High-Performance

Liquid
Chromatography
(HPLC)
 What is HPLC?
• High-Performance Liquid Chromatography (HPLC) is an
advanced form of liquid chromatography.
• Use of high-pressure pumps to push a mobile phase
(solvent) containing the sample through a column packed
with a stationary phase (solid adsorbent).
• Widely used for separating, identifying, and quantifying
components in complex mixtures.
How Does HPLC Work?
 Principle of HPLC
• HPLC operates based on the principle of adsorption
• Each component of the sample interacts with the
stationary phase at different levels, leading to separation.
• Compounds with a higher affinity for the stationary
phase will have longer retention times.
 Types of Liquid
Chromatography
 Particle Size in HPLC
• HPLC columns are packed with small particles, typically
ranging from 3 µm to 10 µm in diameter.
• Smaller particles provide a larger surface area for
interaction, which leads to better resolution of separated
components.
• However, smaller particles create higher back pressure,
requiring robust pumps to maintain flow rates.
 Bonded Phases in HPLC
• Bonded phases are silica particles chemically
modified with functional groups (e.g., C18) to create
the stationary phase.
• They are used to adjust the polarity of the stationary
phase for specific separation needs.
• Silica particles are heated in dilute acid (e.g., HCl) for 24-
48 hours to generate silanol groups.
• Silica is treated with organosilanes, such as C18, to bond
functional groups to the surface.
• The final result is a non-polar stationary phase, ready for
reverse-phase chromatography.
 Column in HPLC
• HPLC columns are typically made of stainless steel to
withstand high pressures.
• Internal Diameter: Usually ranges from 4.5 mm to 5
mm.
• Length: Columns are typically 10–25 cm in length.
• Packing Material: The column is packed with
materials like silica or bonded-phase silica beads,
which serve as the stationary phase.
 Stationary Phases

• The material packed in the column, often made of

silica or bonded phases.
• Separation is achieved as analytes interact differently
with the stationary phase depending on their
polarity.
 Mobile Phase
• The material packed in the column, often made of
silica or bonded phases.
• Separation is achieved as analytes interact differently
with the stationary phase depending on their
polarity.
 Retention Time in HPLC
• The time taken for a specific analyte to pass through
the column and be detected.
• Analytes with stronger interactions with the
stationary phase will have longer retention times,
while those with weaker interactions elute faster.
 Normal-Phase
Chromatography (NPC)
Stationary Phase (SP):
• Polar: Typically unmodified silica gel.
• Retains polar compounds through adsorption.
Mobile Phase (MP):
• Non-polar solvents such as hexane, chloroform, or
ether.
Separation Mechanism:
• Based on adsorption and polarity.
• Polar analytes have a stronger affinity for the polar
stationary phase, resulting in longer retention times.
• Non-polar analytes interact more with the non-polar
mobile phase and elute faster.
• Suitable for polar compounds, such as sugars,
alcohols, and amines.
 Reverse-Phase
Stationary Phase (SP):
• Non-polar: Typically C18-bonded
silica (octadecyl silane).
• Retains non-polar analytes
through hydrophobic interactions.

Mobile Phase (MP):

•Polar solvents, such as water, methanol, or
acetonitrile.
Separation Mechanism:
• Based on hydrophobic interactions.
• Non-polar compounds are retained on the non-polar
stationary phase, leading to longer retention times.
• Polar compounds have higher affinity for the polar
mobile phase and elute more quickly.
Analyzed Compounds:
• Suitable for non-polar analytes, such as lipids,
steroids, and hydrophobic drugs.
Critical Parameters in RPC
Column Length:
• Longer columns provide better resolution but
increase retention time.
• Typical length: 10–25 cm.
Flow Rate:
• Affects the speed of elution and resolution.
• Optimal range: 0.2-1 mL/min.
Temperature:
• Higher temperatures lower retention time by
reducing mobile phase viscosity.
• Helps maintain peak shape and resolution
consistency
 Instrumentation of HPLC
• Solvent Reservoir and Degassing System
• Pressure, Flow, and Temperature Control
• Pumps
• Sample Injection System
• HPLC Columns
• Detectors
• Strip-chart recorder
• Data handling device
 Solvent Reservoir and
Degassing System
Solvent Reservoir:

• Holds the mobile phase,

which can be a single solvent
or a mixture (e.g., water,
methanol, acetonitrile).

• Mobile phases must be free

from impurities to avoid
interference.
Degassing:
• The removal of dissolved gases from solvents is
critical to prevent bubble formation in the system.
• Methods: Degassing is done using vacuum,
ultrasonication, or helium sparging.
 Pumps in HPLC
Pumps deliver the mobile phase at a constant flow rate
and pressure through the column.
Types of Pumps:
• Reciprocating Pumps: Provide a constant flow;
common for modern HPLC systems.
• Syringe Pumps: Provide precise flow, used for small
volume systems.
• Constant Pressure Pumps: Ideal for systems
where pressure must remain stable.
 Sample Injection System
Introduces the sample into the mobile phase with high
precision and minimal volume variation to ensure
reproducible analysis.
Types:
• Manual Injection:
– Involves injecting the sample with a syringe.
– Primarily used for low-throughput analysis or
small volumes.
• Automated Injection:
– Common in modern HPLC systems, using an
autosampler.
– Ensures accurate and reproducible injections,
making it ideal for high-throughput sample
analysis.
Advanced Sample Injection
Systems
Septum Injectors:
• A rubber septum is pierced by a syringe needle to
introduce the sample into the system.
• This method minimizes contamination and ensures
closed system injection.
Stop-Flow Septumless Injectors:
• No septum is used, reducing contamination and pressure
loss during sample injection.
• Ideal for continuous injections without disrupting system
pressure.
Microvolume Sampling:
• Used for very small sample volumes (in the range of
microliters), ensuring minimal dead volume and
preventing sample waste.
• Often used in conjunction with micro-injectors for
specialized, low-volume analyses.
 HPLC Columns
Types of Columns:
• Analytical Columns: Used for standard
separations in pharmaceutical and chemical
analyses.
• Guard Columns: Placed before the analytical
column to protect it from impurities and extend
its life.
• Capillary Columns: Narrow columns (internal
diameters < 0.5 mm) used in high-resolution and
low-flow applications.
 Packing Materials in HPLC
Columns
• Porous Silica Particles:
• Commonly used in reverse-phase and normal-
phase chromatography.
• Offers a large surface area for efficient analyte
interaction and separation.
Porous Layer Beads:
• Solid core particles coated with a thin porous
layer.
• Provide high resolution with low back pressure,
ideal for size-exclusion and ion-exchange
chromatography.
Styrene Divinyl Benzene (S-DVB):
• Polymeric material used in hydrophobic
separations.
• Resistant to high pH; suitable for non-polar
compounds in reverse-phase chromatography.
 Isocratic vs. Gradient
Elution
Isocratic Elution:
• The mobile phase composition remains constant
throughout the run.
• Often used for simple mixtures where all
components have similar affinities for the stationary
phase.
Advantages of Isocratic Elution:
1.Simplicity: Easier to set up and maintain since the
mobile phase does not change.
2.Reproducibility: Consistent mobile phase conditions
lead to reproducible results.
3.Cost-Effective: Requires less solvent compared to
gradient elution.
Gradient Elution:
• The composition of the mobile
phase changes during the run,
typically starting with a weaker
solvent and gradually transitioning
to a stronger solvent.
• Used for complex mixtures where
components have widely varying
affinities for the stationary phase.
Advantages of Gradient Elution:
1.Improved Separation: Enhances the separation of
compounds with different polarities or affinities for the
stationary phase.
2.Faster Analysis: Shortens analysis time for complex
samples by starting with a weaker mobile phase and
progressively increasing the solvent strength.
3.Sharper Peaks: Gradient elution often results in
better resolution and sharper peaks for later eluting
compounds.
Function of HPLC Detectors
• HPLC detectors monitor the eluted analytes from the
column and convert their physical or chemical
properties into an electrical signal.
• They provide quantitative and qualitative data by
detecting changes in the properties of the analytes,
which is presented as a chromatogram.
 Types of HPLC Detectors
Bulk Property Detectors:
• Measure the overall properties of the mobile phase,
which change when analytes are present.

Solute Property Detectors:

• Measure properties specific to the analyte itself
(e.g., UV absorption, fluorescence).
Bulk Property
Detectors
 Refractive Index (RI)
Detector
• Measures changes in the refractive index (how light
bends as it passes through a substance).
• Detects the difference between the refractive index
of the pure mobile phase and the sample.
• Suitable for compounds that do not absorb UV light,
such as sugars, lipids, and polymers.
 Conductivity Detector

• Measures changes in electrical conductivity as ionic

analytes elute from the column.
• Analytes increase or decrease the conductivity of the
mobile phase, which is detected as a signal.
• Best for ionic compounds in ion-exchange
chromatography, including salts and inorganic ions.
 Viscosity Detector
• Detects changes in the viscosity (resistance to flow)
of the mobile phase when analytes elute.
• Measures changes in the flow resistance of the
mobile phase, indicating changes in viscosity due to
the analyte.
• Commonly used in the analysis of polymers and
macromolecules.
 Evaporative Light
Scattering Detector (ELSD)
• Measures light scattering from particles formed after
solvent evaporation.
• The mobile phase is evaporated, and the resulting
analyte particles scatter light, which is detected.
• Non-volatile compounds like lipids, phospholipids,
and polymers are commonly analyzed using ELSD.
 Ultrasonic Detector
• Detects changes in the speed of sound through the
mobile phase as analytes elute.
• Sound waves travel differently through pure mobile
phase vs. a sample-laden mobile phase, which is
detected by changes in the speed of sound.
• Applied in non-chromophoric analytes and
substances where other detectors may not be as
effective.
 Pressure Drop Detector
• Measures pressure differences across the column
when analytes pass through.

• Analytes cause a change in backpressure as they pass

through the column, which is detected.

• Mainly to monitor column performance and detect

potential blockages or flow anomalies.
 UV-Visible (UV-Vis)
Detector
• Measures the absorption of UV or visible light by
analytes.
• Analytes absorb light at specific wavelengths (UV:
190–400 nm; Vis: 400–700 nm), causing a decrease
in the light intensity that reaches the detector.
• Ideal for compounds with chromophores (e.g.,
aromatic compounds, drugs, biomolecules)
 Fluorescence Detector
• Measures fluorescence emission after the analyte
absorbs UV light.
• Analytes are excited by UV light and emit light at a
longer wavelength, which is detected.
• Highly sensitive, used for detecting fluorescent
compounds like vitamins, aromatic hydrocarbons,
and pharmaceuticals at trace levels.
Electrochemical Detector
• Measures the electrical current produced by the
oxidation/reduction of analytes.
• Analytes undergo redox reactions at the detector’s
electrode, generating a current proportional to their
concentration.
• Suitable for redox-active compounds such as
neurotransmitters, antioxidants, and drugs.
 Polarimetric Detector
• Measures the optical rotation of polarized light by
chiral analytes.
• Optically active compounds rotate the plane of
polarized light, and the degree of rotation is
measured.
• Widely used in chiral separations, particularly for the
analysis of enantiomers in pharmaceuticals.
 Nuclear Magnetic
Resonance (NMR) Detector
• Measures the magnetic properties of nuclei (e.g.,
hydrogen).
• Analytes are exposed to a magnetic field, causing
certain nuclei to resonate, and this resonance is
detected.
• Primarily used for structure elucidation and
analyzing complex mixtures.
 Multipurpose Detectors

• These detectors can handle various analyte

properties and are versatile across different
chromatography modes.

• Refractive Index (RI) and UV-Vis detectors are

commonly used multipurpose detectors in HPLC,
suitable for many different compounds.
HPLC Process
 Strip Chart Recorder in
HPLC
• A strip chart recorder is used to provide a
continuous, real-time recording of the detector's
response during an HPLC run.
• As the detector generates a signal, the strip chart
recorder plots it on a moving sheet of paper,
producing a chromatogram.
 Calculation of
Theoretical Plates in HPLC

• N = Number of theoretical plates.

• tR​ = Retention time (time for the analyte peak to elute).
• Wb​ = Width of the peak at its base (in time units).

Interpretation:
• Higher N means the column is more efficient at
separating compounds
 Applications
• Quality Control: Determines purity and detects
impurities in pharmaceutical products.
• Pharmacokinetics: Quantifies drug levels in biological
fluids for bioavailability and drug metabolism studies.
• Drug Development: Analyzes Active Pharmaceutical
Ingredients (APIs) and performs stability studies to
assess shelf life.
• Chiral Separation: Separates enantiomers in chiral drugs
to ensure therapeutic effectiveness and safety.
 Preparative HPLC
Used to purify large quantities of a specific compound,
typically in drug development or chemical synthesis.
Key Differences from Analytical HPLC:
• Analytical HPLC focuses on identifying and
quantifying small amounts of compounds.
• Preparative HPLC uses larger columns and higher
flow rates to isolate and collect significant quantities
of pure compounds.
Process:

• The sample is injected into the column, and the

mobile phase carries the compounds through the
stationary phase.

• Compounds are separated based on their affinities

and then collected in fractions for further use.
 Applications in Pharmacy
• Purification of Active Pharmaceutical Ingredients
(APIs) during drug manufacturing.
• Isolating impurities or degradation products for
further analysis.
• Used in natural product extraction and purification
of biological samples.
 Advantages

• Provides high-purity compounds.

• Capable of handling large sample volumes.

• Ideal for scale-up from lab to industrial production.