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A PRESENTATION ON HIGH PERFORMANCE LIQUID CHROMATOGRAPHY(HPLC) BY ATIYA BANERJEE(M.TECH 1ST YEAR, CAPPD) SANJAY GAUTAM(M.

TECH 1ST YEAR,IPA) ABHISHEK RATNA(M.TECH 1ST YEAR,IPA)

HPLC INSTRUMENT
HPLC AT DRACO

MANUFACTURING FACILITY IN SHANGHAI, CHINA

Definition
HPLC, is a chromatographic technique used to separate the components in a mixture, to identify each component, and to quantify each component. It relies on pumps to pass a pressurized liquid and a sample mixture through a column filled with a sorbent, leading to the separation of the sample components.

PRINCIPLE
Method involves a liquid sample being passed over a solid adsorbent material packed into a column using a flow of liquid solvent Each analyte in the sample interacts slightly differently with the adsorbent material, thus retarding the flow of the analytes. If the interaction is weak, the analytes flow off the column in a short amount of time, and if the interaction is strong, then the elution time is long.

A HPLC SYSTEM
1 Solvent reservoir, 2 Solvent Degasser, 3 Gradient Valve, 4 Mixing Vessel for delivery 5 High-pressure pump 6 Switching valve 7 Sample injection loop 8 Pre Column 9 Analytical Column 10 Detector 11 Data Acquisition 12 Waste or Fraction collector

Working Principle
The

sample mixture to be separated and analyzed is introduced, in a discrete small volume (typically microliters) into the stream of mobile phase percolating through the column. The components of the sample move through the column at different velocities, which are function of specific physical interactions with the sorbent (also called stationary phase). The velocity of each component depends on its chemical nature, on the nature of the stationary phase (column) and on the composition of the mobile phase. The time at which a specific analyte elutes (emerges from the column) is called its retention time. The retention time measured under particular conditions is considered an identifying characteristic of a given analyte.

Typical Chromatograms

Instrumentation-1

Instrumentation-2
Solvent Reservoirs Pump

Sample Injector
Column(s)

Detector
Data System

Mobile Phase Reservoirs


Inert container with inert lines leading to the pump are required. Reservoir filters (2-10 mm) at reservoir end of solvent delivery lines Degassed solvent - Vacuum filtration - Sparge with inert gas (N2 or He) - Ultrasonic under vacuum Elevate above pumps

Description
Mobile

phase reservoir will carry the mobile phase solution and through the pump it will be pumped into the column. This movement is according to the gravitational force. The mobile phase reservoir is a solvent with different polarities such as water, methanol, and acetonitrile. These solvents should be highly pure. For an example, water should be used as de-ionized water. Hygienic conditions are facilitated with the use of 0.45 m filter. The solvent is filtered through this filter mesh to remove all particulate matter. This is termed as Millipore filtering. Degas is done to minimize errors which occur when compounds interact with several gases. If degas or the removal of all the gases, is not done correctly then it may spoil the column.

HPLC Pump Criteria


Constructed of materials inert toward solvents to be used Deliver high volumes (flow rates) of solvent (to 10 mL/min) Deliver precise and accurate flow (<0.5% variation) Deliver high pressure (to 6000 psi) Deliver pulse free flow Have low pump-head volume Be reliable

HPLC Pumps: Types


Reciprocating pumps
Syringe pumps

Constant pressure pumps

Reciprocating Pumps
One, two, or three pump heads - more heads, less pulse Small head volumes (50 to 250 mL)

Short piston stroke


Inert pistons (generally sapphire)

Continuous use (no refill time)


Pulse dampeners

Sample Injectors
HPLC sample injection valves are typically fixed-loop rotary valves, such as the one shown on the right. The valve has an internal rotor with a series of holes or channels etched through it. internal channels connect adjacent pairs of external ports. Rotating the valve through 60 connects alternate ports, allowing the flow through the valve to be diverted through the sample loop.

Auto Injectors
Continuous injections operator free

Comparable precision and accuracy to manual


Much more expensive initially Much more convenient Up 100 samples and standards with microprocessor control

HPLC Column
Liquid-chromatographic

columns range in length from 10 to 30 cm. The inside diameter of liquid columns is often 4 to 10 mm; the most common particle size of packings is 5 or 10 m. The most common column currently in use is one that is 25 cm in length, 4.6 mm inside diameter, and packed with 5 m particles. Columns of this type contain 40,000 to 60,000 plates/meter.

Stationary Phase
Many

different types of columns are available, filled with sorbents varying in particle size, and in the nature of their surface ("surface chemistry"). The use of smaller particle size packing materials requires the use of higher operational pressure ("backpressure") and typically improves chromatographic resolution (i.e. the degree of separation between consecutive analytes emerging from the column). In terms of surface chemistry, sorbent particles may be hydrophobic or polar in nature.

Modes of Elution
Isocratic elution: A separation that employs a single solvent or solvent mixture of constant composition. Gradient elution: Here two or more solvent systems that differ significantly in polarity are employed. After elution is begun; the ratio of the solvents is varied in a programmed way, sometimes continuously and sometimes in a series of steps. Separation efficiency is greatly enhanced by gradient elution. For example, the mobile phase composition may be
kept constant at 5% acetonitrile for 13 min, followed by a linear change up to 95% acetonitrile.

Types
Liquid chromatography based upon highly polar stationary phases such as water or triethyleneglycol supported on silica or alumina particles; a relatively nonpolar solvent such as hexane or I-propylether then served as the mobile phase. This type of chromatography is referred to as normal-phase chromatography.

In reversed-phase chromatography, the stationary phase is nonpolar, often a hydrocarbon, and the mobile phase is relatively polar (such as water, methanol, or acetonitrile). In normal-phase chromatography, the least polar component is eluted first because it is the most soluble in the mobile phase; increasing the polarity of the mobile phase has the effect of decreasing
the elution time.

Chromatograms
Comparison between

Normal-phase and
Reverse-phase chomatography

Guard Column
A

guard column is introduced before the analytical column to increase the life of the analytical column by removing not only particulate matter and contaminants from the solvents but also sample components that bind irreversibly to the stationary phase. The guard column serves to saturate the mobile phase with the stationary phase so that losses of this solvent from the analytical column are minimized. The composition of the guard-column packing is similar to that of the analytical column; the particle size is usually larger. When the guard column has become contaminated, it is repacked or discarded and replaced with a new one.

Detectors
Types

of Detectors: Liquid chromatographic detectors are of two basic types. Bulk property detectors respond to a mobile-phase bulk property, such as refractive index, dielectric constant, or density. In contrast, solute property detectors respond to some property of solutes, such as UV absorbance, fluorescence, or diffusion current, that is not possessed by the mobile phase.

Data System
A chromatography software, also known as a Chromatography data system (CDS), collects and analyzes chromatographic results delivered by chromatography detectors. Many chromatography software packages are provided by manufacturers, and many of them only provide a simple interface to acquire data. They also provide different tools to analyze this data. The most common tool is "integration": It computes simple areas to delimit peaks by adding valleys automatically or not. Areas are computed between two valleys, a signal, and a baseline.

Chromatography software

Name

Publisher

Control

Analysis

Type

Malcom

Schlumberger

No

Yes limited

GC/GC-MS

ChemStation

Agilent

Yes

LC/GC/GC-MS

Chromeleon

Dionex

Yes

Yes

GC/LC/IEX/MS

Empower

Waters

Yes

Yes

GC/LC

OpenChrom

Philip Wenig

Yes

GC/LC

Atlas CDS

Thermo Scientific

Yes

Yes

GC/LC

Retention & Selectivity Factors


Size and shape of the solvated molecule

Mobile phase pH
Concentration & type of competing ion

Addition of organic solvent to mobile phase


Column temperature

Types
Partition chromatography Normalphase chromatography Displacement chromatography Reversed-phase chromatography (RPC) Size-exclusion chromatography Ion-exchange chromatography Bio-affinity chromatography Aqueous normal-phase chromatography

Salient Features of HPLC


In HPLC the mobile phase is a liquid whereas in GC the mobile phase or carrier is a gas. HPLC is useful for analysis of samples which are liable to decompose at higher temperatures. GC involves high temperatures so compounds are stable at such temperatures. GC is applied for analysis of volatile compounds whereas non volatile compounds can be easily analyzed on HPLC GC cannot be used for analysis of high molecular weight molecules whereas HPLC has applications for separation and identification of very high molecular weight compounds HPLC requires higher operating pressures than GC because liquids require higher pressures than gases for transport through the system HPLC columns are short and wide in comparison to GC columns

HPLC Applications
Bioscience
Chemical
polystyrenes dyes phthalates proteins peptides nucleotides

Pharmaceuticals

tetracyclines corticosteroids antidepressants barbiturates

Consumer Products
lipids antioxidants sugars

Environmental
polyaromatic hydrocarbons Inorganic ions herbicides

Clinical
amino acids vitamins homocysteine

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Some worldwide manufacturers


Agilent Cecil Desaga Jasco

Technologies

Knauer
Merck Shimadsu Kromasil

Thank you for your kind attention!