Extraction Technologies for Medicinal and Aromatic ... - Capacity4Dev

EXTRACTION TECHNOLOGIES FOR MEDICINAL AND AROMATIC PLANTS
procedure, the stationary phase is more polar than the mobile phase. Lipophilic
substances like oils, fats and lipids are separated by normal phase
chromatography. Commonly used mobile solvents are n-hexane, heptane,
chloroform, and alcohols. Most biomedical substances are separated by
reverse phase chromatography using aqueous mixture with methanol, acetonitrile
and additives (buffers, ion-pairs).
11.2.2 Important Factors that Influence HPLC Separation
HPLC separation is infl uenced by dead volume, capacity factor,
theoretical plate count and selectivity:
• Dead volume (V 0 ) is the volume at which an un-retained component
elutes.
• Capacity factor (K ’ ) is a measurement of the retention time
of a sample molecule, relative to column dead volume. It
changes with variations in mobile phase composition, column
surface chemistry or operating temperature. Capacity
factor is calculated as follows:
K ' = V 1 – V 0
E
V
i
0
V 1 = Retention volume of peak 1
• Theoretical plate count (N) is a measure of column effi ciency
in terms of band-spreading of a peak. The smaller the bandspread,
the higher the number of theoretical plates, which
indicates good column and system performances.
• Resolution (R s ) is the distance between the peak centres of
two component peaks divided by the average base of the
peaks, as follows:
V
R S = 2 – V 1
√W 1 + W 2
W 1 = width of peak 1
W 2 = width of peak 2
• Selectivity (α) is the relative retention of two peaks in a chromatogram.
α = K' 2
K ' 1
= V 2 – V 0
V 1 – V 0
K 1
and K 2
= capacity factors for retention volume of peak 1
and peak 2 respectively.
183