The document discusses Ultra Performance Liquid Chromatography (UPLC) as an advancement in liquid chromatography, enhancing resolution, speed, and sensitivity using smaller particle sizes and higher pressures compared to traditional HPLC. Key differences between HPLC and UPLC are highlighted, including reduced analysis times, lower solvent consumption, and higher efficiency due to improved instrumentation and column technology. The summary emphasizes UPLC's role in drug discovery, accurate mass determinations, and the analysis of complex samples, positioning it as a modern solution in analytical chemistry.

1. 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

2. CONTENT OF PRESENTATION
Introduction
Principle
Chemistry of small particle
Comparison between HPLC and UPLC
Instrumentation
Advantages
Disadvantages
Advancements in UPLC
Summary
References
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3. 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.
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5. 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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7. PRINCIPLE
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
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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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10. 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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11. CHEMISTRY OF SMALL PARTICLES
FIGURE 1- Van Deemter plot illustrating the evolution of
particle sizes over the last three decades.
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12. 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 pressure
35-40 Mpa
less
103.5 Mpa
more
Resolution Less High
Method development
High Low
cost
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14. UPLC SYSTEM
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15. 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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17. SYSTEM COMPONENTS
 Solvent reservoirs
Auto sampler
Van guard column
Tubing's
Column
 Detectors
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18. 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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21. 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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22. 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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23. RESOLUTION OF PEAKS
HPLC Resolution
UPLC Resolution
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24. 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.
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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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26. 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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27. FACTORS AFFECTING
PRFORMANCE OF UPLC
Pressure
Column
Particle size of packing
Temperature
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28. 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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29. 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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31. 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
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32. UPLC - MS
32
WATERS ACQUITY UPLC-MS
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33. APPLICATIONS(UPLC)
33
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.
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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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36. 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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38. REFERENCES
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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40. THANK YOU...
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