ULTRA PERFORMANCE LIQUID CHROMATOGRAPHY

B.KEERTHANA,B.PHARM.

INTRODUCTION

UPLC means Ultra Performance Liquid Chromatography

UPLC is a chromatographic technique, in which it can separate a mixture

of compounds and is used in biochemistry and analytical chemistry to

identify, quality, purity in the individual components of a mixture utilizing a

small packaging particle and columns with high pack pressure.

Efficiency of Uplc

SPEED

SENSITIVIT Y

R E S OL U T I

O N

A.To work at higher temperaturesB.Use of monolithic columns

Use of Uplc system

Advancements in various areas

Nutrition

Drug Discovery

Toxicology

Natural product discovery

Cancer

Advantages

Operation cost is reduced

Less solvent consumption

Run time and increase

sensitivity

Maintains resolution

performance

Disadvantages

High price of instruments, spare parts

and columns

Number of stationary phases still

limited

Lack of variety in commercial columns

Principle of uplc The UPLC is based on the principle of use of stationary phase consisting of particles less than 2 μm (while HPLC columns are typically filled with particles of 3 to 5 μm). The underlying principles of this evolution are governed by the van Deemter equation, which is an empirical formula that describes the relationship between linear velocity (flow rate) and plate height (HETP or column efficiency).

WhereA ,B,C are constants V - linear velocityA - Eddy mixingB - Axial diffusion

H=A+B/v+CV

Instrumentation of uplc

Various parts present in Uplc instrument are

Columns

Column heater

Sample manager

Binary solvent manager

Pumps

Optional Sample organizer

Binary solvent manager The binary solvent manager uses two individual serial flow pumps to deliver a parallel binary

gradient.

The binary solvent manager is a high pressure pump that moves solvent through the system.

The binary solvent manager delivers solvent at flow rates of 1 ml/min at 103421 Kрa [ 1034 bar, 1500 psi] and up to 2ml/min at reduced pressures to 62053 Kpa [621 bar, 9000 psF] . The solvent manager can pump two solvents immediately.

Sample manager: The Acuity sample manager injects the sample it draws from Micro titer plates or vials in to the

chromatographic flow stream.

A locating mechanism uses a probe to access sample locations and draw sample from them.

The Sample manager can perform an injection in approximately 15 seconds. The sample manager also controls the column heater. Column temperatures up to 65°С can be attained.

Column heater: The column heater is of a modular design and its foot print is identical to that of the sample manager.

Thus it attaches to the top of the sample manager and serves as the instrument’s top cover.

Columns: The UPLC columns are made up of small particles having size less than 2 μm. The role played by small

particle size in UPLC technique has been mentioned above. The particles are bonded in matrix as the bonded stationary phase is required for providing both retention and selectivity.

Micro bore column Optional sample organizer

The optional sample organizer stores

micro miter or vial plates

It transfers them to and from the sample

manages, automating their processing

and increasing throughput

Optional sample organizer holds the

sample

Pumps: The UPLC pump is considered to be one of the most important components in a liquid

chromatography system which has to provide a continuous constant flow of the eluent through the UPLC injector, column, and detector.

The two basic classifications are

Constant pressure pump

Constant flow pump Standard UPLC pump requirements :

Sample injection volume is as less as 3-5 micro liters

Pumps operates at 10000 psi pressure

Particle size in stationary phase packing material is less than 2 micro meter

Types of pumpsReciprocating piston pumps Dual piston pumps

Dual head reciprocating pumps

Detectors

Detectors

Optical & fluorescence

Tunable ultra violet

Evaporate light scattering

Applications of Uplc Rapid analysis of products Bio equivalence studies Dissolution testing Impurity profiling Identification of metabolites High through put qualitative analysis Drug discovery Analysis of dosage form Study of metabolomics Forced degradation studies In finger print analysis ADME screening Determination of pesticides Analysis of amino acids In QA control

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