An introduction to HPLC : 8/19/2010 1 An introduction to HPLC Dr.Mrs. A. S. Tambe CHROMATOGRAPHY : 8/19/2010 2 CHROMATOGRAPHY Chromatography is a process of separation in which the components to be separated are distributed between two phases; one of which is a stationary phase and the other is mobile phase.
If the mobile phase is gas it is gas liquid chromatography if it is liquid it is liquid liquid chromatography, and if the mobile phase is super critical fluid, it is super critical fluid chromatography.
Stationary liquid phase may be distributed on a solid support. How does chromatography work? : 8/19/2010 3 How does chromatography work? The distance between the bands increases with the length traveled but the width of each band increases only with the square root of this length, therefore these bands will be completely separated at some point. The farther they migrate, the better will be the separation. Theory of Chromatography : 8/19/2010 4 Theory of Chromatography The Height Equivalent to a Theoretical Plate (HETP) Characteristic Features of the Chromatogram : 8/19/2010 5 Characteristic Features of the Chromatogram Linear velocity=L/t0
Capacity Factor = k
k = t’R/t0 = tR-t0/t0
k = K*Vs/Vm
K is distribution coefficient
α = k2/k1
α = tR2-t0/tR1-t0
α = K2/K1
α is the separation factor
α is the selectivity Properties of the column : 8/19/2010 6 Properties of the column Selectivity: α
Efficiency: Plate counts, N=L/H=L2/σ2
Resolution: Separation between the two peaks
Can be calculated from chromatogram.
Resolution increases with the square root of the distance traveled by the bands.
Peak Asymmetry: Peak tailing Slide 7: 8/19/2010 7 Peak Asymmetry : 8/19/2010 8 Peak Asymmetry Band Broadening Parameters : 8/19/2010 9 Band Broadening Parameters 1) Eddy diffusion and flow distribution component
2) Longitudinal diffusion component
3) Mass Transfer component
4) The resultant van Deemter curve
H=A+B/u+C*u Resolution : 8/19/2010 10 Resolution Rs= ¼ ((α – 1) / α) (k / (1+k)) (N)1/2
Efficiency High Performance Liquid Chromatography : 8/19/2010 11 High Performance Liquid Chromatography Separation mechanisms used in HPLC : 8/19/2010 12 Separation mechanisms used in HPLC Normal phase Chromatography
Reversed Phase Chromatography
Hydrophilic Interaction Chromatography (HILIC)
Hydrophobic Interaction Chromatography (HIC)
Ion Exchange Chromatography
Size Exclusion Chromatography (Gel Permeation Chromatography) HILIC Chromatography : 8/19/2010 13 HILIC Chromatography Ion Exchange Chromatography : 8/19/2010 14 Ion Exchange Chromatography Slide 15: 8/19/2010 15 Ion Exchange Separation of Biopolymers : 8/19/2010 16 Ion Exchange Separation of Biopolymers Fast Protein Liquid Chromatography (FPLC)
High Performance Separation of Proteins
Strong Cation Exchanger – sulfo propyl SP pKa = 2
Weak Cation Exchanger – carboxymethyl CM pKa=5
Weak Anion Exchanger – diethylaminoethyl DEAE pKa = 9
Strong Anion Exchanger – quaternary methyl groups QMA
Ion exchangers for proteins have a pore size 30-100nm
The ion exchange capacity is smaller for larger pore size Ion Exchange Separation of Biopolymers Protein Binding Capacity : 8/19/2010 17 Ion Exchange Separation of Biopolymers Protein Binding Capacity Slide 18: 8/19/2010 18 Slide 19: 8/19/2010 19 Normal Phase Chromatography and Reversed Phase Chromatography : 8/19/2010 20 Normal Phase Chromatography and Reversed Phase Chromatography Norrmal phase: Stationary phase is polar and the mobile phase is nonpolar. The nonpolar analytes elute faster than the polar.Reversed phase: stationary phase is nonpolar and the mobile phase is polar. The polar analytes elute faster than the less polar.
The separation is achieved by varying the composition of the mobile phase in a controlled way
Isocratic Elution: Constant Composition of the mobile phase
Gradient Elution: Composition of the mobile phase is changed continuously
Buffers are used to control the degree of ionization of the analyte and the tailing of responses and the reproducibility of the retention.
Nonvolatile buffers can not be used in LC/MS. e.g. Phosphate buffers
Volatile buffers are preferred e.g. ammonium acetate Properties of Silica : 8/19/2010 21 Properties of Silica Silica consists of silicon atoms bridged three-dimensionally by oxygen atoms
The lattice is saturated at the surface with OH groups.
Silica is produced by complete hydrolysis sodium silicate or polycondensation of emulsified polyethoxysiloxane followed by dehydration.
The properties of silica depend on the reaction conditions
Metal content of the starting material determines the concentration of acidic silanols Slide 22: 8/19/2010 22 Functional groups in Chemically modified silicas : 8/19/2010 23 Functional groups in Chemically modified silicas Stationary Phases : 8/19/2010 24 Stationary Phases Chemically modified silica stationary phase
Chemical modification determines the polarity of the column
The most commonly used silica column is C18 or ODS
Spherical silica particles give higher efficiencies
Free silanol sites are end capped with trimethylchlorisilane
This increases separation of basic compounds and increases column life
Very pure silica- metal contaminants are removed
Hybrid columns A bridged organo silica coating Particle Diameter : 8/19/2010 25 Particle Diameter The smaller the diameter, higher is the efficiency 10µm - 5µm - 3µm
Backpressure increases with decreasing particle size
<2µm - UPLC Columns based on non silica support : 8/19/2010 26 Columns based on non silica support Problems with silica
It dissolves at pH above 8 at elevated temperatures
Si-O-Si linkage hydrolyzes below pH 2
Useful pH range is 2.5-7.5
Crosslinked polymeric columns – lower efficiency – limited pressure and flow rates
The pH range is 1- 11
Zirconium columns – stable from pH 1-12 Zirconium Bonded Phase Columns : 8/19/2010 27 Zirconium Bonded Phase Columns Can be used at a wide pH range and show anionic, cationic and chelating attraction for amines
Used with MS : polymeric polybutadiene or C18 groups bound by direct C to Zr linkage prepared using diazo compounds
C – Zr supports are not susceptible to the acid hydrolysis and Zr doesn’t dissolve at higher pH.
Similar to ODS but different when separating charged molecules
Chelators (EDTPA) are used to remove irreversibly retained cations and amines. Polymer Bonded Phase Columns : 8/19/2010 28 Polymer Bonded Phase Columns pH 1 to 13
True non polar resolving characteristics
Pressure and temperature limits
Styrene-divinyl benzene copolymerization
The amount of divinyl benzene added determines the degree of cross-linking and the pore structure Polymer Bonded Phase Columns : 8/19/2010 29 Polymer Bonded Phase Columns Detectors used in HPLC : 8/19/2010 30 Detectors used in HPLC Ultraviolet Detector: Fixed wavelength
Photo Diode Array Detector
Refractive Index Detector
Electron Capture Detector
Mass Spectrometer Detector
Evaporative Light Scattering Detector (ELSD)
Corona Charged Aerosol Detector Ultraviolet Detector : 8/19/2010 31 Ultraviolet Detector Based on the absorption of ultraviolet light
Specific detector; not universal
Analyte should have chromophores
High sensitivity; 1 ng can be detected
Wide linear dynamic range (104)
Solute property is measured, can be used for gradient elution Refractive Index Detector : 8/19/2010 32 Refractive Index Detector Continuously monitors the difference in refractive index between pure mobile phase and the mobile phase and sample.
Bulk property detector; universal
Can not be used with gradient elution as it is sensitive to changes in the mobile phase
It takes long time for stabilization
The change in the room temperature also gives noise Electron Capture Detector : 8/19/2010 33 Electron Capture Detector Compounds are oxidized or reduced at the ECD cell and the electric current is measured during the redox reaction
The resultant current is directly proportional to the concentration of the analyte.
Amperometric detection and caulometric detection
AD: highly sensitive, easy maintenance
CD: For preparatory electrolysis
ECD allows specific compound detection by choosing the appropriate analytical conditions for each analyte
Highly sensitive; femtogram quantity can be detected Evaporative Light Scattering Detector (ELSD) : 8/19/2010 34 Evaporative Light Scattering Detector (ELSD) After nebulization and evaporation of the mobile phase, the light scattered from the solid solute particles is measured.
Useful for online quantification of native lipids (without derivatization) Corona Charged Aerosol Detector : 8/19/2010 35 Corona Charged Aerosol Detector Properties of the Detector : 8/19/2010 36 Properties of the Detector Detector Selectivity and Sensitivity : 8/19/2010 37 Detector Selectivity and Sensitivity The selectivity of a detector is it’s ability to determine an analyte of interest without interference from other materials (sample matrix)
LOD is the amount of analyte which produces a signal greater than the SD of the noise by a defined factor (3 or 5)
The sensitivity is the degree of response obtained from the detector.