Basic Principles of HPLC
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Vinay PatelBasic Principles of HPLC
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Vinay PatelChromatography -- what does it mean?
To write with colors -- literally translated from its Greek roots
chroma and graphein ,
chromatography was first developed by the Russian botanist
Mikhail Tswett in 1903 as he produced a
colorful separation of plant pigments through a column of
calcium carbonate.
Mikhail Tswett
First Chromatographic Separation
Chromatography -- what does it mean?
Tswett stated: Chromatography is a
method in which the components of a
mixture are separated on an adsorbent
column in a flowing system. Chromatography: Method
Chromatograph: Machine
Chromatographer: Person
Analytical Nomenclature of IUPAC:
Chromatography is a physical method Chromatogram: Data
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).
Chromatographic Tree
Chromatography
Super Critical Fluid Liquid Chromatography
Gas Chromatography
Chromatography
Column
Planer Chromatography High Performance
Chromatography
Liquid Chromatography
High Performance Liquid Chromatograph
(HPLC)
Shimadzu Prominence
Chromatogram
Separation Technique
• Compounds are separated due to the molecules
moves at different rates in the column.
Separation Technique
• Due to different interaction between stationary
phase and different sample, the molecules move at
different rate, therefore separation can be done.
HPLC Phases
Normal Phase Mode
Reverse Phase Mode
Reverse Phase Ion Pairing Mode
Ion Exchange Mode
Chiral Separation Mode
Normal Phase
Stationary Phase is Polar
Mobile Phase is Non Polar
Stationary Phase for Normal Phase
Stationary Phase:
Base material for Stationary Phase
SiO2
O=Si=O Si OH
Polar Group
Silica Gel
Silica gel
Stationary Phase for Normal Phase
Si Si-OH Unmodified Silica (USP-L3)
Si Si-CH2-CH2-CH2-NH2 Amino (USP-L11)
Si Si-CH2-CH2-CH2-CN Cyano (USP-L10)
OH
Si Si-CH2-CH2-CH2-OCHCH2 Diol (USP-L)
OH
Mobile Phase
Primary solvents(non-polar)
– -Hydrocarbons (Pentane, Hexane, Heptane, Octane)
– -Aromatic Hydrocarbons (Benzene, Toluene, Xylene)
– -Methylene chloride
– -Chloroform
– -Carbon tetrachloride
Secondary solvents
– -Methyl-t-butyl ether (MTBE), Diethyl ether, THF, Dioxane, Pyridine,
Ethyl acetate, Acetonitrile, Acetone, 2-propaol, ethanol, methanol
– A primary solvent is used as mobile phase. Addition of secondary
solvents is to adjust retention time.
What is Interaction
HO
Si Si-OH
A
OH U
Minutes
Interaction
Reverse Phase
Normal Phase
Reverse Phase
Column for Reverse Phase
Si Si-CH2-CH2CH2-----CH3 (C18) ODS (USP-L1)
Si Si-CH2 -CH2 -CH2 -CH2-CH2-CH2-CH2-CH3 C8 (USP-L7)
Si Si-CH2-CH2-CH2-CH3 C4 (USP-L26)
CH3
Si Si-CH3 C1 (USP-L-13)
CH3
Column for Reverse Phase
Si Si-CH2-CH2CH2NH2 Amino
Si Si-CH2-CH2CH2CN Cyano
Si Si-CH2-CH2CH2- Phenyl
Mobile Phase for RP
• Water (buffer) + Organic Solvents
-When buffer is used, the concentration and
pH are important factors
-Methanol, Acetonitrile or THF are common
organic solvents for RP HPLC
4 Optimization of water (buffer) and organic
solvents ratio is very important
Interaction of RP
Interaction of RP
Interaction of RP
CH3
7 Carbons H 3C CH3
8 Carbons
6 Carbons
Increase of Solvent Polarity
Effect of pH
• The Effect of pH on the Retention of Acids and Bases in Reversed Phase HPLC
As acids lose a proton and become ionized (with
increasing pH), their retention decreases.
As bases gain a proton and become ionized
(with decreasing pH) their retention increases.
Effect of pH
• The Robustness of an HPLC Method Is Often Dependent on Mobile
Phase pH
• A change in mobile phase pH from 7.0 to 7.2 will cause the
retention of methyl-amphetamine to increase from 13.6
minutes to 14.8, an almost 9% increase.
Effect of pH
• An Example of the Effect of Changes in Mobile Phase pH on Resolution
• Column: C8, 4.6 x 250 mm
Mobile Phase:
27% CH 3 OH
73% Phosphate buffer
pH 2.5 and 2.6
Temperature: 50°C
• Flow Rate: 1.0 mL/min
• Sample:
1. p-anisidine
2. m-toluidine
3. 4-chloroaniline
4. 3-aminobenzonitrile
• Some separations are extremely sensitive to changes in mobile phase pH. This example shows how
resolution goes from an unacceptable 1.4 to an acceptable 3.0 when the mobile phase pH is decreased by
only 0.1.
HPLC Instrumentation
• 1. Pump
• 2. Column
• 3. Injector
• 4. Detector
• 5. Data Processor
HPLC configuration by
eluting mode
• Isocratic
• Binary
• Quaternary
Gradient VS Isocratic
Isocratic
Low Pressure Gradient
High Pressure Gradient
Pump
• Reciprocating Pump
Injector
• Manual Injector
• Auto Injector (Sampler)
• Advantage of Auto Injector (Sampler)
Detectors
• 1. UV-Vis /Photodiode Array Detector
• 2. Refractive Index Detector
• 3. Fluorescence Detector
• 4. Conductivity Detector
• 5. Electro Chemical Detector
• 5. Evaporate Light Scattering Detector
• 6. Mass Detector
Data Processor
• Software or Integrator or Recorder
Column Particle Physical
Characteristics
•Column Dimensions
• • Length and internal diameter of packing bed
•Particle Shape
• • Spherical or irregular
•Particle Size
• • The average particle diameter, typically 3-20µm
•Surface Area
• • Sum of particle outer surface and interior pore
surface, in m2/gram
Column Particle Physical
Characteristics
•Pore Size
• • Average size of pores or cavities in particles, ranging from
60-10,000Å
•Bonding Type
• • Monomeric - single-point attachment of bonded phase
molecule
• • Polymeric - multi-point attachment of bonded phase
molecule
•Carbon Load
• • Amount of bonded phase attached to base material,
expressed as %C
•Endcapping
• • “Capping” of exposed silanols with short hydrocarbon
chains after the primary bonding step
Column length
•Effect on chromatography
• • Short (30-50mm) - short run times, low backpressure
• • Long (250-300mm) - higher resolution, long run times
Column ID
• Narrow ( 2.1mm) - higher detector sensitivity, Sharp peak
• Popular ID – 4.6 mm
• Wide (10-22mm) - high sample loading
System Suitability
Repeatability,%RSD (CV)
Capacity factor, K’
Selectivity/Relative Retention Time
Theoretical Plates, N (Efficiency)
Resolution
Asymmetry/Tailing Factor
Repeatability, CV (%RSD)
X= (X1+X2……..Xn-1+Xn)/n
N= : Number of Analysis
X1..Xn : Retention time (or area or heights)
X : Average
C.V : Coefficient of Variation
Capacity Factor, K’
Selectivity/Relative Retention Time,
Theoretical Plate
Resolution
Tailing Factor,T
T=W0.05/2f
Thank You
