Chromatography Techniques mainly include two basic sub-divisions: Separation Science and Analytical Science. In 2004, separation science was revolutionized with the introduction of ‘Ultra High-Performance Liquid Chromatography which provides improved resolution, increased separation efficiency, shorter analysis time and lower operating costs. It uses HPLC columns with a mean particle size less than 2μm and pressures up to 15,000 psi which drastically increases the number of theoretical plates of the column and results in enhanced column efficiency.

1. Presented By:
Darewin
Mendonsa
1st Mpharm
ULTRA HIGH PERFORMANCE
LIQUID CHROMATOGRAPHY
(UHPLC)

2. Analytical
Science
Deals with methods for
determining chemical
composition of samples of
matter using various
techniques
Separation
Science
Most commonly carried out by
chromatography and
electrophoresis, particularly
with samples that are
multicomponent and complex
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3. Electromagnetic
Radiation
Electric
Current
Mass
Rate of
Reaction
Thermal
Characteristics
Radioactivity
• Gravimetry (Quartz crystal balance)
• Mass Spectrometry
• Turbidimetry
• Nephelometry
• Raman Spectroscopy
• Refractometry
• Interferometry
• Nuclear Magnetic Resonance
• Potentiometry
• Chronopotentiometry
• Coulometry
• Amperometry
• Polarography
• Conductometry
• Emission spectroscopy
• Florescence
• Phosphorescence
• Kinetic Methods
• Thermal Gravimetry and Titrimetry
• Diffrential Scanning Calorimetry
• Diffrential Thermal Analysis
• Thermal Conductance
Methods
• Activation and Isotope
Dilution Methods
3

4. CHROMATOGRAPHY
ADSORBTION
Solid - Liquid
Column
Chromatography
Thin Layer
Chromatography
HPTLC
Solid – Gas
(GSC)
PARTITION
Liquid – Gas
(GLC)
Liquid – Liquid
Thin Layer
Chromatography
Paper
Chromatography
Column
Chromatography
HPLC
ION EXCHANGE
Ion Exchange
Chromatography
MOLECULAR SIEVE
Gel
Chromatography
4

5. High Performance Liquid Chromatography (HPLC)
Involves separation of compounds on a stationary phase column
Solvent
Reservoir
Vacuum
Degasser
Gradient
Proportionating
Valves
Manifold
AutosamplerColumnDetectorCDS
5

6. Solvent Reservoir :
1. Quaternary Solvent Manager (QSM)
2. The Mobile Phase
3. Maintains slight head pressure on the pump inlet
4. Solvent Filter – Removes particles that can damage the
system
5. Very useful in method development, method transfer
and validation
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7. 13%
10%
6%
71%
14%
10%
5%
71%
13%
10%
6%
71%
12%
10%
7%
71%
pH 4.33pH 4.0pH 3.3 pH 5.25
Water
125 mM Formic Acid
Acetonitrile
125 mM Ammonium Hydroxide
Mobile phase of varying
pH prepared by adjusting
the percentage of
solvents mixed
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8. Vacuum Degasser :
Eliminates any dissolved gases that can interfere with the
flow or compromise the automated proportioning
8

9. Gradient Proportioning Valves :
They open in sequence, where the proportion of time
that each valve is open corresponds to the percentage of
the solvent that is specified in the gradient table.
9

10. Manifold :
1. This is where the four solvents first meet
2. Opens into the intelligent intake valve that leads to
the pump
10

11. Auto-sampler :
1. Holds the sample vials in a temperature controlled
environment
2. Injects the sample in the system when instructed
using high pressure pumps (Displacement,
Reciprocating or Pneumatic)
3. Allows the user to perform multiple sequences of
runs, unattended
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12. The Column :
1. Heart of the system
2. Packed with a stationary phase
3. Three major types – Guard, Preparative and Analytical
Longer the column – Higher the efficiency
Shorter the column – Faster the separation
Larger the diameter – Greater the loading capacity
Narrower the column – Greater the mass sensitivity
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13. Detectors :
Receives the result of the sample separation from the
column and monitors a physical property which changes
as the sample elutes
Photo Diode Array
Refractive Index
Ultraviolet
Visible
Florescence
Evaporative Light Scattering
Mass Spectrometer
Optical Rotation
Electrochemical
Conductivity
PDA
RI
UV
FL
VIS
ELS
MS
EC
OR
CD 13

14. Chromatography Data System (CDS) :
Translates the signal from the detector into a chromatographic
spectrum, that provides qualitative and quantitative data about
the sample.
Allows complete control of the pump, autosampler and detector
14

15. UHPLC
15

16. Ultra High Performance Liquid Chromatography
Column ‘mean-
particle-size’ less
than 2 micron
Almost double the
conventional
operating pressures
(~15,000 psi)
Optimal Linear
Flow Rates
achieved
Increased
Efficiency
Higher
Resolution
Shorter Analysis
Time
Lower Operating
Costs
16

17. Ultra High Performance Liquid Chromatography
17

18. Theoretical Principle :
Plate Theory:
𝑁 =
𝐿
𝐻
N = Number of theoretical plates
L = Length of column packing
H = Plate height
Higher the number of theoretical plates, better the column efficiency
Column
efficiency
increased
Number of
theoretical
plates
increased
Plate
height
greatly
decreased
Particle
size lower
than 2μm
18

19. Column Chemistry :
Ethylene Bridged Hybrid
Particle Technology
High Strength Silica
Particle Technology
Charged Surface Hybrid
Particle Technology
Bridged-ethanes
within a silica
matrix
A mechanically-
tolerant silica
based material
to withstand
high UHPLC
pressures
Particles posess
low-level surface
charge that
enhances
selectivity
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21. Product Vendors :
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22. Conclusion :
• In 2004, separation science was revolutionized with the
introduction of ‘Ultra High Performance Liquid
Chromatography.
• This technology provides improved resolution, increased
separation efficiency, shorter analysis time and lower operating
costs.
• It uses HPLC columns with mean particle size less than 2μm
and pressures up to 15,000 psi which drastically increases
number of theoretical plates of the column and result in
enhanced column efficiency.
22

23. 1. Douglas A Skoog, F James Holler, Stanley R Crouch. Principles of instrumental
analysis. Thomson brooks. International student edition. 2007. p.762-784.
2. Kasture A V, Wadokar S G, Mahadik K R, et al. Pharmaceutical analysis
instrumental methods. Volume 2. Nirali prakashan. August 2013. p.2.4-7.10.
3. Remington, The science and practice of pharmacy. 20th edition, vol-1, p.587-612.
4. https://www.chromatographytoday.com/news/hplc-uhplc/31/breaking-
news/what-is-the-difference-between-uhplc-and-uplc/30754 (accessed on : 16
September 2017)
5. https://www.shodex.com/en/kouza/f.html#! (accessed on : 16 September 2017)
6. http://www.thermofisher.com/us/en/home/industrial/chromatography/liquid-
chromatography-lc/hplc-uhplc-systems.html?ce=E.17CMD.AP120.02970.01
(accessed on : 16 September 2017)
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