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UPLC
1. Ultra Performance
Liquid
Chromatography
Prepared by:
SHAIMAA OBAID AHMADEEN
Supervised by:
DR / NOuRAH AL ZOMAN
2. Presentation's outlines:
• Introduction
• Definition
• UPLC vs HPLC
• efficiency of UPLC
• Switching from HPLC to UPLC
• Examples showing different UPLC
instruments
• Applications of UPLC in pharmacy
• Conclusion
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3. Introduction :
The pharmaceutical industry is
under intense pressure to increase
productivity and put new drugs onto
the market in a shorter period of
time. Analytical chemists are
challenged to find faster ways of
delivering quality data across a
range of project needs. A number of
approaches are being employed to
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5. Ultra : means not within
reasonable limits or far
beyond the normal
Ultra high pressure
• Equal to or more than 1500psi
(1034 pa =1000 ba )
Ultra small packaging material
• Particle size 1.7 , 1.8µm
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6. Definition :
UPLC is a chromatographic technique that can
separate a mixture of compounds, and is used
in biochemistry and analytical chemistry to
identify, quantify and purify the individual
components of the mixture. Utilizing a small
packaging particle size's columns and ultra high
pack pressure
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8. HPLC vs UPLC
HPLC UPLC
• id (3-10) µm • id (0.75-1.8)µm
• inlet pressure 400 ba • inlet pressure more
• Lower comparative than1000ba
precision in sample • Higher precision in sample
introduction introduction
• Detectors that use larger • Detectors that use small
flow rates and larger flow rates and low
detection cells detection limits
• Some are equipped with • Most of equipments with
automated sampling automated sampling
devices
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devices 8
9. Why is UPLC more
efficient
• Peak capacity (P) is the number of
peaks that can be resolved in a
specific amount of time.
• P is proportional to the inverse of the
square root of the Number of
theoretical plates (N): N = L/H
• Lower plate heights generate a greater
number of plates (N : efficiency )
• Plate heights are correlated through
the Van Deemter equation
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10. Van Deemter Eqn.
• H = A +B/µ +C x µ
• A is related to the mobile phase
movement through paths in the
stationary phase.
• B describes longitudinal diffusion
• C relates the analyte to mass transfer
between the pores of the stationary
phase
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12. Chemistry of Small Partic
So as the particle size decreases to
increase N and subsequently Rs, an
increase in sensitivity is obtained, since
narrower peaks are taller peaks.
Narrower peaks also mean more peak
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16. Scaling the Separation
The gradient is scaled from HPLC to UPLC
using:
L2/L1 x tg1 = tg2
Where L1and L2 are the lengths of the HPLC and UPLC
columns, and tg1 and tg2 are the times of each gradient step
respectively.
Flow rate is scaled taking into account the
difference in the diameter of the two columns:
(d2)2/(d1)2 x F1 = F2
Where d2 and d1 are the column diameters and F1 and F2 the
flow rates.
The injection volume is scaled taking into
account the volumes of the two columns:
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1 2 2 2 1 2
19. Nano-UPLC SYSTEM
FEATURES:
•Maximum resolution to yield maximum peak capacity
for complex mixtures.
•Maximum reproducibility – Direct nano-flow control
and a novel, moveable heating and trapping module for
< 0.25 minute standard deviation run-to-run
reproducibility.
•Maximum throughput –with faster gradient
separations for mixtures
•Robustness – Nano-scale columns and fittings
provide reliable operation for up to 10,000 psi, with
novel high pressure trapping.
•Ease-of-use – Direct gradient control enables
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22. Hydrogen Deuterium Exchange (HD-x) is an experimental
technique to obtain structural data on proteins. It relies on
the accurate measurement of the degree of labeling of the
protein by deuterated hydrogen.
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24. Contrasting HPLC and
• UPLC gives UPLC with better
faster results
resolution
• UPLC uses less of valuable solvents
like acetonitrile which lowers cost
• The reduction of solvent use is more
environmentally friendly
• Increased sensitivity
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25. Applications of UPLC
1.Higher Speed
separation of caffeic
HPL
acid derivatives from C
Echinacea Purpurea
UPL
C
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27. 2.Higher performance
100 %
Intensity
5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00 50.00 55.00
Time (minutes)
Total ion chromatogram of rat urine obtained with a 2.1 × 150 mm, 1.7 μm
Acquity BEH C18 column and a gradient over 60 minutes at 90°C at 0.8 mL/min. Gradient:
0-95% B; A = 0.1% formic acid in water, B = acetonitrile with 0.1% formic acid
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29. 4.Bio-Separations with high resolution
Gain in separation performance and in analysis time when switching from a 25 cm 3 μm
column to a 15 cm 1.7 μm column. Calculation for a typical protein digest.
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30. 5.Baseline enhancement
(HPLC-top) and 3 (UPLC-Bottom) showing increased UPLC resolution
and signal to noise (sensitivity).
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31. Conclusion :
(UPLC) is a relatively new technique
giving new possibilities in liquid
chromatography :
• Decreasing time of analysis.
• Decreasing solvent consumption.
• Increasing the resolution.
• Increasing the sensitivity.
UPLC chromatographic system is
designed in a special way:
• to withstand high system back-pressures
• Special analytical columns packed with 1.7 μm
particles are used
• The quality control analyses of various
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