HPLC
Presented by
JASPREET SINGH
Overview
HPLC is
A physical separation technique in which a sample dissolved
in liquid is injected into a column packed with small
particles and is separated into its constituent components
The most important and widely used analytical technique
for the quantitative analysis of organics and biomolecules
Applicable to many sample types
– Most useful for pharmaceuticals, biomolecules, and labile
organics (also some ionic compounds)
– Annual sales of HPLC equipment worldwide > $1 billion;
>100,000 HPLC's are in use worldwide today
History of Liquid Chromatography
Chromatography was first discovered by Russian
botanist Mikhail Tswett who separated plant
pigments on chalk columns in 1903
British chemists A.T. P Martin and R. L. M. Synge
developed partition chromatography in 1942; Martin
and A. T. James developed gas chromatography (GC)
in 1952
Since 1940’s, chemists used gravity-fed silica columns
to purify organic materials
In late 60’s, LC turned high performance with small-
particle columns that require high-pressure pumps
Development of on-line detectors allows HPLC to
become a sensitive and quantitative technique for
diverse applications
Classical LC (Gravity flow
Advantages of HPLC
Amenable to diverse samples including labile organics, biomolecules, and
ions - Can handle > 60% of all existing compounds vs. 15% for GC
High-resolution
– Resolves hundreds of components in complex samples
High sensitivity detection
– pg - ng detection limits
Rapid and precise analysis
– 1 - 60 min analysis, Precision < 1% RSD
Automated analysis
– Using autosampler and data system for unattended analysis and
report generation
Quantitative sample recovery
– Preparative technique from mg to kg quantities
The Chromatographic Process
Mobile Phase
Analyte
In LC, analytes to be separated is distributed between two
phases Stationary Phase
– Mobile phase (a flowing liquid)
– Stationary phase (a column packed with porous particles)
– Components is separated by differential interactions
(repetitive sorption/desorption) with the porous support
An on-line detector monitors the concentration of each eluting
component and generates a trace called the chromatogram
The
chromatogram
An HPLC Chromatogram
Column : PE Reduced activity 3x3
C18 (32 x 4.6 mm i.d.)
Mobile Phase : 80% Methanol in water
Flow rate : 1 mL/min
Pressure : 1000 psi
Sample : A mixture of organics
0.01 - 2%
pyridine
T-butylbenzene
Absorbance
260 nm
uracil
0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8
Time (min)
HPLC: Basic Terminology
Retention time (tR)
Peak width (Wb)
Capacity Factor (k’)
Column Efficiency
– Plate number (n)
– Height Equivalent of a Theoretical Plate (HETP)
Selectivity (a)
Resolution (Rs)
Retention Time (tR)
tR = Retention time t0 = Retention time of an unretained solute
Wb = Base width of peak (4 s width)
ak tends to be gaussian and broadens with time - Wb becomes larger with longe
Capacity Factor (k’)
k’ is a measure of peak retention or how many times
the peak is retained vs an unretained peak (to )
k’ = (tR - to ) / to = tR ‘ / to
Separation Factor or Selectivity
(α )
a (separation factor) = k2’ / k1’ = 2.1” / 1.5” = 1.4
a is dependent on the column and mobile phase
a is measure of differential retention of two analytes
by the column and a must be > 1.0 for peak separation
Column Efficiency (n)
Plate number (n) is a measure of column efficiency
n = (tR / s) 2 = 16 (tR / Wb ) 2 = 16 (135 / 10)2 = 2916
n is determined by particle size (dp ), column length (L) and
flow rate (F)
Wb is base width by the tangent method. Alternately, use n = 5.54 (tR / W0.5 ) 2
Resolution (Rs)
Rs (resolution) = 2(tR1 - tR2 ) / Wb = D t / Wb = 23 / 14 =1.5
Rs is a measure of the degree of separation of two peaks.
Rs = 0 (no separation); Rs = 1 (partial separation);
Rs = 1.5 (baseline separation)
Rs = 1.5
The Resolution Equation
Rs = (k’/1+k’) (a -1/ a) (n)0.5 / 4
retention selectivity efficiency
The goal of most LC separations is to achieve baseline
resolution for all key analytes (Rs > 1.5)
k’ should be kept between 1 to 10
a is maximized with by selecting the column and mobile phase
that resolve all critical analytes
n is increased using high-efficiency columns of a reasonable
length
Keep analysis time < 1 hour and operating pressure < 3000 psi
HPLC Equipment
Components of an HPLC System
Waste
Solvent
Reciprocating Pump
Schematics Most HPLC pumps are
reciprocating
A motor driven cam
drives the piston to deliver
solvent through the outlet
check valve
Gradient are formed by
using 2 or more pumps
(high-pressure mixing)
or solenoid-actuated
proportioning valves (low-
pressure mixing)
Detectors for HPLC
An HPLC detector is equipped with a small
flow cell
(< 8-mL) connected to the column outlet and
monitors the concentration (or mass) of the
analyte component
The most common detector is:
UV/Vis absorbance (UV/Vis)
UV/Vis Absorbance Detector
Schematic
Characteristics of a UV/Vis
Detector
UV/Vis absorbance detector typically consists of :
– A deuterium source (190 - 600 nm)
– A monochromator involving a moveable grating controlled by
stepper motor to select a certain wavelength through the exit slit
to a small flow cell (about 8 µ L)
– Two photodiodes to measure the light intensity of the sample
and reference beam
Principle for absorbance detector is the Beer’s Law
Absorbance = molar absorptivity x pathlength x concentration
A = ε b c = - Log I / I0 where I0 = Initial light intensity
Is the most common detector for HPLC, capable of ng detection
– Noise/drift characteristics important for sensitivity
HPLC Applications
Applications Categories
Pharmaceutical
Environmental
LifeSciences and Biotechnology
Food applications
GPC/Plastics/Chemicals
