An introduction to HPLC


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Dear Madam, I found your presentation on HPLC very useful and would like to use it as a teaching tool if you would allow complete access. Thanks Mrs. Rana Zainuddin

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An introduction to HPLC : 

8/19/2010 1 An introduction to HPLC Dr.Mrs. A. S. Tambe


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 RS= ΔL/0.5(W1+W2) Can be calculated from chromatogram. Resolution increases with the square root of the distance traveled by the bands. Peak Asymmetry: Peak tailing

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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 Selectivity Capacity Factor 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

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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

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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

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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 Cross-linked Polystyrene 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 Fluorescence 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.