1. ULTRA PERFORMANCE LIQUID CHROMATOGRAPHY
(UPLC)
Presented By, Guided By,
Miss. Mohini Patil Dr. Rajesh J Oswal
Prof.Sandip Kshirsagar
Department of Pharmaceutical Chemistry
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2.  INTRODUCTION
 PRINCIPLE
 INSTRUMENTATION
 EXAMPLE OF UPLC SEPRATION
 COMPARISON BETWEEN HPLC & UPLC
SEPRATION
 APPLICATIONS
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3.  INTRODUCTION:
 UPLC provides improved resolution, speed, and
sensitivity
 By using conventional particle
sizes, speed, pressures and peak capacity can be
extended to new limits and compromises with
sacrificing retention time.
 As the particle size is 1.7µm there is a significant
gain in efficiency along with resolution and without
diminish efficiency. (Figure 1)
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4. Figure1. Van Deemter plot, illustrating the evolution of
particle
According to figure 1 ,
column efficiency (N) is inversely proportional to particle
size (dp).
Thus, smaller particles provide higher resolution and lower
retention time.
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5. PRINCIPLE:
The UPLC is based on the principal of HPLC.
 It involves stationary phase consisting of particles sub 2
μm, as HPLC columns are typically filled with particles of 3
to 5 μm.
 Efficiency is the primary separation parameter behind
UPLC since it relies on the same selectivity and retentivity as
HPLC.
 In the fundamental resolution equation resolution is
proportional to the square root of N.
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6. Where,
N is number of theoretical plates,
α is Selectivity factor and
k is mean retention factor.
But since N is inversely proportional to particle size (dp);
N α 1/dp
As the particle size is lowered by thrice i.e. from 5 mm to1.7
mm, N is increased by three and the resolution by square root
of three i.e 1.7
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7. 1. Binary Solvent
Manager
2. Sample Manager
3. Column Heater
4. Optional Sample
Organizer
5. Optical Detectors
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9. 4. Optional Sample Organizer: Sample organizer stores
micro titer or vial plates & transfer them to & from sample
manager.
5. Optical Detectors: The system can be configured with an
TUV (Tunable Ultraviolet), PDA (Photodiode Array) or ELS
(Evaporative Light Scattering) optical detector or any
combination of the three.
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10. Column: 2.1 by 30mm 1.7 mm ACQUI-TY UPLC BEH C18
at 358C.
A 20–40% B linear gradient over 1.0 minute, at a flow rate of
0.86mL/min was used.
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11. Mobile phase: A was 0.1% formic acid, B was Acetonitrile.
UV detection: at 254 nm and 40pts/sec.
Peaks are in order: 1: 7-hydroxycoumarin glucuronide, 7-
hydroxycoumarin, 4-hydroxy coumarin, coumarin, 7- methoxy
coumarin, 7-ethoxycoumarin, and 4-ethoxycoumarin.
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12. Figure shows an HPLC versus UPLC separation comparison
of a ginger root extract sample where both speed and
resolution are improved, as well as an increase in sensitivity.
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13. 1. UPLC with MS: UPLC coupled to quadrupole tandem mass
spectrometry which operates with rapid, generic gradients and
shows increase in analytical throughput and also shows
sensitivity in high throughput pharmacokinetics or bioanalysis
studies, the rapid measurement of potential p450
inhibition, induction, and drug-drug interactions had been
studied by UPLC/MS/MS.
2. UPLC in Pharmaceutical Development: Now a day’s UPLC
is a very attractive tool for the pharmaceutical development
laboratory because of high resolution obtained in extremely
short period of analysis times, as UPLC provides high
throughput, high productivity and high resolution
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14. 3. UPLC used in Identification of Metabolite: Biotransformation of
new chemical entities (NCE) is necessary for drug discovery. When a
compound reaches the trial stage, metabolite identification is required
and it is necessary for lab to successfully detect and identify all
circulating metabolites of a contender drug.
4. UPLC used. in Bioanalysis / Bioequivalence Studies: For
Pharmacokinetic, Bioequivalence and toxicity studies, the quantitative
analysis of a drug in biological samples is an important part of drug
development process and this is carried out by UPLC
5. UPLC used in stressed degradation Studies: The most common
analytical technique for monitoring forced degradation experiments is
HPLC with UV and/or MS detection for peak purity, mass balance, and
identification of degradation products but these HPLC-based
methodologies are time-consuming and provide only medium
resolution
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15. to ensure that all of the degradation products are accurately detected.
PDA/MS (photodiode array and MS) used along with UPLC, which
allows for faster and higher peak capacity separations of complex
degradation product profiles.
7. UPLC used in Impurity Profiling: UPLC PDA detector involves
two analytical flow cells with maximum flexibility and according to
application requirements, as one for maximum chromatographic
resolution and a second for high sensitivity. UPLC also ensure the
latest peak detection algorithms and custom calculations to optimize
data processing and reporting. It also assertively detects impurities in
compounds even at trace levels. To characterize impurities, it is often
necessary to perform several analytical runs to obtain the necessary
MS and MS/MS data.
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