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