High Performance Liquid

Chromatography
HPLC
Yamen Alkhateeb

1
 Agenda
• Introduction
• Advantages
• Applications
• Component of HPLC
• Normal Phase vs. Reverse Phase
• Calibration
• Method Validation

2
 The History of HPLC
• Liquid chromatography is a separation technique
• The components of the sample are separated because of
the different interactions between the sample components
and the stationary phase.

3
 The History of HPLC
• Beginning of the 60’s: start of HPLC as High
Pressure Liquid Chromatography
• End of the 70’s improvements of column material
and instrumentation – High Performance Liquid
Chromatography
• Since beginning of the 80’s: “boom” in HPLC
started
4
 Introduction
• HPLC is a form of liquid chromatography used to separate
compounds that are dissolved in solution.
• A powerful tool in analysis, it yields high performance and
high speed analysis.
• It is used in biochemistry and analytical chemistry to
identify, quantify and purify the individual components of a
mixture.

5
 Advantages of HPLC
• High separation capacity, enabling the batch analysis of multiple
components
• Superior quantitative capability and reproducibility
• Moderate analytical conditions
– Unlike GC, the sample does not need to be vaporized.
• Generally high sensitivity
• Low sample consumption
• Easy preparative separation and purification of samples

6
 HPLC Applications
Bioscience
Chemical proteins
peptides
HPLC is one of polystyrenes
dyes
nucleotides

the most widely phthalates

applied analytical
Pharmaceuticals tetracyclines
separation corticosteroids Consumer Products
antidepressants
techniques. barbiturates
lipids
antioxidants
sugars
Environmental
polyaromatic hydrocarbons Clinical
Inorganic ions amino acids
herbicides vitamins
homocysteine
7
The 5 Major components of
HPLC
solvent

pump

injector

column

detector
8
 Mobile Phase
• The type and composition of the • Isocratic system: Constant
mobile phase affects the separation eluent composition
of the components.
• Gradient system: Varying
• Different solvents are used for
different types of HPLC. For
eluent composition
normal-phase HPLC, the solvent is
usually nonpolar, and, in reverse-
phase HPLC, the solvent is
normally a mixture of water and a
polar organic solvent.

9
 Aim of Gradient System

Isocratic mode Gradient system

10
 Pump
• The role of the pump is to force
the mobile phase through the
liquid chromatograph at a
specific flow rate
• Normal flow rates in HPLC are in
the 1- to 2-mL/min range.
• Typical pumps can reach
pressures in the range of 400 to
600 bar.

11
 Injector
• to introduce the liquid sample into the
flow stream of the mobile phase.
• Typical sample volumes are 5 to 100
microliters (µL).
• The most accurate injections are made
with a technique known as Overfilling.
injection has to be 3 to 5 times larger
than the volume of the injection loop.
• Autosampler removes human error and
increases the precision and accuracy.

12
 Column
Several column types
• Normal phase
• Reverse phase
• Size exclusion
• Ion exchange

13
 Guidelines for Selecting
Separation Mode
• Reversed phase mode using an ODS column is the first choice.
• Exceptions
– Large molecular weight (> 2,000)  Size exclusion
– Optical isomers  Chiral column
– Stereoisomers, positional isomers  Normal phase /
adsorption
– Inorganic ions  Ion chromatography
– Sugars, amino acids, short-chain fatty acids
 Special column
14
 Reversed phase
• C18 (ODS) type • Phenyl type
• C8 (octyl) type • Cyano type
• C4 (butyl) type

CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2

Si -O-Si
CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH3

C18 (ODS)
15
 Relationship Between
Retention Time and Polarity

C18 (ODS) OH

Weak
Strong
CH3

16
Relationship between Polarity of Eluent and
Retention Time in Reversed Phase Mode
Eluent: Methanol / Water

60/40

70/30

80/20
17
 Detector
The detector can detect the individual molecules that elute
from the column and convert the data into an electrical
signal.
• UV-VIS detector
• Photodiode array-type UV-VIS detector
• Fluorescence detector
• Refractive index detector
• Evaporative light scattering detector
• Electrical conductivity detector
• Electrochemical detector 18
• Mass spectrometer
 UV-VIS Absorbance Detector

C: Concentration
Detection cell

Ein Eout

A
l

A = e·C·l = –log (Eout / Ein) C

(A: absorbance, E: absorption coefficient)
19
Spectrum and Selection of Detection
Wavelength

The longer wavelength

is more selective.

200 250 300 350

Wavelength [nm] 20
Photodiode Array Detector

21
 Calibration
Calibration of HPLC is done to check the performance of its
instrument.
• Flowrate(pump calibration)
• Detector and injector linearity
• System precision
• Column oven temperature
• Detector wavelength accuracy: Absorbance of Potassium
dichromate at (235 nm, 257 nm, 313 nm, 350 nm) wavelenth are
measured and compared against vendor specification 22
 Method Validation
• Trueness
Accuracy Accuracy
• Precision
• Selectivity
• Linearity
• Limit of Detection(LOD)
• Limit of Quantitation (LOQ)
• Robustness and Ruggedness
23
Linearity

24
Four sets of data with the same correlation
of 0.816

25
26