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Liquid Chromatography,
an introduction
Parts Provided by
Dr. David Lavorato, MDS SCIEX
Problem
How can one separate an analyte of interest from other analytes or potential interferences?
Example:
A food additive in a chocolate bar?
A pollutant in lake water or sediment?
Drugs (steroids or medication) in urine?
Principle
Components of a mixture are carried througha stationary phase by the flow of amobile phase
Separations are based on differences inmigration rates among the samplecomponents
Tswett
Discovered by Russianbotanist Mikhail Tswettat the turn of the century. He separated various plantpigments by passingsolutions through a glasscolumn containing calcium carbonate.
Chroma meaning “color” + graphein meaning “to write”
Tswett Device
Tswett Device
2 Dyes:
Red No 2 (banned)Yellow No 5
LC Column
Experiment 1
Take the strip of filter paper, and put an ink dot from a pen on the line
Keep the the vial vertical, and put thepaper in the glass with the dot in the bottom
Chromatography
Bonded Phase Chromatography
Stationary phase is chemically bonded to a backbone, such as siloxane
Si OH + SiCl
R
R
R’ Si O Si
R
R
R’
R = CH3
R’ = functional group, ex: CH3 chain, CN, phenol...
Stationary phase
Reverse Phase Mechanism
To elute those that really stick, you make the mobile phase more similar to those sticky molecules
SeparationS
ign
al
Time, t
Packedcolumn
A+B
detector
Solvent
t0
SeparationS
ign
al
Time, t
Packedcolumn
A+B
detector
Solvent
A
B
t0 t1
SeparationS
ign
al
Time, t
Packedcolumn
A+B
detector
A
B
Solvent
A
B
t0 t1 t2
SeparationS
ign
al
Time, t
Packedcolumn
A+B
detector
A
B
Solvent
A
B
t0 t1 t2
A
B
t3
A
SeparationS
ign
al
Time, t
Packedcolumn
A+B
detector
A
B
A
B
t0 t1 t2
A
B
t3
A
Solvent
B
t4
B
Advantages of HPLC
Fast: use small particles, short columns.
High Resolution: good efficiency, lots of column types
Wide range of compounds
Versatile: it is easier to dissolve a sample than to volatilise it for GC.
Non destructive (detector dependent)
Good for quantitiation
Variety of separation mechanisms
Adaptable from small to large scale
Operates at near ambient temperature
Basic HPLC Instrumentation
Instrumentation
SolventReservoir
Pump
Injector
Column Detector
Data System
Core HPLC
Detector System
Cost?
HPLC Pump $ 15 000 to 20 000
Injector $ 1 000 to 2 000
Autosampler $ 12 000 to 15 000
Detector UV $ 6 000 to 8 000
Diode Array $ 15 000 to 20 000
Mass Spectrometer $ 100 000 to 500 000
Data treatment package $ 5 000 to 15 000
HPLC Column $ 200 to 5 000
Solvents, Vials, peripherals $ 1 000 and up
MINIMUM: $ 30 000 (approx.)
Typical HPLC Stack (Agilent 1100)
Mobile Phase Properties
Dissolve Sample
High purity
Must allow interaction of sample with column
Cost, viscosity, toxicity, boiling point
Typical Solvents
hexane, methylene chloride, chloroform, methanol, acetonitrile (normal phase)
methanol, acetonitrile, water (reverse phase)
tetrahydrofuran, toluene, chloroform (gel permeation)
aqueous buffer (ion exchange)
Solvent Filtering
Pump Requirements
Chemically Inert (ex: Teflon, Ceramic, Sapphire, S 316*, PEEK*)
High Pressure (6000 psi)
Flow rate (1 l - 10 ml/min)
Pulse free or dampened
Flow control and flow reproducibility
Gradient, rapid solvent change
Types of Pumps
Reciprocating piston (PE LC 200)
Dual piston reciprocating piston(Shimadzu SL10AD, Agilent 1100)
Displacement pump (ABD 140, Harvard 22) – A motor pushing a very big syringe.
Pneumatic pumps - “pushing liquid out a hose by blowing into the other end”.
Simple Reciprocating Piston Pump
In this design, the solvent flow passes by a check-valve and into the pump chamber.
Notice that the second check-valve is closed
(Draw Stroke)
Simple Reciprocating Piston Pump
As the solvent is drawn into the cylinder, the check-valves momentarily float
(end of the draw stroke)
Simple Reciprocating Piston Pump
On the exhaust or flow stroke, solvent is pushed past the second check-valve generating flow
(end of the flow stroke)
Agilent Dual Reciprocating Piston Pump
Principle of Dual Reciprocating Pumps
Reciprocating Piston Pumps
Advantages
Small internal volume
High output pressure
External reservoir
Adaptable to gradient elution
Disadvantages
Seal and valve maintenance
Pulse noise (reduced with dual head)
Operating Modes
Isocratic: Separation of componentswith constant composition ofmobile phase
Gradient: Separation achievedthrough timed alteration of the mobile phase composition.
100%
20%
B
100%
20%
B
Low Pressure Mixing
Proportioning valve
Mixing chamber
Pump To column
• Mixing chambers are static or dynamic• Susceptible to air bubbles (degassing required)• Less expensive• Not suitable for very low flow rates
Detectors
Ideal: sensitive, reproducible, linear response, temperature stability, short response time, small internal volume, non-destructive, non-selective, and insensitive to changes in mobile phase (gradients).
Ultraviolet Detection ($ 5 000 minimum)
Refractive Index Detection ($ 5 000 )
Fluorescence Detection ($ 10 000)
Electrochemical Detection ($ 5 000 )
Mass Spectrometry ($ 80 000 minimum)
Factors Affecting Chromatography
Retention Parameters
Detectorresponse
time
Baseline
Injection
t0 Solvent “peak”
Peak height
Peak Area
Retention time tR(A)
Solute A Solute B
t’R(A)
t’R(B)
tm
k(A) = t’R(A) / tm
Selectivity Parameters ()
shoes perfume toys video
Mall A
dessertstereo
F
F M
M shoes shoes shoesperfume perfume perfume
Mall B
stereoshoes shoes shoesperfume stereo
Mall C
Poor selectivity
Equal affinity for all
Equal affinity for FNo affinity for M
Partial selectivity
Different affinity for all
Good selectivity
Efficiency versus Selectivity
Reference
Increased EfficiencyUnchanged Selectivity
Increased SelectivityUnchanged Efficiency
Capacity Factor (k’)
Relates to the time it takes a compound to run through the column
As time , k’
Ex
Has a higher capacity factor than
Resolution
timeSolute A Solute B
t
WA WB
RS = t
1/2 (WA + WB)
RS > 1.5
Resolution
Capacity: Optimize k (2-10) by adjusting solvent polarity
Selectivity: Maximize by changing solvent system or packing material
Efficiency: Maximize by decreasing flow rate, using a longer column, usinga higher quality column, using a column with a smaller particle size.
Most desirable resolution: RS > 1.5
RS = k
1 + k
- 1
4
Capacity Selectivity Efficiency
Resolution
RS=0.6
RS=1.0
RS=1.5
Other types of Chromatography
Column Selection
Sample
MW > 1500 MW < 1500
Water solubleOrganic Soluble Water solubleOrganic Soluble
GPC GFC
Hexane Soluble Methanol Soluble Non-electrolytes Electrolytes
LSC BPC-NP BPC-RP IEC IPC
Adsorption Chromatography
+-
-
-
-
-
-
-
-
+
+
+
+
+
+
+
Flow+
-+
+--
(Polar Stationnary Phase)
Solute interacts directly by adsorption with the stationary phase.
Solute and solvent compete for adsorption sites.
Ex. stationary phases: Silica, Alumina, Charcoal, Cellulose
Non-polar molecules interact weakly with polar adsorbents.Polar molecules interact strongly with polar adsorbents.
Column Selection
Sample
MW > 1500 MW < 1500
Water solubleOrganic Soluble Water solubleOrganic Soluble
GPC GFC
Hexane Soluble Methanol Soluble Non-electrolytes Electrolytes
LSC BPC-NP BPC-RP IEC IPC
Partition Chromatography
Two types: liquid-liquid chromatography
bonded-phase chromatography
Separation is mainly dependent upon the relative solubility of the compounds (Solutes) in the liquid attached to the support (Stationary phase) and in the liquid flowing through the stationary phase (Mobile phase)
If the affinity of the solute for the stationary phase is high, the solute does not elute.
Ex: Fat dissolves in oil but not in water. If oil is the stationary phase, a fatty molecule will only elute with an oily mobile phase.
Therefore, one goes from a weak solvent (water) to a strong solvent (oil).
Silica Support
Macroporous irregular
50 - 100 m
Pellicularspherical
35 - 45 m
Microporous
Impervious glass
Poroussilica
1.5 -10 m
Preparative
high capacityeasily packedinexpensive
low efficiency
Guard
low capacityeasily packed
expensiveefficient
Analytical
high capacitydifficult to pack
expensivevery efficient
Typically used columns
Stationary phases: C 18, C 8
Partical sizes: 3, 5 m
Internal diameter: 1 mm (average flow rate 50 l/min)
2.1 mm (average flow rate 200 l/min)
4.6 mm (average flow rate 1 ml/min)
Column length: 50 to 250 mm
Mobile phases: Acetonitrile/Water
Methanol/Water
Column Selection
Sample
MW > 1500 MW < 1500
Water solubleOrganic Soluble Water solubleOrganic Soluble
GPC GFC
Hexane Soluble Methanol Soluble Non-electrolytes Electrolytes
LSC BPC-NP BPC-RP IEC IPC
Size Exclusion Chromatography
Smallest molecules penetrate the smallest pores, retained longest
Larger molecules are excluded, so they elute first
Separation on basis of MW, volume in solution, solvent, temperature
Packing is a cross-linked polystyrene (GPC), or a silica based gel (GFC).
Characteristics of SEC
Handles high M.W. and has short run times
Predictable elution order
Simple method development
Low resolution
Solubility of compounds can be problematic
LC throughout the world
Market: >20 companies sell complete systems
>50 companies manufacture/sell HPLC columns
Numerous specialty column manufacturers (>100)
Even more suppliers of chemicals and accessories
> $ 1 000 000 000
Ratio of LC systems to Mass Spectrometers: 10 to 1
Ancillary Equipment
Column heaters: maintain the column at a constant temperature, leads to more reproducible retention times, less peak tailing, and faster desorption.
Autosampler: used to automate the injection of multiple samples.
Fraction collectors: used frequently with preparative LC columns to collect substances
after they have been separated and identified by a detector.
Automated sample preparation units: useful when processing a large number of samples. The units can perform extractions, preconcentrations, derivatizations, sample dilutions, standard additions, sample clean up.
References
D. A. Skoog, Principles of Instrumental Analysis, third edition, Saunders Publishing (1985)
H. M. McNair, L. N. Polite, HPLC, ACS Publication (1997)
L. R. Snyder, J. J. Kirkland, Introduction to modern liquid chromatography, second edition, John Wiley and Sons, (1979)
L. R. Snyder, J. J. Kirkland, J. L. Glach, Practical HPLC method development, second edition, John Wiley and Sons, (1997)