ULTRA PERFORMANCE LIQUID

CHROMATOGRAPHY (UPLC)`

Prepared by

Mr. MANOJ R. INGALE

M.Pharm .

Department of Quality Assurance Technique.

Sinhgad Technical Education Society’s

SINHGAD INSTITUTE OF PHARMACY

Narhe, Pune - 411041

CONTENT OF PRESENTATION

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Introduction

Principle

Chemistry of small particle

Comparison between HPLC and UPLC

Instrumentation

Advantages

Disadvantages

Advancements in UPLC

Summary

References

INTRODUCTION

Chromatography-

Separation technique involving mass transfer between

stationary and mobile phase.

In Greek chromo meaning “color”, Graphic meaning

“writing”.

Since the 1970’s chromatographers have been limited to LC

systems that were capable of operating at maximum

operating system pressure of only 6000 psi (400bar).

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This pressure limitation coupled with large system

volumes & slow data acquisition rates hindered the

ability of separation scientist to fully realize speed &

efficiencies.

INTRODUCTION TO UPLC

Ultra Performance Liquid Chromatography or

Ultra Pressure Liquid chromatography.

It improves in three areas:

Resolution,

Speed,

Sensitivity.

It can withstand high system back-pressure.

Special analytical columns UPLC BEH C18 packed with

1.7µm particles are used in connection with system.

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The factor responsible for development of UPLC technique

was evolution of packing material used to effect the

separation.

The technology takes full advantage of chromatographic

principles to run separations using columns packed with

smaller particles.

It decreases analysis time and solvent consumption.

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The principle of UPLC is based on Van Deemeter

equation which describes the relationship between flow

rate and HETP or column efficiency

H=A+B/µ + Cµ

Where,

A = Eddy diffusion

B = Longitudinal diffusion

C = Equilibrium mass transfer

µ = Flow rate

PRINCIPLE

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Chromatographic resolution is described by

Where,

Rs = Resolution

N = Separation efficiency (theoretical plate )

α = Selectivity factor

k = Retention factor (Capacity factor)

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Separation efficiency (N)

Where,

L = Column length

H = Height of theoretical plate

h = Reduced plate height

dp = Particle diameter

Therefore,

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CHEMISTRY OF SMALL PARTICLES

As the particle size decreases to less than 2.5µm, not only

there is significant gain in efficiency, but the efficiency

doesn't diminish at increased flow rates.

By using smaller particles, speed and peak capacity

(number of peaks resolved per unit time in gradient

separation) can be extended to new limits, termed ultra

performance liquid chromatography.

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CHEMISTRY OF SMALL PARTICLES

FIGURE 1- Van Deemter plot illustrating the evolution of

particle sizes over the last three decades.

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COMPARISON BETWEEN

HPLC AND UPLC Parameters HPLC Assay UPLC Assay

Column XTerra,C18,50 × 4.6mm AQUITY UPLC BEH

C18,50 ×2.1mm

Particle size 4µm particles 1.7µm particles

Flow rate 3.0 ml per min 0.6 ml per min

Injection volume 20 µl 3 µl partial loop fill or

5 µl full loop fill

Total run time 10 min 1.5 min

Theoretical Plate count 2000 7500

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Parameters HPLC Assay UPLC Assay

Lower limit of

quantization

0.2 µg/ml 0.054µl/ml

Total solvent

consumption

Acetonitrile:10.5ml,

water:21ml

Acetonitrile:0.53ml,

water:0.66ml

Delay volume 720 µl 110 µl

Column temperature 30 °C 65 °C

Maximum back pressure35-40 Mpa

less

103.5 Mpa

more

Resolution Less High

Method development

cost

High Low

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UPLC SYSTEM

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INSTRUMENTATION

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A completely new system design with advanced

technology in the pump, auto sampler, detector, data

system, and service diagnostics was required.

The ACQUITY UPLC system has been designed for low

system and dwell volume.

Achieving small particle, high peak capacity separations

requires a greater pressure range than that achievable by

HPLC system.

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SYSTEM COMPONENTS

Solvent reservoirs

Auto sampler

Van guard column

Tubing's

Column

Detectors

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UPLC COLUMN

To maintain retention and capacity similar to the

HPLC,UPLC uses novel porous particles that can

withstand high pressure.

Xterra TM :-

First generation technology

Introduced by Waters in year 2000.

Have high efficiency and operates over extended pH

range.

They produced by incorporating carbon in the form of

methyl groups.

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AQUITY UPLC BEH COLUMN

BEH-Bridged Ethane Hybrid.

Second generation technology

Have particle size 1.7µm.

Enhanced mechanical stability by bridging the methyl

group in the silica matrix .

It can withstand at high pressure and high pH.

All AQUITY UPLC BEH Column include eCordTM

microchip technology that capture manufacturing

information for each column.

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AQUITY UPLC BEH Column chemistries

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PUMPS

Pumps are capable of delivering solvent smoothly and

reproducibly.

Calculated pressure drop at optimum flow rate for

maximum efficiency across the 15cm long column packed

with 1.7µm particle is approximately 15000 psi.

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DETECTORS

1. UV detectors

2. Fluorescent detector

3. Refractive index detector

4. Light scattering detector

5. Electrochemical detector

6. Mass spectrometric detector

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RESOLUTION OF PEAKS

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HPLC Resolution

UPLC Resolution

ADVANTAGES OF UPLC

Shortening analysis time up to nine times.

Provides the selectivity, sensitivity, and dynamic range

of LC analysis.

Maintains resolution performance.

Fast resolving power quickly quantifies related and

unrelated compounds.

Operation cost is reduced.

Less solvent consumption.24 7-Dec-14

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Improves the quality of data , resulting in more definitive

map.

Separation on UPLC is performed at very high pressures up

to 15000psi.

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DISADVANTAGES OF UPLC

Due to increased pressure requires more maintenance and

reduces the life of the columns.

In addition, the phases of less than 2 μm are generally

non-regenerable and thus have limited use.

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FACTORS AFFECTING

PRFORMANCE OF UPLC

Pressure

Column

Particle size of packing

Temperature

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EFFECT OF TEMPERATURE

Improve protein separation.

Modify properties of column surface alter protein structure.

Increase column efficiency.

Reduces mobile phase viscosity decrease in backpressure.

Reduced run times in UPLC experiments

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WHAT IS UPLC –Q- TOF?

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Hybrid Quadrupole Orthogonal Time of Flight

(Q-TOF) Mass Spectrometer.

Highly sophisticated

High resolution mass spectrometry system, which

employs a time-of-flight mass spectrometry technology.

Used to analyze compounds in samples with a mass

accuracy of typically 5 ppm (or better) between the found

and the theoretical molecular weight of the analyte.

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Excellent chromatographic separation at the cost of high

operating system pressure.

Overcome by a high-precision plumbing and fittings

system design, in which exotic materials, such as gold, etc.,

are used to efficiently resist high liquid pressure.

(i.e., 14,000 psi or more) and provide a leak-free

environment.

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APPLICATIONS

OF THE UPLC/Q-TOF

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High resolution mass spectrometry

Accurate mass determinations

Bioanalytical method of validation

Application to a pharmacokinetic study

Comprehensive screening and quantification of

veterinary drugs in milk

Peptide and Protein Research (MW approx. <12 kDa)

with Accurate Mass 7-Dec-14

UPLC - MS

32WATERS ACQUITY UPLC-MS

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APPLICATIONS(UPLC)

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Drug Discovery : UPLC improves the drug discovery

process by means of

High throughput screening

Combinational chemistry

High throughput screening determines physicochemical and

drugs pharmacokinetics.

High throughput quantitative analysis :

UPLC coupled with time of flight mass spectroscopy give

the metabolic stability assay.7-Dec-14

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Analysis of dosage form :

It provides high speed, accuracy and reproducible results

for isocratic and gradient analysis of drug. Thus method

development time decreases.

Determination of pesticides :

UPLC couples the triple Quadra-pole tandem mass

spectroscopy will help in identification of trace level of

pesticide from water.

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Analysis of amino acids :

UPLC used to accurate, reliable and reproducible

analysis of amino acids in the areas of

Protein characterization,

Cell culture monitoring,

The nutritional analysis of food.

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SUMMARY

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New materials and smaller particles are now available

which gives improved separations, mostly following

expected trends.

For UPLC, some reduction in sample size, significantly

show reduction in flow rate.

As we go small we need matching of particle size,

chemistry , analytes , and separation methods.

UPLC sets new standard in the science of chromatography.

Working range with 15000 to 16000 psi pressure and

column packed with size less than 2µm.

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Due to very narrow and sharp peaks, more number of

peaks may appear in less time which may facilitate in

analysis of complex mixtures and it may give more

information regarding sample to be analysed.

Now a day’s pharmaceutical industries are in search of

new ways to reduce cost and time for analysis of drugs

therefore UPLC serve as best substitute for previous

technologies.

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REFERENCES

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Preeti Vinod Gaikwad*, Sanjay Dinkar Sawant, Minal

Rushikesh Ghante and Neha Manish Munot,(2010),Ultra

Performance Liquid Chromatography: A Recent Novel

Development In HPLC, International Journal of

Comprehensive Pharmacy(sep 5,2010) Page no:1-3

Maulik Acharya*, Mandev Patel, Darshan Patel, Mahesh

Chaudhary, Divyang Patel,(2012), Ultra Performance

Liquid Chromatography, American Journal of Pharmatech

Research,2(3),Page no:327-336

Dr. Michael E. Swartz and Brian J. Murphy,(2004), Ultra

performance liquid chromatography: Tomorrow's HPLC

technology today, LPI,(june,2004),Page no-1-3

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Dr.P.D.Sethi, (2001),High Performance Liquid

Chromatography, First edition, New Delhi, CBS Publishers

and distributor, Page no-6-8,69.

Dr. Michael E. Swartz,(2005), Ultra Performance Liquid

Chromatography: An introduction,massachusetts,Separation

Science Redefined, Page no:8-14

Ecord System-

http://www.waters.com/webassets/cms/support/docs/WA4000

1.pdf(Accessed on-09-11-2014)

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THANK YOU...