High Performance Liquid Chromatography

(HPLC)

Winda Trisna W, M.Si

Prodi S1 Farmasi Fakultas Farmasi
Universitas Bakti Tunas Husada
2023/2024
What is HPLC?

• Chromatography is a technique used to separate the

components contained in a sample
• High-performance liquid chromatography (HPLC) is a type
of chromatography that, because of its wide application
range and quantitative accuracy, is regarded as an
indispensable analytical technique, particularly in the field of
organic chemistry. It is also widely used as a preparation
technique for the isolation and purification of target
components contained in mixtures.
Separation Process and Chromatogram for
Column Chromatography

concentration

Chromatogram
Output

Time
Chromatogram
Intensity of detector signal

tR
Peak tR : Retention time
t0 t0 : Non-retention time
h
A A : Peak area
h : Peak height

Time
From Liquid Chromatography to High Performance
Liquid Chromatography
• Higher degree of separation!
→ Refinement of packing material (3 to 10 µm)
• Reduction of analysis time!
→ Delivery of eluent by pump
→ Demand for special equipment that can
withstand high pressures

The arrival of high-performance liquid chromatography!

High Performance Liquid Chromatography
Flow Channel Diagram for High Performance
Liquid Chromatograph

Detector

Column

Pump Column oven

(thermostatic column
chamber)
Sample injection unit Drain
(injector)
Data processor
Degasser
Advantages of High Performance Liquid
Chromatography
• High separation capacity, enabling the batch analysis of
multiple components
• Superior quantitative capability and reproducibility
• Moderate analytical conditions
• Unlike GC, the sample does not need to be vaporized.
• Generally high sensitivity
• Low sample consumption
• Easy preparative separation and purification of samples
Fields in Which High Performance Liquid
Chromatography Is Used
• Biogenic substances • Food products
• Vitamins, food additives,
• Sugars, lipids, nucleic acids, sugars, organic acids, amino
amino acids, proteins, acids, etc.
peptides, steroids, amines, etc.
• Medical products • Environmental samples
• Inorganic ions
• Drugs, antibiotics, etc.
• Hazardous organic
substances, etc.
• Organic industrial
products
• Synthetic polymers,
additives, surfactants, etc.
 Polarity of Substances
• Polarity • Miscibility of solvents
• Property of a substance • Solvents of similar polarities
whereby the positions of the can be easily dissolved
electrons give rise to positive together.
and negative poles • Polar and nonpolar molecules
• Water: Polar have a similar relationship to
Methane: Nonpolar that of water and oil.

H –
H O
O
C H C C –
H H O
+ H
H H
H
Methane Water Acetic acid
Nonpolar (Hydrophobic) Functional Groups
and Polar (Hydrophilic) Functional Groups
• Nonpolar Functional • Polar Functional Groups
Groups • -COOH
• -(CH2)nCH3 • Carboxyl groups
• Alkyl groups • -NH2
• -C6H5 • Amino groups
• Phenyl groups • -OH
• Hydroxyl groups
Normal Phase / Reversed Phase

Stationary
Mobile phase
phase
Normal High polarity Low polarity
phase (hydrophilic) (hydrophobic)

Reversed Low polarity High polarity

phase (hydrophobic) (hydrophilic)
 Reversed Phase Chromatography

• Stationary phase: Low polarity

• Octadecyl group-bonded silical gel (ODS)
• Mobile phase: High polarity
• Water, methanol, acetonitrile
• Salt is sometimes added.
Separation Column for Reversed Phase
Chromatography
• C18 (ODS) type • Phenyl type
• C8 (octyl) type • TMS type
• C4 (butyl) type • Cyano type

CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2

Si -O-Si
CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH3

C18 (ODS)
Effect of Chain Length of Stationary Phase

C8

Medium
C18 (ODS)

Strong C4

Weak
 Hydrophobic Interaction
H2O H2O H2O
H2O
H2O
H2O Nonpolar solute
H2O
H2O
H2O If a nonpolar H2O
H2O H2O substance is added... H2O H2O
Network of hydrogen bonds …the network is broken and...

H2O H2O
H2O
H2O H2O …the nonpolar substance
H2O H2O is pushed to a nonpolar
location.
Nonpolar solute

Nonpolar stationary phase

Relationship Between Retention Time and
Polarity

C18 (ODS) OH

Weak
Strong
CH3
 Basic Settings for Eluent Used in Reversed
Phase Mode
• Water (buffer solution) + water-soluble organic solvent
• Water-soluble organic solvent: Methanol
Acetonitrile
Tetrahydrofuran etc.
• The mixing ratio of the water (buffer solution) and organic solvent has the
greatest influence on separation.
• If a buffer solution is used, its pH value is an important separation
parameter.
Difference in Solute Retention Strengths for
Water and Water-Soluble Organic Solvents
Tightly packed network Loose network

H2O H2O CH3OH CH3OH

H2O H2O
H2O CH3OH
H2O H2O CH3OH CH3OH

CH3OH
Nonpolar solute CH3OH
Nonpolar solute

Nonpolar stationary phase

 HPLC Separation Modes

• Adsorption (liquid-solid) chromatography

• Partition (liquid-liquid) chromatography
• Normal phase partition chromatography
• Reversed phase partition chromatography
• Ion exchange chromatography
• Size exclusion chromatography
 Normal Phase (Partition) Chromatography

• Partition chromatography in which the stationary phase has a high

polarity (hydrophilic) and the mobile phase has a low polarity
(hydrophobic)
• Essentially based on the same separation mechanism as adsorption
chromatography in which the stationary phase has a hydrophilic
base, such as silica gel
 Normal Phase (Partition) Chromatography

• Partition chromatography in which the stationary phase has a high

polarity (hydrophilic) and the mobile phase has a low polarity
(hydrophobic)
• Essentially based on the same separation mechanism as adsorption
chromatography in which the stationary phase has a hydrophilic
base, such as silica gel
 Stationary Phase and Mobile Phase Used in
Normal Phase Mode
• Stationary Phase
• Silica gel: -Si-OH
• Cyano type: -Si-CH2CH2CH2CN
• Amino type: -Si-CH2CH2CH2NH2
• Diol type: -Si-CH2CH2CH2OCH(OH)-CH2OH
• Mobile Phase
• Basic solvents: Aliphatic hydrocarbons, aromatic hydrocarbons, etc.
• Additional solvents: Alcohols, ethers, etc.
Relationship between Hydrogen Bonding and
Retention Time in Normal Phase Mode
SiOH HO
Strong
SiOH
Weak
Very weak
OH

Steric hindrance
Relationship between Polarity of Eluent and
Retention Time in Reversed Phase Mode
Eluent: Methanol / Water

60/40

70/30

80/20
Relationship Between Eluent Polarity and
Retention Time in Normal Phase Mode
Eluent: Hexane/methanol

100/0

98/2

95/5
Comparison of Normal Phase and Reversed
Phase
• Normal Phase • Reversed Phase
• Effective for separation of • Wide range of applications
structural isomers • Effective for separation of
• Offers separation selectivity homologs
not available with reversed • Stationary phase has long
phase service life
• Stabilizes slowly and is • Stabilizes quickly
prone to fluctuations in • Eluents are inexpensive and
retention time easy to use
• Eluents are expensive
 Gradient System

• Isocratic system
• Constant eluent composition
• Gradient system
• Varying eluent composition
• HPGE (High Pressure Gradient)
• LPGE (Low Pressure Gradient)
 Qualitative Analysis
• Identification based on retention time
• Acquisition of spectra with detector
• UV spectra
• MS spectra
• Transfer to other analytical instruments after
preparative separation
 Quantitative Analysis

• Quantitation performed with peak area or height.

• Calibration curve created beforehand using a
standard.
• Absolute calibration curve method
• Internal standard method
• Standard addition method
Calibration Curve for Absolute Calibration
Curve Method
Area
Concentration
A1 Calibration curve
C1
A4

A2

Peak area
A3
C2

A2
A3
C3
A1

A4
C4 C1 C2 C3 C4
Concentration
 Calibration Curve for Internal Standard
Method
Concentration Area

Area for target substance / Area for internal standard

Target Internal
substance standard A1 AIS Calibration curve
C1 CIS A4 /AIS

A2 AIS A3 /AIS
C2 CIS

A2 /AIS
A3 AIS
C3 CIS
A1/AIS

A4 AIS
C4 CIS C1/CIS C2 /CIS C3 /CIS C4 /CIS
Concentration of target substance /
Concentration of internal standard
Advantages of Internal Standard Method (1)
• Not affected by inconsistencies in injection volume.

IS
X AX / AIS

10 µL
injected

Same area
ratio
IS
X
9 µL
injected
CX / CIS
Advantages of Internal Standard Method (2)

• Not affected by the pretreatment recovery rate.

IS
X

AX / AIS
100%
recovery
rate

Same area
ratio
IS
90% X
recovery
rate
CX / CIS
 Selection Criteria for Internal Standard
• It must have similar chemical properties to the target
substance.
• Its peak must appear relatively near that of the target
substance.
• It must not already be contained in the actual samples.
• Its peak must be completely separated from those of
other sample components.
• It must be chemically stable.
 Objectives of Pretreatment

• To improve the accuracy of quantitative values

• To improve sensitivity and selectivity
• To protect and prevent the deterioration of
columns and analytical instruments
• To simplify measurement operations and
procedures
• To stabilize target substances
 Substances That Must Not Be Injected into
the Column
• Insoluble substances (e.g., microscopic particles
and precipitation)
• Substances that are precipitated in the eluent
• Substances that irreversibly adsorb to the
packing material
• Substances that dissolve, or chemically react,
with the packing material