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CHROMATOGRAPHIC THEORY ORIGINS OF BAND BROADENING IN HPLC

VAN DEEMTER EQUATION IN LIQUID CHROMATOGRAPHY


INTRODUCTION - Chromatographic peaks have width
Chromatography Molecules of a single compound, despite
- Most frequently used analytical technique in having the same capacity factor, take
pharmaceutical analysis different lengths of time to travel through
the column
VOID VOLUME AND CAPACITY FACTOR - travel time of analyte to column, spread out of
- If a compound does not partition appreciably into the indiv molecules, broad the band becomes
the stationary phase, it will travel through the - rapid the band broadens, less efficient column
column at the same rate as the solvent. - Equation 3: Van Deemter equation
Void volume of the column 1
= + + + 2
length of the time it takes an unretarded
1+ 1
molecule to flow through the column 2
length of time it measure of the efficiency of the column
length of time it takes a retarded compound to (smaller term, more efficient column)
pass through the column linear velocity of the mobile phase
measure of the degree to which it partitions how many cm/s an unretained molecule travels
(adsorbs) into the stationary phase from the through the column
mobile phase: eddy diffusion term

=

Retention volume of the analyte
Void volume of the column
time taken for the analyte to pass through
the column
time taken for an unretained molecule to
pass through the void volume

CALCULATION OF COLUMN EFFICIENCY


- Broader chromatographic peak, the less efficient the
column it is eluting form
- Equation 1:

= 5.54 ( )2
1
2
number of theoretical plates
- Column efficiency expressed in theoretical plates
per meter:
100


column length (cm)
- Equation 2: stricter measure of the column
efficiency, esp. if the retention time of the analyte is
short

= 5.54 ( )2
1
2
number of effective plates
# of times the analyte partitions between
the mobile phase and stationary phase during its
passage through the column and =
- Height if a theoretical plate
Length of column required for one partition step
to occur

=