CHROMATOGRAPHIC THEORY ORIGINS OF BAND BROADENING IN HPLC

VAN DEEMTER EQUATION IN LIQUID CHROMATOGRAPHY

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

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

=