Objectives, Introduction, Advantages, Disadvantages, Principle, Instrumentation, Basic difference in HPLC and UPLC and Applications of UPLC,

1. ULTRA PERFORMANCE LIQUID
CHROMAROGRAPHY

2. CONTENT
•Objective
•Introduction
•Advantages
•Disadvantages
•Principle
•Instrumentation
•Basic difference in HPLC and UPLC
•Applications
2

3. OBJECTIVE
• To focus on ultra performance liquid
chromatography technique.
• To study its instrumentation.
• To know its various application
3

4. INTRODUCTION
CHROMATOGRAPHY-
Chroma-color, logos-writing
 “It is Separation technique involving mass transfer
between stationary phase and mobile phase”
Separated species appeared as colored bands on
columns.
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5. UPLC (Ultra Performance Liquid Chromatography) :
It improves three areas-Speed, resolution, sensitivity.
Speed-1.5min
Pressure-15000psi
Sensitivity-3-5µl
5

6.  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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7. ADVANTAGES
Decreases run time and increases sensitivity.
Provides the selectivity, sensitivity, and maintaining
resolution performance.
UPLC’s fast resolving power quickly quantifies related and
unrelated compounds.
Use of multi residue method.
Reduces process cycle time.
Less solvent consumption.
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8. DISADVANTAGES
Due to increased pressure, requires more
maintenance and Reduces the life of the columns.
The phases of less than 2μm are generally non-
regenerable.
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9. 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) 9

10. UPLC INSTRUMENTATION
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11. SOLVENT RESERVOIR
 The most common type of solvent reservoir is glass
bottle.
 Most of manufacturers supply these bottles with
special caps, tubing and filters to connect to the
pump inlet and so the purge gas (Helium) used to
remove dissolved air.
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12. 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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13. i. Constant pressure pump- constant pressure is used
only for column packing.
ii. Constant flow pump -This type is mostly used in all
common UPLC application.
iii. Reciprocating piston pump.
iv. Dual piston pump.
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14. Sample injection system
• To protect the column from extreme back pressure
fluctuations, the injection system must be relatively
pulse free and fast injection cycle time is needed
that’s why low volume injection system are used
which can deliver 3-5 microliter sample.
• Low volume injection also require to increase
sensitivity.
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15. COLUMNS
 Particle size of packing material is approximately 1.7
μm. Which increase speed of separation and efficiency
of column.
 Separation of the component of sample require
bonded phase that provide both selectivity and
retention.
That bonded phases are-
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16. ACQUITY UPLC BEH C18 AND C8 columns:
 These are straight alkyl chains.
 Columns were designed to be the universal columns of choice for
most UPLC separation by providing widest pH range.
 They incorporate tri-functional ligand bonding chemistries which
produce superior low pH stability.
 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
ACQUITY UPLC BEH Shield RP18:
 Columns are designed to provide selectivity that complement the
ACQUITY UPLC BEH C18 and C8 phases.
 Unique bonding chemistry provides complementary selectivity and
enhances the peak shape for basic compounds and
 Yielding compatibility with 100% aqueous mobile phases.
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17. BEH phenyl columns:
 Phenyl group attach with C6 alkyl.
 Each column chemistry provides different combination of
hydrophobicity, hydrolytic stability and chemical interaction
with analyte.
Acquity UPLC BEH Amide columns:
 BEH particle technology, in combination with a
trifunctionally bonded amide phase, provides exceptional
column life time, thus improving assay robustness.
 BEH Amide columns facilitate the use of a wide range of
phase pH (2 –11).
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18. ACQUITY UPLC BEH COLUMN
CHEMISTRIES
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19. 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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20. 1. ULTRA VIOLET DETECTORS
UV detectors are most frequently used to measure
components showing an absorption spectrum in the
ultraviolet or visible region.
A UV detector uses deuterium discharge lamp
(D2 lamp) as a light source, with the wavelength of its
light ranging from 190 to 380 nm
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21. 21

22. 2. FLUORESCENCE DETECTOR
The advantage
of
fluorescence
method is its
high sensitivity
for selective
groups of
compounds.
By using a
specific
wavelength,
analyte atoms
are excited
and then emit
light signal
(fluorescence).
The intensity
of this emitted
light is
monitored to
quantify the
analyte
concentration.
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23.  Most pharmaceuticals, natural products, clinical
samples, and petroleum products have fluorescent
absorbance.
 For some compounds which do not have
fluorescence absorbance or low absorbance, they
can be treated with fluorescence derivatives such as
dansylchloride.
 The system is easy to operate and relatively stable.
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24. 3. REFRACTIVE INDEX DETECTOR
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, 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.
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25. 4. Evaporative light scattering detector
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26. ELSD provides good sensitivity for non-volatile
analytes.
↓
The column effluent is nebulized and then evaporated
to make it form fine particles.
↓
The analyte is then radiated with a laser beam and
the scattered radiation is detected.
↓
The target sample includes lipids, sugar, and high
molecular weight analytes.
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27. 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 27

28. APPLICATIONS
•Drug Discovery- UPLC improves the drug discovery
process by means of high throughput screening,
combinational chemistry.
•Analysis of natural products and herbal traditional
medicines.
•Analysis of Dosage form- It provides high speed,
accuracy and reproducible results for analysis of
drugs and their related substance. Thus method
development time decrease.
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29. • Analysis of amino acids- UPLC used for accurate,
reliable and reproducible analysis of amino acids in
area of protein characterisation, cell culture
monitoring and nutritional analysis of food.
• Study of metabonomics/ metabolomics-
Metabonomics studies are carried out in labs to
accelerate development of new medicines.
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30. • Identification of metabolites
• Bio analysis/bioequivalence studies.
• Manufacturing/QA/QC
• Impurity profiling.
• Forced Degradation Studies.
• Dissolution Testing.
• Food analysis
• Toxicity Studies.
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