This document provides an overview of the instrumentation of high performance liquid chromatography (HPLC). It discusses the main components of HPLC including mobile phase reservoirs, pumping systems, sample injection systems, columns, detectors, and data collection devices. The pumping systems are usually reciprocating pumps that can generate high pressures. Columns separate components via stationary and mobile phases. Common detectors discussed are based on properties like UV absorption.
4. INTRODUCTION
CHROMATOGRAPHY
physical method in which separation of components takes place between two
phases-a stationary phase and a mobile phase•
STATIONARY PHASE
The substance on which adsorption of the analyte (the substance to be separated
during chromatography) takes place. It can be a solid, a gel, or a solid liquid
combination
MOBILE PHASE : solvent which carries the analyte (a liquid or a gas)
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5. HISTORY
• The late 1960s, was the technology developed for producing and using
packings with particle diameters as small as 3 to 10 mm.
• This technology required instruments capable of much higher pumping
pressures than the simple devices that preceded them. Simultaneously,
detectors were developed for continuous monitoring of column effluents.
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6. HPLC
• HPLC stands for “High-performance liquid chromatography”
• sometimes referred to as High-pressure liquid chromatography
• HPLC is a chromatographic technique that can separate a mixture of
compounds
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7. BLOCK DIAGRAM
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8. INSTRUMENTATION OF HPLC
Mobile-Phase Reservoirs and Solvent Treatment Systems
Pumping Systems
Sample Injection systems
Columns for HPLC
HPLC Detectors
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9. Mobile-Phase Reservoirs and Solvent
Treatment Systems
• HPLC instrument is equipped with one or more glass reservoirs, each of
which contains 500 mL or more of a solvent.
• Provisions are often included to remove dissolved gases and dust from the
liquids.
• Dissolved gases can lead to irreproducible flow rates and band spreading
• In addition, both bubbles and dust interfere with the performance of most
detectors.
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10. • An isocratic elution in HPLC is one in which the solvent composition
remains constant.
• A gradient elution in HPLC is one in which the composition of the
solvent is changed continuously or in a series of step
• gradient elution frequently improves separation efficiency, just as
temperature programming helps in gas chromatography
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11. PUMPING SYSTEMS
• PROPERTIES
The requirements for liquid chromatographic pumps include
(1) the generation of pressures of up to 6000 psi (lb./in2 ),
(2) pulse-free output,
(3) flow rates ranging from 0.1 to 10 mL/min,
(4) flow reproducibility of 0.5% relative or better, and
(5) resistance to corrosion by a variety of solvents.
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12. TYPES OF PUMPING SYSTEM
RECIPROCATING PUMP
• Reciprocating types are used in almost
all commercial instruments.
• commercial instruments are equipped
with computer-controlled devices for
measuring the flow rate by
determining the pressure drop across a
restrictor located at the pump outlet.
SCREW DRIVEN SYRINGE
• Syringe-type pumps produce a
pulse-free delivery whose flow rate
is easily controlled.
• They suffer, however, from
relatively low capacity (,250 mL)
and are inconvenient when solvents
must be changed
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13. Douglas A. Skoog, Donald M. West, F. James Holler, Stanley R. Crouch - Fundamentals of Analytical Chemistry-Cengage Learning (2013) 13
14. SAMPLE INJECTION SYSTEMS
• The most widely used method of sample introduction in liquid
chromatography is based on a sampling loop.
• an integral part of liquid chromatography equipment and have
interchangeable loops capable of providing a choice of sample sizes ranging
from 1 to 100 mL or more.
• The reproducibility of injections with a typical sampling loop is a few tenths
of a percent relative.
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15. COLUMNS FOR HPLC
• Liquid chromatographic columns are usually constructed from stainless steel
tubing, although glass and polymer tubing, such as polyetheretherketone
(PEEK), are sometimes used. In addition, stainless steel columns lined with
glass or PEEK are also available.
• TYPES OF COLUMNS
1. Analytical Columns
2. Precolumns
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16. ANALYTICAL COLUMNS
• Most columns range in length from 5 to 25 cm and have inside diameters of
3 to 5 mm. Straight columns are invariably used.
• The most common particle size of packings is 3 or 5 mm. Commonly used
columns are 10 or 15 cm long, 4.6 mm in inside diameter, and packed with 5-
mm particles. Columns of this type provide 40,000 to 70,000 plates/m.
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17. PRECOLUMNS
• Two types of pre columns are used.
SCAVENGER COLUMN
A pre column between the mobile phase reservoir and the injector is used for
mobile-phase conditioning and is termed a scavenger column. The solvent
partially dissolves the silica packing and ensures that the mobile phase is
saturated with silicic acid prior to entering the analytical column. This
saturation minimizes losses of the stationary phase from the analytical column.
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18. GUARD COLUMN
positioned between the injector and the analytical column. A guard column is a
short column packed with a similar stationary phase as the analytical column.
The purpose of the guard column is to prevent impurities, such as highly
retained compounds and particulate matter, from reaching and contaminating
the analytical column. The guard column is replaced regularly and serves to
increase the lifetime of the analytical column
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19. COLUMN PACKINGS
• Two types of packings are used in HPLC, pellicular and porous particle.
• The original pellicular particles were spherical, nonporous, glass or
polymer beads with typical diameters of 30 to 40 mm.
• The typical porous particle packing for liquid chromatography consists of
porous micro particles having diameters ranging from 3 to 10 mm; for a
given size particle, a very narrow particle size distribution is desirable.
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20. DETECTOR
• HPLC detector must have low internal volume (dead volume) to minimize
extra-column band broadening. The detector should be small and compatible
with liquid flow.
• Unfortunately, no highly sensitive, universal detector system is available for
high-performance liquid chromatography. Thus, the detector used will
depend on the nature of the sample
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21. DATA COLLECTION DEVICES
• Signals from the detector might be gathered on graph recorders or electronic
integrators that fluctuate in many-sided quality and in their capacity to
process, store and reprocess chromatographic information.
• The PC coordinates the reaction of the indicator to every part and places it
into a chromatograph that is anything but difficult to interpret.
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22. CONCLUSION
• The HPLC is mostly used analytical technique. It is having several
advantages. With the use of HPLC one can produce extremely pure
compounds. It can be used in both laboratory and clinical science. With the
use of HPLC the accuracy, precision and specificity can be increased. The
only disadvantage of HPLC is high cost.
Douglas A. Skoog, Donald M. West, F. James Holler, Stanley R. Crouch - Fundamentals of Analytical Chemistry-Cengage Learning (2013) 22
23. REFERENCES
• Rogatsky E. Modern high performance liquid chromatography and HPLC
2016 International Symposium. J Chromatogr Sep Tec.
• Douglas A. Skoog, Donald M. West, F. James Holler, Stanley R. Crouch -
Fundamentals of Analytical Chemistry-Cengage Learning (2013)
Douglas A. Skoog, Donald M. West, F. James Holler, Stanley R. Crouch - Fundamentals of Analytical Chemistry-Cengage
Learning (2013)
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