HPLC High Pressure Liquid Chromatography High Performance Liquid Chromatography

Advantages of LC vs GC Separation of compounds that are soluble in a liquid phase. ie. biological compounds, synthetic or natural polymers and inorganic compounds Liquid mobile phase Lower temperatures - Compounds that may be thermally labile Retention of solutes depend on their interaction with both mobile and stationery phase Most LC detectors are non-destructive Disadvantage of LC vs GC Greater peak or band-broadening

Traditional LC vs HPLC Liquid chromatography (LC) Use large, non-rigid support material Particles size : > 150 μm, column size: 10 ~ 50 mm, column length L: 50 ~ 500 cm, flow rate F: < 1mL/min Gravity. Large height, small pressure Poor system efficiencies and large plate heights High-performance liquid chromatography (HPLC) Use small, uniform, rigid support material Particle size dia < 40 μm, usually 3-10 μm Good system efficiencies and small plate heights, narrow peaks, shorter separation times

What is HPLC ? Most widely used analytical separation technique Martin and Synge (1941) - liquid-liquid partition chromatography Got Nobel Prize in chemistry (1952) Most widely used analytical separation technique Utilizes a liquid mobile phase to separate components Uses high pressure to push solvent through the column Popularity because of: sensitivity ready adaptability for quantitative analysis suitability for separating non- volatile species or thermally fragile ones widespread applicability in industry, to many fields of science, and to the public Liquid- liquid extraction

HPLC is…. Commonly used in analysis of Biological compounds - Amino acids, proteins, nucleic acids, hydrocarbons, carbohydrates, pigments Pharmaceuticals – antibiotics, steriods Low- or Non-volatile environmental compounds- Pesticides A variety of inorganic substances HPLC can be widely applied to substances that are of prime interest to industry, to many fields of science, and to the public

Diameters: 150-200 m Earlier LC carried out in glass columns Diameters: 1-5 cm Lengths: 50-500 cm Size of solid stationary phase Diameters: 150-200 m Flow rates still low! Separation times long! Decrease of column material particle size increased in column efficiency Diameters 3-10 m -Early LC carried out in glass columns -to assure reasonable flow rates, diameters… -BUT were still low (a few tenths of a mL/min) separation times long, taking several hours -attempts to speed up the classic procedure by application of vacuum or by pumping were not effective, because increases in flow rates acted to increase plate heights beyond the minimum -early on, scientists realized that major increases in column efficiency could be brought about by decreasing the particle size of packings -1960s particle sizes 3-10 m----required sophisticated instruments

Types of chromatography Normal Phase: Separation of polar analytes by partitioning onto a polar, bonded stationary phase. Reversed Phase: Separation of non-polar analytes by partitioning onto a non-polar, bonded stationary phase. Adsorption: In Between Normal and Reversed. Separation of moderately polar analytes using adsorption onto a pure stationary phase (e.g. alumina or silica) Ion Chromatography: Separation of organic and inorganic ions by their partitioning onto ionic stationary phases bonded to a solid support. Size Exclusion Chromatography: Separation of based in the paths they take through a “maze” of tunnels in the stationary phase.

Selection of LC Modes: Methods can be chosen based on solubility and molecular mass. In most cases for non-ionic small molecules ( < 2000) reversed phase methods are suitable. Techniques toward the bottom are best suited for species of high molecular mass ( >2000)

Parts of HPLC 1. Solvent treatment system 2. Pumping system Solvent reservoir Pre column filter Inlet solvent filter Inline solvent filter Pump 1. Solvent treatment system 2. Pumping system 3. Sample injection system 4. Column 5. Detector Sample injection valve Guard column Column Back pressure regulator Waste reservoir Detector Recorder

Schematic diagram of HPLC

Mobile phase reservoir Reservoirs (> 500mL) Degassing: Removal of dissolved gas - band spreading and interfere detection Sparging: fine bubble of gas Vacuum pumping, distillation, heating Dust removal: Interference with detection, column clogging, damage pumping system Millipore filter under vacuum Isocratic elution: Constant composition Gradient elution: Different solvent systems during elution, continuous change or step wise, solvent proportion valve

Isocratic vs Gradient elution Isocratic elution has a constant mobile phase composition Can often use one pump Simpler, no mixing chamber required Limited flexibility, Not used much in research Mostly used in process chemistry or routine analysis. Gradient elution has a varying mobile phase composition Uses multiple pumps (2-4) whose output is mixed together Changing mobile phase components changes the polarity index can be used to subsequently elute compounds that were previously “stuck” on the column Some additional wear on the stationary phase Column has to re-equiluibrate to original conditions after each run (takes additional time).

High-pressure pump Pumping systems High Pressure (6 k psi) Pulse-free, constant flow (0.1 ~ 10 mL/min.) Reproducibility (0.5%) Resistant to corrosion Displacement pump (Screw-driven syringe pump) Pulse free Small capacity (250 mL) No gradient elution Reciprocating pump Most widely used. Small internal volume (35 ~ 400 μL), High pressure (105 psi) Gradient elution, constant flow. Need pulse damper

Sample Injection system Sample size: 0.5 ~ 500 μL No interference with the pressure 6-PORT Rotary Valve is the standard manual injector - Sample loop, 1 ~ 100 μL Reproducibility: 0.1%, P < 7000 psi Auto sampler: Inject continuously variable volume 1 μL – 1 mL Controlled temperature environment for derivatization reaction A sampling loop for HPLC

LOAD (the sample loop) Inject (move the sample loop into the mobile phase flow)

Column ($200 ~ $1000) Stainless steel tubing to withstand high pressure Heavy-wall glass or PEEK tubing for low pressure (< 600 psi) Analytical column: Straight, L (5 ~ 25 cm), dc(3 ~ 5 mm), dp(3 ~ 5 μm) N (40 k ~ 70 k plates/m) Microcolumn: L (3 ~ 7.5 cm), dc (1 ~ 5 mm), dp: 3 ~ 5 μm, N: ~ 100k plates/m, high speed and minimum solvent consumption Guard column: Remove particulate matter and contamination protect analytical column. Have similar packing as analytical column Temperature control: < 150 °C± 0.1 °C

Bonded Phases C-2 Ethyl Silyl -Si-CH2-CH3 C-8 Octyl Silyl -Si-(CH2)7-CH3 C-18 Octadecyl Silyl -Si-(CH2)17-CH3 CN Cyanopropyl Silyl -Si-(CH2)3-CN

Column packing (Stationery phase) Solid support : Silica, alumina, polystyrene divinylbenzene. Synthetic or an ion-exchange resin Pellicular particle:(dp: 5 μm) Original, spherical, non-porous beads For proteins and large biomolecules separation Porous particle: (dp: 3 ~ 10 μm) Commonly used. Narrow size distribution, Porous micro-particle coated with thin organic films Bonded Phases Functional groups firmly linked (chemically bound) to the solid support

Stationery phase and functional groups Polar (“Normal” Phase): Silica, alumina Cyano, amino or diol terminations on the bonded phase Non-Polar (“Reversed Phase”) C18 to C8 terminations on the bonded phase Phenyl and cyano terminations on the bonded phase Packed particles in a column require: Frits at the ends of the column Filtering of samples to prevent clogging with debris High pressure pumps and check-valves Often a “Guard Column” to protect the analytical column

Normal and Reverse phases Stationary phase: liquid, immiscible with the mobile phase. Non-ionic polar compounds (Mr < 3k), bonded (C8 or C18) Normal-phase partition chromatography: Highly polar stationary phase - triethylene glycol or water Non-polar solvent (mobile phase) - hexane or i-propyl ether Compound polarity ↓ or solvent polarity ↑ - Retention time ↓ Reversed-phase partition chromatography: common Non-polar stationary phase: Hydrocarbon Polar mobile phase: Water, ethanol, acetonitrile or tetrahydrofuran Compound polarity ↓ or solvent polarity ↑ Retention time ↑ Mobile phase polarity: – Water > acetonitrile > methanol > ethanol > tetrahydrofuran > propanol > cyclohexane > hexane

Detectors Low dead volume: ↓ extra-column band broadening Small and compatible with liquid flow Not highly sensitive and universal detector as GC Types: Bulk-property detector and solute-property detector

Absorption detectors UV-Visible Most widely used Z-shape, flow-through cell (V, 1 ~ 10 μL and b 2 ~ 10 mm) Photometer: Hg 254 nm and 280 nm line for organic, D2 or W filament + interference filter Spectrophotometer: more versatile Infra Red Filter instrument or FTIR Similar cell (V, 1.5 ~ 10 μL and b, 0.2 ~ 1.0 mm) Limit: No suitable solvent, special optics Fluorescence: Hg or Xe lamp Fluorometer and spectrofluorometer Fluorescing species or fluorescent derivatives

Refractive index detectors (RI): General, unaffected by flow rate Disadvantages: Limited sensitivity (1 ng/μL) Highly temperature dependent (0.001°C) No gradient elution Evaporative light scattering detector: New, laser beam Nebulizer - fine mist in N2 - solvent evaporation in drift tube - fine particles - scattered radiation Advantages : Same for all nonvolatile solutes, More sensitive than RI, 0.2 ng/μL Disadvantage: Mobile phase must be volatile

Electrochemical detectors: Amperometry, voltammetry, coulometry and conductormetry Advantages Simplicity High sensitivity, Convenience Wide-spreading application Thin-layer flow cell of Teflon : 50 μm thick, 1 ~ 5 μL volume Indicators: E: Pt, Au, C Multi-electrode: Simultaneous detection or sample purity indication

Advantages to HPLC Higher resolution and speed of analysis HPLC columns can be reused without repacking or regeneration Greater reproducibility due to close control of the parameters affecting the efficiency of separation Easy automation of instrument operation and data analysis Adaptability to large-scale, preparative procedures Two major advances: stationary supports with very small particle sizes and large surface areas appliance of high pressure to solvent flow