3. Objective
• To focus on ultra performance liquid chromatography
technique.
• To study its instrumentation.
• To know its various application.
4. What is
chromatography ???
• Chromatographic process can be defined as
separation technique involving mass transfer
between stationary and mobile phase.
• The separated species appeared
as a colored bands on the column,
which account for the name for
the method,
In Greek chromo meaning “color”,
Graphic meaning “writing”
5. 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.
6. 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)
7. 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.
8. 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.
9. 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
11. 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.
12. Cont…
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.
13. 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).
14. Cont…
• 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
15. Cont…
• 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].
17. 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
18. 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.
19. 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
20. 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.
22. 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.
efractive index detector
23. Common UPLC chromatographic
conditions
1. Columns: ACQUITY UPLC BEM C18, BEH Shield RP18, BEH C8 OR BEH
Phenyl Column.
2. Dimensions: 2.1 X 50mm 1.7μm.
3. Mobile Phase A1: 20mM NH4COOH in H2O, pH 3.0.
4. Mobile Phase A2: 20mM NH4HCOOH in H2O, pH 10.0.
5. Mobile Phase B1: Acetonitrile .
6. Mobile Phase B2: Methanol.
7. Flow rate: 0.5ml/min.
8. Injection Volume: 10.0μl.
9. Week needle wash: 3% methanol.
10. Strong needle wash: 90% acetonitrile.
11. Temperature: 30°C.
12. Detection: UV 254 nm.
13. Sampling rate: 20pts/sec.
24. 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.
