Ultra Performance Liquid Chromatography (UPLC) is an analytical technique that improves chromatographic resolution, speed, and sensitivity compared to traditional HPLC. It uses columns packed with smaller particles less than 2.5 μm. This allows for higher pressures, faster flow rates, and shorter run times. UPLC provides increased peak capacity and sensitivity for applications like metabolite identification, impurity profiling, and dissolution testing in pharmaceutical analysis and quality control.

1. Ultra Performance Liquid
Chromatography
By Mohammad Mudassar
Department of Quality Assurance, M- Pharm First Sem
R. C. Patel Institute of pharmaceutical education & Research, Shirpur

2. Contents
 Introduction
 Principal
 Comparison
 Instrumentation
 Advantages
 Disadvantages
 Application
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3. Introduction
 UPLC refers to Ultra Performance Liquid Chromatography.
 UPLC is one mode of chromatography, used in analytical techniques.
 It improves in three areas: chromatographic resolution, speed and
sensitivity.
 UPLC is a rising chromatographic separation technique whose packing
materials have smaller particle size lesser than 2.5μm.
 The technology takes full advantage of chromatographic principles to run
separations using columns packed with smaller particles and higher
flow rates.
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4. Principle
 The principle of UPLC is based on Van Deemter equation which describes the
relationship between flow rate and HETP or column efficiency
H=A+B/v + Cv
Where,
 A = Eddy diffusion
 B = Longitudinal diffusion
 C = Equilibrium mass transfer
 v = flow rate
 van Deemter equation, that describes the relationship between linear velocity (flow
rate) and plate height (HETP or column efficiency)
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5. Comparison between HPLC and UPLC
CHARACTERISTICS HPLC UPLC
Particle size 3-5μm Less than 2μm
Maximum back pressure
35-40 Mpa
less
103.5 Mpa
more
Column Alltima C18
Acquity UPLC BEH
C18
Column dimension 150 X 3.2 mm 150 X 2.1 mm
Column temperature 30 °C 65 °C
Volume injection 5μL (Std. In100% MeOH) 2μL (Std.In100% MeOH)
Sample throughput less more
Sample preparation simple tedious
Column coagulation Does not takes place Takes place
Analysis time more less
Sensitivity less higher
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6. UPLC
Instrumentation
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7. Instrumentation
1. Pumping systems
 Achieving small particle, high peak capacity separations requires a
greater pressure range.
 Both the gradient and isocratic separation modes are used.
 The binary solvent manager uses two individual serial flow pumps to
deliver a parallel binary gradient.
 There are built-in solvent select valves to choose from up to four
solvents.
 There is a 15,000-psi pressure limit (about 1000 bar) to take full
advantage of the sub-2μm particles.
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8. 2. Sample injection
 In UPLC, sample introduction is critical. Conventional injection
valves, either automated or manual, are not designed and hardened to
work at extreme pressure.
 To protect the column from extreme pressure fluctuations, the
injection process must be relatively pulse-free and the swept volume
of the device also needs to be minimal to reduce potential band
spreading.
 Low volume injections with minimal carryover
required to increase sensitivity.
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9. UPLC columns
 Resolution is increased in a 1.7 μm 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:
1. ACQUITY UPLCTM BEH C18 & C8 (straight chain alkyl columns),
2. ACQUITY UPLC BEH Shield RP18 (embedded polar group column)
3. ACQUITY UPLC BEH Phenyl (phenyl group tethered to the silyl
functionality with a C6 alkyl)
4. ACQUITY UPLC BEH Amide columns (trifunctionally bonded amide phase).
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10.  ACQUITY UPLCTM BEH C18 & C8 .
1. These are considered as the universal columns of choice for most
UPLC separations by providing the widest pH range.
2. They incorporate trifunctional ligand bonding chemistries which
produce superior low pH stability.
3. This low pH stability is combined with the high pH stability of the
1.7μm BEH particle to deliver the widest usable pH operating range
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11.  Acquity UPLC BEH Shield RP18
These are designed to provide selectivity's that complement the
ACQUITY UPLC BEH T M C18 and C8 Columns.
 Acquity UPLC BEH phenyl columns
These utilize a trifunctional C6 alkyl ethyl between the phenyl ring.
 Acquity UPLC BEH Amide columns
1. BEH particle technology, in combination with a
trifunctionally bonded amide phase, provides
exceptional column life time, thus improving
assay robustness.
2. BEH Amide columns facilitate the use of a wide
range of phase pH [2 –11].
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12. Detectors
 Detectors used are
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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13. Phototube
 Consist of high sensitive cathode in a form of half cylinder in evacuated tube.
 Anode is also present along the axis of the tube.
 Inside layer is coated with light sensitive layer.
 When light is incident surface coating emits electron this is attracted and
collected by anode.
 Current which is created between cathode and anode is regarded as measure of
radiation falling on the detector.
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14. Photomultiplier
Ejected photoelectron strikes dynode,
secondary e- released
Voltage accelerates e- to next dynode and so
Result is large charge packet
hitting anode
High Gain & detected
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15. Fluorescence detector
The light from an excitation source passes through a filter or monochromator,
and strikes the sample.
A proportion of the incident light is absorbed by the sample, and some of the
molecules in the sample fluoresce. The fluorescent light is emitted in all
directions.
Some of this fluorescent light passes through a second filter or monochromator
and reaches a detector, which is usually placed at 90° to the incident light beam
to minimize the risk of transmitted or reflected incident light reaching the
detector.
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16. Evaporative light scattering detector
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17. This detector based on the deflection principle of refractometry, where the
deflection of a light beam is changed when the composition in the sample flow-
cell changes in relation to the reference side (as eluting sample moves through
the system).
As sample elutes through one side, the changing
angle of refraction moves the beam.
This results in a change in the photon current
falling on the detector which unbalances it.
The extent of unbalance (which can be related
to the sample concentration) is recorded on a
strip chart recorder.
Refractive index detector
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18. Advantages…
 Decreases run time and increases sensitivity.
 Reducing analysis time so that more product can be produced with
existing resources.
 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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19. Disadvantages
 Due to increased pressure requires more maintenance and reduces the
life of the columns of this type.
 In addition, the phases of less than 2 μm are generally non-regenerable
and thus have limited use.
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20. Application
 Analysis of natural products and traditional herbal medicine.
 Identification of metabolite.
 Study of metabonomics/metabolomics.
 Bio analysis/bioequivalence studies.
 Manufacturing/QA/QC
 Impurity profiling.
 Forced Degradation Studies.
 Dissolution Testing.
 Toxicity Studies.
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21. Reference
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