Pradeep Kuamr Pal M.Pharm (Pharmaceutics) ISF College of Pharacy Moga

1. Ultra pressure liquid
chromatography(UPLC)
Presented by: Pradeep Kumar Pal
M.Pharma (Pharmaceutics)
I.S.F.C.P. Moga, Punjab

2. Table of content
1.Introduction
2.Principle
3.Instrumentation
4.Advantages
5.Disadvantages
6.Application
7.HPLC Vs UPLC
8.References 2

3. Introduction
Ultra-performance liquid chromatography (UPLC) is an
important liquid chromatography (LC) technique used for
the segregation of different components in mixtures. It is
also used for the identification and quantification of
compounds in the process of drug development
UPLC, improve in three areas: ‘‘speed, resolution, and
sensitivity’’.
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4. Introduction
As column packing particle size decreases, efficiency and
thus resolution also increases. As particle size decreases to
less than 2.5μm, there is a significant gain in efficiency.
•Pressure:1500psi
•Sensitivity:3-5µl
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5. Principle
•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 principles is based on 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).
H=A+B/v+Cv
•Where A, B and C are constants. 5

6. Principle
•v = linear velocity of the carrier gas flow rate.
•The A term is independent of velocity and represents
"eddy" mixing.
•The B term represents axial diffusion or the natural
diffusion tendency of molecules.
•The C term is due to kinetic resistance to equilibrium in
the separation process.
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7. Instrumentation
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8. 8

9. Instrumentation
1. Injection
•The use of the injector is to add precisely measured, a small
volume of solution containing the sample in the mobile
phase.
•The injection process must be comparatively pulse-free.
•A quick injection cycle time is required to fully avail the
speed afforded by UPLC.
•To increase the sensitivity, low volume injections with
minimal carryover are required. 9

10. Instrumentation
•The volume of the sample in UPLC is usually 2-5 μL.
•Now a days, direct injection approaches are utilized for
the biological samples.
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11. Instrumentation
2. UPLC COLUMNS
• Resolution is increased in a 1.7μm particle packed
column because efficiency is better. Separation of the
components of a sample requires a bonded phase that
provides both retention and selectivity.
Four bonded phases are available for UPLC separations.
• ACQUITY UPLCTM BEH C18.
• ACQUITY UPLCTM BEH C8 (straight chain alkyl
columns).
•
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12. Instrumentation
•ACQUITY UPLC BEH Shield RP18 (embedded polar
group columns).
•ACQUITY UPLC BEH Phenyl (phenyl group tethered to
the silyl functionality with a C6 alkyl).
•ACQUITY UPLC BEH C18 and C8 columns are
considered the universal columns of choice for most
UPLC separations by providing the widest pH range.
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13. UPLC Column
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14. Instrumentation
Pumps
•The most important advantages of UPLC pumps are
higher pressure, faster analyses, and increased sample
load capacity.
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15. Instrumentation
Types
•Isocratic pump - delivers constant mobile phase
composition, solvent must be pre-mixed, best for simple
preparations.
•Gradient pump - delivers variable mobile phase
composition, best for analysis of complex mixtures.
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16. Instrumentation
Detector
• 1. Spectroscopic Detection.
• 2. Refractive Index Detection.
• 3. Fluorescence Detection.
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17. Advantages
•Decreases run time and increases sensitivity.
•Provides the selectivity, sensitivity, and dynamic range of
LC analysis.
• Maintaining resolution performance Operation cost is
reduced.
• Less solvent consumption
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18. Disadvantages
•Due to increased pressure requires more maintenance and
reduces the life of the columns of this type.
• The phases of less than 2 μm are generally non-generable
and thus have limited use.
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19. Applications
•Analysis of natural products and traditional herbal
medicine.
•Identification of metabolite.
•Bio analysis/bioequivalence studies.
•Manufacturing/QA/QC
•Impurity profiling.
•Dissolution Testing.
•Toxicity Studies. 19

20. HPLC Vs UPLC
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21. References
1. Patil A.S, A review on ultra performance liquid chromatography
(UPLC), Published aby asian journal of pharmaceutical technology
& innovation.
2. Naresh K, Ultra performance liquid chromatography, Published
by international journal of pharma medicine and biological sciences.
3. Chawla G, Principle, instrumentation, and applications of uplc: a
novel technique of liquid chromatography, published by open
chemistry journal.
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22. References
4. Taleuzzaman M, Ultra performance liquid
chromatography(UPLC)-A review, published by Austin
journal of analytical and pharmaceutical chemistry.
5. www.slideshare.com
6. www.slideserve.com
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