Pos Charge: Quaternary amines [(C4H9)4N+]. The Basic HPLC Apparatus.
Undergraduate Instrumental Analysis, 6th ed. Robinson, Skelly-Frame, & Frame.
LC III: HPLC
What is HPLC?
Originally referred to as High-Pressure Liquid Chromatography
Now more commonly called High Performance Liquid Chromatography
In general: The instrument controlled version of LC, utilizing very small particles, small column diameters, and very high fluid pressures.
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Why is HPLC Needed? The highest resolution separations require complete mobile/stationary equilibrium To reach complete diffusion equilibrium using liquid mobile phase requires:
Extremely small support particles Extremely small stationary phase layers Very tight packing of these particles Small column diameters
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Forcing viscous liquid through these columns would require very high pressure
Stationary Phase
Support Particles { { { {
Silica: monodisperse, 2-10 µm, porous, pH=2-8 Polymers: larger, subject to swelling Pellicular: solid core, porous coat, large particle Zirconia: pH 1-14, thermostable, ↑$
Stationary Phases {
Normal Phase: Hydrophillic
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Water, Amino, Cyano, Diol
Reverse Phase: Hydrophobic
C18, C8, C2, Phenyl
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Ion Suppression/Ion Pairing
Ionized/charged molecules don’t play well with partition chromatography Ion Suppression {
Change in pH to shift dissociation equilibrium
Ion Paring Chromatography (IPC) { { { { {
Addition of a counter-ion to sample Counter-ions have large, non-polar substituents Form tightly bound “ion pairs” with analytes Neg Charge: Alkyl sulfonic acids [C12H25SO3-] Pos Charge: Quaternary amines [(C4H9)4N+]
The Basic HPLC Apparatus
Undergraduate Instrumental Analysis, 6th ed. Robinson, Skelly-Frame, & Frame. 2005.
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Mobile Phase Handling (1&2)
Filtering { {
Degassing {
{
Particlulates will clog your column Most HPLCs have inlet filters Dissolved gasses can come out of solution during pressure changes, causing bubbles We all know that bubbles = bad
Sparging {
Driving out dissolved gasses by forcing in a poorly soluble inert gas (He)
Mobile Phase Mixing (3)
Usually 3-4 pure mobile phases are stored in separate reservoirs
Isocratic mixtures can be generated
Multi-eluent gradients can be generated just as easily – Elution Programs { {
Precisely controlled Highly reproducible gradient
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The Pump (4)
Reciprocating Piston {
{
Single Piston
Oscillations in flow rate
Requires a pulse dampener
Dual/Triple Piston
No need for pulse dampener
Must generate 500+ psi { {
Typically 5000-6000 psi Newer pumps can generate 15,000+ psi – UPLC Undergraduate Instrumental Analysis, 6th ed. Robinson, Skelly-Frame, & Frame. 2005.
Fill / Drain Valve (5)
Lots of tubing and associated valves between reservoirs and column
This valve allows priming of an empty system with new mobile phase
Air bubbles in the system can be removed
The fluid transfer lines can also be cleaned without having to pump against the column
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The Sample Loop Injector (6)
Must introduce sample into column without introducing air or disrupting fluid flow
Two-position rotary injection valve
Load position: { {
Sample is injected into sample loop (50-100µL) Pumping lines go directly to column not sample
Inject position: { {
Pump flow is redirected through sample loop Sample loop now outputs to column flow lines
Sample Loop Injector
Undergraduate Instrumental Analysis, 6th ed. Robinson, Skelly-Frame, & Frame. 2005.
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The Column(s) (7) The Guard Column
Short (5.0 cm) and Cheap Often uses larger pellicular particles (~40µm) Stationary phase should be same/similar When clogged or contaminated, it is replaced Saves having to replace analytical column ($$$)
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The Analytical Column
The workhorse Temperature control - ↑Temp, ↓Viscosity
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Not subject to ambient temperature variations Must pre-heat mobile phase
Column Diameter
Always given as Internal Diameter (ID) ID > 10 mm {
ID ~ 5 mm (4.6 mm is common) {
Analytical columns, short, fast and accurate
ID ~1-2 mm {
Preparative, lower resolution, larger sample
For small sample high sensitivity detection
ID < 0.5 mm {
Capillary columns, almost always used w/ MS
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The Detector (8)
Ideal Detector Properties: { { { { { { { {
High Sensitivity (≤ 0.1 µg/mL minimum) Universality or predictable specificity Large linear response range (>102) Low dead volume Non-Destructive Insensitive to temperature & mobile phase Continuous operation Reliable and easy to use
No single detector fits all these criteria
HPLC Detectors
UV/Vis
Refractive index
Fluorescence
Evaporative light scattering
Electrochemical
Conductivity
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UV/Visible Detectors
Properties: { { { {
Somewhat compound specific Non-destructive Concentration sensitive over wide range Compatible with most mobile phases, salts, buffers, and elution types
Three main forms { { {
Fixed wavelength Variable wavelength Diode array
Refractive Index (RI)
Properties: {
Universal
{
Non-destructive
{
Concentration dependent, not very sensitive
{
Can only be used with isocratic elutions
{
Requires temperature control
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Fluorescence
Properties: {
Compound specific
{
Non-destructive
{
Concentration dependent
{
Sensitivity differs by compound
{
High signal-to-noise ratio
{
Compatible with most mobile phases, salts, buffers, and elution types
Evaporative Light Scattering
Properties: {
Universal
{
Destructive
{
Mass-flow detector, very sensitive
{
Uniform response to different compounds
{
High signal-to-noise ratio
{
Compatible with isocratic and gradient elution but not buffers/salts
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ELS Diagram
Undergraduate Instrumental Analysis, 6th ed. Robinson, Skelly-Frame, & Frame. 2005.
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