1. HPLC High Pressure Liquid Chromatography High Performance Liquid Chromatography

2. 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

3. 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

4. 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

5. 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

6. Diameters: 150-200 m Earlier LC carried out in glass columns
Diameters: 1-5 cm
Lengths: cm
Size of solid stationary phase
Diameters: 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

7. 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.

10. 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)

12. 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

13. Schematic diagram of HPLC

14. 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

15. 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).

17. 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

18. 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

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

20. 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

21. 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

22. 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

23. 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

24. 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

26. 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

27. 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

28. 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

29. 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

30. 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