Imagine a race where the runners are molecules from a mysterious mixture. HPLC sets the stage for this separation marathon. The sample, dissolved in a solvent, acts as the starting line. A pressurized stream of solvent pushes the molecules through a packed column, like an obstacle course. Each molecule interacts uniquely with the obstacles and the solvent, causing them to separate along the way. Finally, a detector acts as the finish line, identifying and measuring each molecule as it emerges. This powerful technique allows scientists to unmask unknown competitors (compounds), determine the number of each type of runner (quantify components), and even check if anyone cheated (assess purity).

1. MATERIAL
CHARACTERIZATION
TECHNIQUES
High Performance Liquid Chromatography
Presenters
Amina Khan – 2020-CH-07
Muhammad Hamza – 2020-CH-11
Salman Mohsin – 2020-CH-09

2. CONTENTS
What is MCT ?
1
What are materials
What is characterization
Classification of techniques
2
3
4
5
6
Chromatography
What is Chromatography
What is HPLC
Working Principle
& Construction
Working Principle
Components of HPLC
Classification
Types of HPLC
Comparison of Different Types
Applications
Applications of HPLC
In Various Fields
Results
Interpretation
Form in which result is obtained
The interpretation of the results

3. Materials
 Derived from term MATTER
 Matter is anything that has mass and occupy
some space
 Materials are the building blocks of our world.
 Everything around us, from the clothes we wear to
the buildings we live in, is made from materials.
 In simple words, materials is the MATTER IN USE.

4. Natural Materials
Occurring in nature without
human intervention
(e.g., wood, stone, metals like
gold or copper)
Classification
On the basis of Nature
Synthetic Materials
Man-made materials created
through chemical processes
(e.g., plastics, nylon, advanced
ceramics)
1
2

5. Classification
On the basis of Engineering

6. Key Points
•Properties: Each material has unique properties that
determine its suitability for different applications. These
properties can be:
• Physical: Density, strength, hardness, conductivity
(heat and electricity), melting point, etc.
• Chemical: How a material reacts with other
substances
• Mechanical: How a material responds to forces
(deformation, fracture, etc.)

7. Key Points
Selection: Choosing the right material for a specific job
is crucial. Engineers and designers consider factors like:
Required properties for the application (e.g., strength
for a bridge, insulation for a building)
•Cost of the material
•Availability of the material
•Environmental impact of the material (sustainability)

8. Characterization
•Material characterization is the
process of determining the physical,
chemical, and structural properties of
a material.
•It involves a variety of techniques to
analyse the material's composition,
microstructure, and performance.

9. Importance
Material characterization is essential for several reasons:
• Material selection: Helps engineers choose the right material for a
specific application based on its properties.
• Material development: Enables scientists to develop new materials
with improved properties.
• Quality control: Ensures that materials meet the required
specifications.
• Troubleshooting: Helps identify the cause of material failures.
• Understanding material behavior: Provides insights into how
materials will perform under different conditions.

10. Classification
On the basis of Information TO be Obtained
Chemical
Composition
•X-Ray Fluorescence
(XRF): Identifies and
quantifies elements
present in a material.
•Energy-Dispersive X-ray
Spectroscopy (EDS):
Analyzes the elemental
composition of a specific
region within a sample
(often used in conjunction
with Scanning Electron
Microscopy - SEM).
•Mass Spectrometry
(MS): Determines the
mass of molecules in a
sample, aiding in chemical
identification.
Microstructure:
•Scanning Electron
Microscopy (SEM): Creates
high-resolution images of a
material's surface, revealing
its morphology and
topography.
•Transmission Electron
Microscopy (TEM): Provides
even higher resolution
images, allowing
visualization of a material's
internal structure at the
atomic level.
•X-Ray Diffraction (XRD):
Analyzes the crystal
structure of a material by
identifying the arrangement
of atoms and molecules.
Physical and
Mechanical Properties:
•Mechanical Testing:
Measures a material's
strength, stiffness,
hardness, and other
mechanical properties
through techniques like
tensile testing,
compression testing, and
hardness testing.
•Thermal Analysis:
Evaluates a material's
response to changes in
temperature, including
techniques like
Differential Scanning
Calorimetry (DSC) and
Thermogravimetric
Analysis (TGA).
Surface and Interfacial
Properties:
•Atomic Force
Microscopy (AFM):
Creates high-resolution
images of a surface,
revealing its topography
and measuring forces at
the atomic level.
•X-ray Photoelectron
Spectroscopy (XPS):
Analyzes the chemical
composition and
electronic state of
elements at a material's
surface.

11. HPLC
High Performance Liquid Chromatography

12. Mobile Phase
A solvent or solvent mixture
that continuously flows through
the system.
Chromatography
is a separation technique that
separates components of a
mixture based on their differential
interaction with two phases Stationary Phase
A solid material packed within a
column.
1
2

13. Historic Timeline
First Ever Presented
1903
The official date of birth of chromatography is
the 21 March 1903 in Warsaw when Mikhail
Semenovitch TSWETT has presented at the
Congress of the Polish Natural Sciences Society a
communication entitled: « A new class of
adsorption phenomena and their applications in
biochemical analysis » about the separation and
purification of vegetal pigments (a mixture of
chlorophylls and xantophylls) on a chalk column
1938
Elution Theory
REICHSTEIN proposes a theory
for the elution and separation
of solutes on a column
1967
HPLC
beginning of HPLC after the works of HUBER
and HUZSMAN, this technique was first
named « High Speed Liquid Chromatography »
then « High Pressure Liquid Chromatography
» and finally « High Performance Liquid
Chromatography »
1969
Progress
after the 5th International
ymposium International «
Advances in Chromatography »
the development of HPLC was
very fast

14. Classification
On the basis of Separation Technique
Adsorption
Chromatography
• Stationary phase
is polar.
• Mobile phase is
nonpolar.
• Separation
based on the
differing
adsorption
strengths of
components
onto the polar
stationary phase.
Partition
Chromatography
• Stationary phase
is nonpolar.
• Mobile phase can
be polar or
nonpolar.
• Separation
based on the
partitioning of
components
between the
polar mobile
phase and the
nonpolar
stationary phase.
Ion-Exchnage
Chromatography
•Stationary
phase contains
charged groups.
•Separation
based on the
ionic interaction
between charged
components in
the sample and
the oppositely
charged groups
on the stationary
phase.
Size-Exclusive or Gel
Permeation
Chromatography
•Stationary phase is
porous with pores of
different sizes.
•Separation based
on the size and
shape of the sample
components.
•Larger components
are excluded from
the smaller pores
and elute first, while
smaller components
can enter the pores
and elute later.

15. High Performance Liquid Chromatography
• It is also referred to as high pressure liquid chromatography ,is a
technique in analytical chemistry used to separate, Identify, and
quantify specific components in mixtures.
• The mixtures can originate from food, chemicals , pharmaceuticals,
biological, environmental and agriculture etc. which have been
dissolved into liquid solutions.
• High performance means high resolution: ability to distinguish
between two components

16. 01 - BRANDING
02 - WEBSITE
03 - SOCIAL MEDIA
High pressure liquid chromatography(HPLC):
• Type of liquid chromatography
• Conducted in a column
• Characterized by the use of high pressure & small particle size to push a
mobile phase solution through a column of stationary phase allowing
separation of complex mixtures with high resolution.

17. Basic principle
• Based on distribution of solute between a liquid
mobile phase and a stationary phase.
• The small diameter particles are used as stationary
phase support.

18. 1
2
3
4
5
6
Components of HPLC:
1. Solvent Reservoir
2. Pumps
3. Sample Injection System
4. Columns
5. Detectors
6. Computer
7. Waste collector

19. 1
2
3
4
5
6
HPLC PUMP
• To produce an appropriate pressure to push
solvent & sample into the column.
• Ideal pump
 Deliver high pressure (upto 50MPa)
 Deliver pulse free flow
 Constant volume delivery
 Deliver high volumes (flow rates) of solvent
(to 10 mL/min)
 Solvent replacement is easy

20. 1
2
3
4
5
6
Types of HPLC Pump
Constant Pressure
• A steady pump pressure
• (usually about 1000–2000 psi) is needed to
ensure reproducibility & accuracy.
Constant displacement Pump
 Reciprocating pump: constant flow rate
through the column
 Slight cyclical variation in pressure→pulse
dampeners.

21. 1
2
3
4
5
6
Analyte Columns in HPLC:
• Considered the “heart of the chromatograph” the
column’s stationary phase separates the sample
components of interest using various physical and
chemical parameters.
• The small particles inside the column are what cause
the high back pressure at normal flow rates.
• The pump must push hard to move the mobile phase
through the column and this resistance causes a high
pressure within the chromatograph.
• Standard Column : 3-25cm long, ID(4.6 mm) ;optimum
flow volume = 1 ml/min. • Narrow Bore : 3 mm(ID); flow
volume 0.6 ml/min
• Microbore /Open tubular : 25-50cm long, 1 mm(ID); 50
microliter/min.

22. 1
2
3
4
5
6
HPLC columns
Within the Column is where separation occurs.
Key Point –Proper choice of column is critical for success in HPLC
Materials of construction for the tubing
• Stainless steel (the most popular; gives high pressure capabilities)
• Glass (mostly for biomolecules)
• PEEK polymer (biocompatible and chemically inert to most solvents
Packing material:
The packing material is prepared from SILICA particle, ALUMINAparticle and
ion exchange RESIN.
Porous plug of stainless steel or Teflon are used in the end of the columns to
retain the packing material.
According to the mode of HPLC , they are available in different size ,
diameters, pore size or they can have special materials attached ( such as
antigen or antibody ) for immuno affinity chromatography.

23. Types of columns in HPLC:
• Guard Column
• Fast Column
• Preparative(i.d. > 4.6 mm; lengths 50 –250
mm)
• Capillary(i.d. 0.1 -1.0 mm; various lengths)
• Nano(i.d. < 0.1 mm, or sometimes stated as <
100 μm)
• Analytical[internal diameter (i.d.) 1.0 -4.6-
mm; lengths 15 –250 mm]

24. Daniel Gallego
“Lorem ipsum dolor sit amet,
consectetur adipiscing elit. Etiam
mattis, nunc vitae eleifend posuere,
turpis mauris vestibulum purus, in
pellentesque tellus elit vel nisl.”
Lorna Alvarado
Detectors in HPLC
The detector can see (detect) the
individual molecules that come out
(elute) from the column.
•A detector serves to measure the
amount of thosemolecules so that the
chemist can quantitatively analyze the
sample components.
•The detector provides an output to a
recorder or computer that results in the
liquid chromatogram(i.e., the graph of
the detector response).

25. Common HPLC detectors:
• UV-VIS
•DiodeArray
•Multiple Wavelength
•Variable Wavelength
•Mass Spectrometers
•Refractive Index
•Fluorescence
•Light Scattering
•Electrochemical
•Radioactivity
•Conductivity

26. Daniel Gallego
“Lorem ipsum dolor sit amet,
consectetur adipiscing elit. Etiam
mattis, nunc vitae eleifend posuere,
turpis mauris vestibulum purus, in
pellentesque tellus elit vel nisl.”
Lorna Alvarado
COMPUTERS:
• Electronic signals generated by detectors
are recorded inthe form of
chromatograghic peak at varied function
of time
• Peak Area, height, retention time, base
width of chromatograghic peak is
measured to compute analyte
concentration of each peak.

27. How can we analyze the
sample?
For example:
1.Carbohydrates
2. fructose
3. Glucose
4. Saccharose
5.Palatinose
6. Trehalulose
7. isomaltose

28. “Lorem ipsum dolor sit amet,
consectetur adipiscing elit. Etiam
mattis, nunc vitae eleifend posuere,
turpis mauris vestibulum purus, in
pellentesque tellus elit vel nisl.”
Lorna Alvarado

29. 03 - SOCIAL MEDIA
Working of HPLC:
 Equilibration stage: (to ready column for purification)
Only buffer pass not sample, detector detect the buffer and
straight line obtain called baseline.
 Hydrophilic curve
 Hydrophobic curve

30. 01 - BRANDING
02 - WEBSITE
03 - SOCIAL MEDIA
Types of HPLC
1. Normal phase HPLC
Mobile phase is non
polar
Stationary phase is
polar
2. Reverse phase HPLC:
Mobile phase is polar
Stationary phase is
non polar
3. Size exclusion HPLC:

31. 4. Ion Exchange HPLC:

32. 01 - BRANDING
02 - WEBSITE
03 - SOCIAL MEDIA
Factors which influence HPLC performance
1. Internal diameter of column - the smaller in diameter, the higher
in sensitivity
2. Pump pressure - the higher in pressure, the higher in separation
3.Sample size
4. The polarity of sample, solvent.
5. .Temperature - the higher in temperature, the higher in
separation

33. Applications Of
HPLC
Pharmaceutical
Analysis
Biological Analysis
Environmental
Analysis
Food And Beverages

36. Equipment Specification
Operating Conditions
• Solvent A: This is a mixture of water and acetic acid in a 25:1 ratio by
volume. Water is a polar solvent, while acetic acid is a weak acid with
some polarity.
• Solvent B: Pure methanol, which is a more organic and less polar
solvent compared to water and acetic acid.
• Binary Gradient: This indicates that the HPLC system uses two
pumps to deliver a mixture of Solvent A and B. The proportion of each
solvent changes gradually throughout the analysis (gradient elution).
• Flow Rate: 1.000 mL/min. This is the rate at which the mobile phase is
pumped through the column.
• Pressure Limits:
• Maximum Pressure (P.Max): 400.0 kgf/cm² (kilograms-force per
square centimeter).
• Minimum Pressure (P.Min): 0.0 kgf/cm². (Atmospheric Pressure)
• Column Oven:
• Temperature: 40 °C. Maintaining the column at this temperature
can influence analyte retention times and separation efficiency.
• Detector: A UV/Vis detector model with designation A for
identification within the system.
• Lamp: D2. This refers to the type of lamp used in the detector.
Deuterium lamps emit light in the ultraviolet (UV) region, commonly
used for detecting aromatic compounds that absorb UV light.
•HPLC-DAD: This refers to the analytical technique used. HPLC
separates compounds based on their interaction with a stationary
phase and a mobile phase. DAD allows detection of compounds at
various wavelengths, providing a more comprehensive analysis.
•Flavonoid standards: The sample analysed likely contains known
flavonoid compounds used for comparison with unknown samples.
•Gradient HPLC: This indicates that the composition of the mobile
phase changed gradually during the analysis, potentially improving the
separation of various flavonoids.
•C18 column: This refers to the type of stationary phase used in the
HPLC system. C18 columns are commonly used for separating non-polar
and moderately polar compounds, which many flavonoids are.
•Monitored at 280 nm: This specifies the wavelength of light used for
detection. Many flavonoids absorb UV light around 280 nm, allowing for
their identification.

37. THANK YOU