Chapter 25 High-Performance Liquid Chromatography
(HPLC)
p. 608-634 Harris (6th)
p. 556 Harris (7th)
Advantages of HPLC

two interaction phases  stationary and mobile

not limited by molecular weight or volatility of analytes

room-temperature operation
 can accommodate thermally unstable samples
Limitation of HPLC
separation efficiency has a practical upper limit of 100,000 plates/m
Experiment 6
Monitoring of Carbonyls by 2,4-Dinitrophenylhydrazine
Derivatization and Reversed-Phase High Performance Liquid
Chromatography
HPLC Solvents
21 solvents of uncompromised quality are available to meet the
most demanding applications

high assay

clean UV trace with a smooth curve throughout

low UV absorbance at critical wavelengths

UV cut-offs at or approaching the theoretical limit

low residue after evaporation
Water
$63 for a 4 L bottle
Solvents (or Mobile Phase)
 degassed by evacuation, boiling, or purging with He (very insoluble).
 otherwise, gas bubbles are formed by pressure changes or the mixing of certain
solvents
Guard column
 short, expendable (periodically discarded).
 contains same stationary phase as analytical column.
 removes impurities in the solvent and sample
Sample loop
 a steel tube with a narrow bore.
 in various fixed sample volumes (2 μL - 1 mL).
LOAD position
 a syringe is used to wash and load the loop with sample solution at
atmospheric pressure.
 high-pressure flow from the pump to the column passes through the
valve
Valve handle is rotated 60o
INJECT position
 the contents of the sample loop are injected into the column at high
pressure.
p. 566 Harris (7th)
Selecting the Separation Mode
Normal-phase HPLC is the method of choice for analysis of organic
solutions (in chloroform).
p. 561 Harris (7th)
Reverse-phase HPLC for analysis of aqueous solutions (in water)
non-polar bonded stationary phase
R = -(CH2)17CH3
octadecyl
ODS
most hydrophobic
R = -(CH2)7CH3
octyl
intermediate hydrophobicity
R = -(CH2)3CH3
butyl
R = -(CH2)3C6H5
phenyl
polar solvent as mobile phase
eluent strength of mobile phase is increased by addition of a
less polar solvent
Reverse-phase HPLC of non-polar solutes in water
Solvents in order
of increasing polarity
Solvent strength (Eluting power)
Hexane, heptane
Petroleum ether
Cyclohexane
.
.
.
.
.
Ethyl acetate
Acetone
Acetonitrile
n-Propanol
Ethanol
Methanol
Acetic acid
Water
Highest solubility, shortest tR
Non-polar, high eluent strength
Polar, low eluent strength
Lowest solubility, longest tR
pH effect on retention time (and elution order)
Octanoic acid and 1-aminooctane were passed through a reversephase HPLC column, using an eluent of 20% methanol in water
adjusted to pH 3.0 with HCl.
Which compound is expected to be eluted first, and why?
Octanoic acid
CH3CH2CH2CH2CH2CH2CH2CO2H
1-Aminooctane
CH3CH2CH2CH2CH2CH2CH2CH2NH2
p. 565 Harris (7th)
Isocratic and Gradient Elution
Isocratic elution
using a single solvent (or constant solvent mixture composition)

maintains the column in equilibrium

no re-equilibration time between analyses

sample throughput is maximised

preferred for routine analysis
Gradient elution
using two or more solvents of different eluent strengths (or polarities)

the volume ratio is varied in a programmed way

mixed in solvent chamber before introduction to
column

enhances resolution of early peaks
shortens elution times of late peaks
p. 580-583 Harris (7th)
Pharmaceutical Analysis by HPLC
To look for an impurity in a pharmaceutical formulation/
preparation by analyzing the major component is
inherently an unsound approach
EXPLOSIVE ANALYSIS by HPLC on Ultracarb 5μ ODS
Canadian Explosives Research Laboratory
http://www.nrcan.gc.ca/mms/cerl/home_e.htm
Qualitative GC HPLC Analysis for peak identification
p. 541 Harris (7th)

Comparison of retention time
 with that of an authentic sample of
suspected compound.

Co-chromatography
 authentic sample of suspected compound is
added to the unknown
if the added compound is identical to one
component of the unknown, the area of that
one peak will increase
 or with tabulated Kovats retention
indexes.
For isocratic elution on a single column at different flow rates
 Capacity factor
k = ( tr - tm ) / tm
 Retention volume
Vr = tr F
where F = flow rate of mobile phase
( mL/min )
Vr = Vm + K Vs
or
K = (Vr - Vm) / Vs
where K = partition coefficient
Vr = retention volume
Vm = volume of mobile phase
and Vs = volume of stationary phase
= volume of solvent inside the gel particles (in MEC)
 23-4
Efficiency of Separation
p. 511 Harris (7th)
Resolution
a measure of how well two compound peaks are separated from
each other by chromatography
R = tr / wav
where tr is the difference in retention times between the two peaks
and wav is their average base width.