is around 20 s and the K value of the analyte is small, a lower fl ow rate is recommended. 13.5.1.7 Revolution Speed The optimum revolution speed (revolution and planetary rotation speeds are always same) for the commercial HSCCC-CPC instrument for preparative separation ranges between 600 and 1400 rpm. • Use of a lower speed will reduce the volume of the stationary phase retained in the column, leading to lower peak resolution. • On the other hand, the higher speeds may produce excessive sample band broadening by violent pulsation of the column because of elevated pressure. 13.5.1.8 Filling the Column with the Stationary Phase • In each separation, the column is fi rst entirely fi lled with the stationary phase. • Before introducing the stationary phase, the column may be fl ushed with a column volume of a solvent miscible with the two phases used in the previous run (e.g. ethanol or methanol) to wash out materials remaining in the column. • This will also ensure a stable, clean baseline before the solvent front emerges. • Avoid trapping the air in the column, especially in a preparative column. This can be easily tested as: i) If no air is present in the column, the flow from the column outlet is ceased shortly after stopping the pumping. ii) If the solvent keeps fl owing from the outlet for more than several seconds, the air trapped in the column should be eliminated by resuming the pumping of the stationary phase under low speed column rotation (100–200 rpm) in a tail to head elution mode to accelerate air movement toward the outlet of the column. 13.5.1.9 Sample Loading There are two ways to load samples; both are satisfactory for HSCCC-CPC separations: • In the first method, the column is entirely filled with stationary phase and this is immediately followed by sample injection. 225
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EXTRACTION TECHNOLOGIES FOR MEDICINAL AND AROMATIC PLANTS<br />
ii) 2–3 ml/min <strong>for</strong> a semi preparative column with 1.6 mm i.d.<br />
PTFE tubing (800–1000 rpm) (up to 500 mg sample load);<br />
iii) 1 ml/min <strong>for</strong> an analytical column with 0.85–1.0 mm i.d.<br />
PTFE tubing (1000–1200 rpm) (up to 50 mg sample load).<br />
• These fl ow rates should be modifi ed according to the settling<br />
time of the two-phase solvent system as well as other factors.<br />
When the settling time is around 20 s <strong>and</strong> the K value<br />
of the analyte is small, a lower fl ow rate is recommended.<br />
13.5.1.7 Revolution Speed<br />
The optimum revolution speed (revolution <strong>and</strong> planetary rotation<br />
speeds are always same) <strong>for</strong> the commercial HSCCC-CPC instrument <strong>for</strong><br />
preparative separation ranges between 600 <strong>and</strong> 1400 rpm.<br />
• Use of a lower speed will reduce the volume of the stationary<br />
phase retained in the column, leading to lower peak resolution.<br />
• On the other h<strong>and</strong>, the higher speeds may produce excessive<br />
sample b<strong>and</strong> broadening by violent pulsation of the column<br />
because of elevated pressure.<br />
13.5.1.8 Filling the Column with the Stationary Phase<br />
• In each separation, the column is fi rst entirely fi lled with the<br />
stationary phase.<br />
• Be<strong>for</strong>e introducing the stationary phase, the column may be<br />
fl ushed with a column volume of a solvent miscible with the<br />
two phases used in the previous run (e.g. ethanol or methanol)<br />
to wash out materials remaining in the column.<br />
• This will also ensure a stable, clean baseline be<strong>for</strong>e the solvent<br />
front emerges.<br />
• Avoid trapping the air in the column, especially in a preparative<br />
column. This can be easily tested as:<br />
i) If no air is present in the column, the flow from the column<br />
outlet is ceased shortly after stopping the pumping.<br />
ii) If the solvent keeps fl owing from the outlet <strong>for</strong> more than<br />
several seconds, the air trapped in the column should<br />
be eliminated by resuming the pumping of the stationary<br />
phase under low speed column rotation (100–200 rpm)<br />
in a tail to head elution mode to accelerate air movement<br />
toward the outlet of the column.<br />
13.5.1.9 Sample Loading<br />
There are two ways to load samples; both are satisfactory <strong>for</strong><br />
HSCCC-CPC separations:<br />
• In the first method, the column is entirely filled with stationary<br />
phase <strong>and</strong> this is immediately followed by sample injection.<br />
225