High Performance Liquid
Chromatography (HPLC)
Principle,
Instrumentation and
Application of HPLC
Department of Pharmacy, IUB
Course: PHA 305
History Of Chromatography
• The history of chromatography spans
from the mid-19th century to the 21st.
Chromatography, literally "color
writing", was used—and named— in
the first decade of the 20th century,
primarily for
• the separation of plant pigments such
as chlorophyll
• New forms of chromatography
developed in the 1930s and 1940s
made the technique useful for a wide
range of separation processes.
Nomenclature of Chromatography
According to IUPAC:
Chromatography is a physical method of separation in
which the components to be separated are distributed
between two phases, one of which is stationary (stationary
phase) while the other (the mobile phase) moves in a
definite direction (IUPAC, 1993).
What is chromatography ?
• Chromatography is a separation process in which the solute equilibrates between
stationary (solid)and mobile (liquid) phases
• Two types of chromatographic separation methods are used in quantitative analysis -
gas chromatography with small diameter are used to accomplish efficient separation.
• In liquid chromatography, packed columns are used to conduct the separation where
the mobile phase pumped through the separation column (stationery phase).
• Since the particles in the column are of small size ( 3-5µm), for efficient separation,
high pressure is applied.
• The function of the column is similar to that of a fractional distillation column where
separation efficiency depends on the number of fractions.
• In HPLC, efficiency depends, apart from other factors, on the number theoretical plates
the column has as it is calculated as :
N = 3 000 L(cm)/dp( µm) where L is the length of the column,
and dp the particle diameter. (equivalent of fractions in distillation column)
Concept of Chromatography
• Chromatography is an analytical method where the compounds are physically separated
prior to measurement.
• The main purpose of chromatography is to separate and quantify the target compounds in a
sample matrix.
• For quantitative analysis, the analytes are identified by the time it came out of the column,
known as the retention time, t R the quantity is measured by the. peak area measured by the
detector
Liquid
Chromatography
Gas
Chromatography Chromatography
Supercritical-fluid
Chromatography
Flow Diagram of HPLC
Column
Detector
Injector
Pump Oven
Mobile Phase
Chromatogram
tR
Peak tR : Retention time
Signal
h A : Area
A h : Height
Time
Some Important Terms
• Chromatogram: A plot of detector signal output versus time or elution volume.
• Mobile phase: The liquid that moves the solute through the column.
• Stationary phase: The packing material of the column, which is the immobile phase involved in the
chromatographic process.
• Peak: The visual representation on the chromatogram based on the detector's electrical response due to
the presence of a sample component inside the flow cell.
• Retention time: The time taken by the analyte peak to reach the detector after sample injection.
• Qualitation: An analysis process which is designed to identify the components of a substance or mixture.
• Quantitation: An analysis process which is designed to determine the amounts or proportion of the
components of a substance.
Advantages of HPLC Use
• Simultaneous Analysis
• High Resolution
• High Sensitivity (ppm-ppb)
• Good repeatability
• Small sample size
• Moderate analysis condition - no need to vaporize the sample like GC
• Easy to fractionate the sample and purify
• Non destructive
Scope of HPLC
Field Typical mixtures
Pharmaceuticals Antibiotics, sedatives, steroids, analgesics, crude drugs, cosmetics
Amino acids, proteins, peptides, carbohydrates, lipids, enzymes,
Biochemical
medicines, hormone
Mycotoxins, additives, saccharides, amino acids, vitamins, fatty acid,
Food products
coloring agents, anti-bacterials
Condensed aromatics, surfactants, propellants, dyes, polymers,
Industrial chemicals
plasticizers
Forensic chemistry Drugs, poisons, blood alcohol, narcotics
Inorganic ions, organic acids, agricultural chemicals, pesticides, herbicides,
Environmental field
phenols,
Clinical medicine Bile acids, drug metabolites, urine extracts, estrogens
Separation Mechanism
Compounds are separated because the molecules move at different rates in the
column.
1
2
column
Separation Mechanism
cont’d…
Due to different interaction between stationary phase and different sample, the
molecules move at different rate, therefore separation can be done.
Separation Modes
• Normal phase chromatography
• Reversed phase chromatography
• Ion chromatography
• Size exclusion chromatography
• Affinity chromatography
Normal Phase Chromatography
The combination of a polar stationary phase and a non-polar mobile
phase is called normal-phase chromatography.
To prevent unwanted interactions between the solutes and any unreacted –
SiOH groups, the silica frequently is “capped” by reacting it with Si(CH3)3Cl;
such columns are designated as end-capped.
Column Materials
Bulky isobutyl groups protect siloxane bonds from
hydrolysis at low pH.
Reversed Phase Mode
Stationary phase: Non-polar property
Mobile phase : Polar property
This combination is defined as
Reversed Phase Mode
Stationary Phase in
Reversed Phase Column
• C18 (ODS) type
• C8 (octyl) type CH3
• C4 (butyl) type Si O Si C18H37
CH3
• Phenyl type
Non-polar
• TMS type
• Cyano type Reversed phase HPLC
• Stationary phase: Non-polar property
• Mobile phase: Polar property
Mobile Phase for Reversed Phase
HPLC
• Water / buffer + Organic solvent
• Organic solvents:
• Methanol
• Acetonitrile
• THF
• Buffer:
• Phosphate buffer
• Acetate buffer
• etc
• Ratio of aqueous and organic solvents is
important
What Is the Interaction?
Hydrophobic Interaction
Less
A polar analyte
More
B polar analyte
B B
A
A
B
A B
A
A B
A B
Support Nonpolar Interstitial area
particle bonded phase (mobile phase)
Less polar (more hydrophobic) analytes are more attracted and spend more
time associated with the hydrophobic bonded phase, therefore, they are
eluted last.
Hydrophobicity
• If the sample has more
• CH3CH2CH2--- : Carbon chain
• : Aromatic group
• Hydrophobicity is stronger
• If the sample has more ionizable
group
• -COOH : Carboxyl group
• -NH2 : Amino group
• -OH : Hydroxyl group
Hydrophobicity is weaker
Retention Time and Hydrophobicity
Increase of Solvent
Polarity
Effect of Stationary Phase
Effect of Stationary Phase
Polarity of Common Organic Functional Groups & Solvents
HPLC Column
• An HPLC typically includes two columns: an analytical column responsible for the
separation and a guard column.
• The guard column is placed before the analytical column, protecting it from contamination.
• Analytical Columns most commonly used in HPLC are constructed from stainless steel
with internal diameters between 2.1 mm and 4.6 mm, and lengths ranging from approximately
30 mm to 300 mm.
• These columns are packed with 3–10 mm porous silica particles that may have an irregular
or spherical shape.
• Typical column efficiencies are 40,000–60,000 theoretical plates/m.
• Guard columns usually contain the same particulate packing material and stationary
phase as the analytical column.
HPLC Column
Introduction of Samples
• HPLC analysis is carried out under high pressure.
• The sample is introduced using a loop injectors which is interchangeable, and available
with volumes ranging from 0.5 µL to 2 mL.
• In the load position the sampling loop is isolated from the mobile phase and is open to
the atmosphere.
• After loading the sample, the injector is turned to the inject position.
• In this position, the mobile phase is directed through the sampling loop, and the
sample is swept onto the column
Schematic diagram of a loop injector
6 Port Valve System
Typical sample loop volume is 5-200 µl.
Column Dimension
Possible Configuration
Isocratic system
Low-pressure gradient system
High-pressure gradient system
Elution Modes
( Column : ODS )
Isocratic System
Low-pressure Gradient System
Scope of HPLC
Quantitative Analysis
Separation of Enantiomers
Baseline separation of enantiomers of the drug
Ritalin by HPLC with a chiral stationary phase.
Detectors for HPLC
• UV-VIS Ultraviolet / Visible detector
• PDA Photodiode Array detector
• RF Fluorescence detector
• CDD Conductivity detector
• RID Refractive Index detector
• ECD Electrochemical detector
• ELSD Evaporative light scattering detector
• MS Mass spectrometer detector
Ultraviolet / Visible Detector
Ultraviolet / Visible Detector
Ultraviolet / Visible Detector
Advantage:
• Sensitivity is high
• Relative robust to temperature and flow rate change
• Compatible with gradient elution
Disadvantage:
• Only compounds with UV or visible absorption could be detected.
Additional Functions
• Dual Wavelength mode
• Wavelength Time Program mode
• Wavelength Scan mode
Photodiode Array Detector
Photodiode Array Detector
3-D Data
Example of PDA Application
- Identification by Spectrum
ACN/H2O=30/70 1. Azelaic acid
2. Benzoic acid
1 2 3. Nitrobenzoic acid
ACN/H2O=15/85
min
Example of PDA Application
- Identification by Spectrum
Elution sequence
Peak 1 Peak 2 Peak 3
Azelaic acid Benzoic acid Nitrobenzoic acid
ACN/H2O
=30/70
Benzoic acid Azelaic acid Nitrobenzoic acid
ACN/H2O
=15/85
Peak 1 Peak 2 Peak 3
Example of PDA Application
- Checking Peak Purity
UV-Vis Spectrum of Acetyl
Salicylic Acid
Sample
Standard
PDA Detector
Advantages:
• PDA Detector could analyze a sample simultaneously at many different wavelengths.
• UV Visible spectra are useful for compound identification, checking peak purity, as well as
finding the optimum absorbance for the compounds.
• UV Visible spectra of many compounds could be stored in the spectrum libraries, which are
useful for compound identification.
• Relatively robust to temperature and flow rate fluctuations
• Compatible with gradient elution.
Disadvantages:
• Slightly less sensitive than UV-Visible detector.
