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

through
a stationary phase by the flow of a
mobile phase

Separations are based on differences in

migration rates among the sample
components
Tswett

Discovered by Russian
botanist Mikhail Tswett
at the turn of the century.
He separated various plant
pigments by passing
solutions through a glass
column 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 the

paper in the glass with the dot in
the bottom
Chromatography
Bonded Phase Chromatography

Stationary phase is chemically bonded to a backbone, such as siloxane

R R

Si OH + Cl Si 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
Separation
Solvent

A+B

Packed
column

detector

t0
Signal

Time, t
Separation
Solvent

A+B

Packed A
column

detector

t0 t1
Signal

Time, t
Separation
Solvent

A+B

Packed A
B
column

detector

t0 t1 t2
Signal

Time, t
Separation
Solvent

A+B

Packed A
B
column

A
B

detector A

t0 t1 t2 t3
Signal

Time, t
Separation
Solvent

A+B

Packed A
B
column

A
B

detector A B

t0 t1 t2 t3 t4
Signal

A B

Time, t
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

Detector System

Solvent
Reservoir

Column Detector

Pump

Data System

Injector

Core HPLC
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 µ 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 Disadvantages

Small internal volume Seal and valve maintenance

High output pressure Pulse noise (reduced with dual head)
External reservoir
Adaptable to gradient elution
Operating Modes

100%
Isocratic: Separation of
components
with constant composition of
mobile phase B

20%

100%

Gradient: Separation achieved

through timed alteration of the B
mobile phase composition.

20%
Low Pressure Mixing

Proportioning Mixing Pump To column

valve chamber

• 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

t’R(B)

t’R(A)
k(A) = t’R(A) / tm
Retention time tR(A)

tm
Detector
response

Peak Peak Area

height
Injection

Baseline

t0 Solvent Solute A Solute B time

“peak”
 Selectivity Parameters (α )

shoes stereo perfume toys video dessert Equal affinity for all

Mall A
Poor selectivity

shoes perfume shoes perfume shoes perfume Equal affinity for F

F M
No affinity for M
Mall B
F M Partial selectivity

shoes perfume shoes stereo shoes stereo Different affinity for all
Mall C
Good selectivity
Efficiency versus Selectivity

Reference

Increased Efficiency
Unchanged Selectivity

Increased Selectivity
Unchanged 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

∆t
RS =
1/2 (WA + WB)

RS > 1.5

WA WB

Solute A Solute B time

Resolution

k α -1 √ Ν
RS =
1+k α 4

Capacity Selectivity Efficiency

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, using

a higher quality column, using a column with a smaller particle size.

Most desirable resolution: RS > 1.5

Resolution

RS=0.6

RS=1.0

RS=1.5
Other types of Chromatography
 Column Selection

Sample
MW > 1500 MW < 1500

Organic Soluble Water soluble Organic Soluble Water soluble

GPC GFC

Hexane Soluble Methanol Soluble Non-electrolytes Electrolytes

LSC BPC-NP BPC-RP IEC IPC

Adsorption Chromatography

Solute interacts directly by adsorption with

the stationary phase.
++-- Flow
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. -
- +
+ -

(Polar Stationnary Phase)

 Column Selection

Sample
MW > 1500 MW < 1500

Organic Soluble Water soluble Organic Soluble Water 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 Pellicular Microporous

irregular spherical

Impervious
glass

Porous
silica

50 - 100 µ m 35 - 45 µ m 1.5 -10 µ m

Preparative Guard Analytical

high capacity low capacity high capacity

easily packed easily packed difficult to pack
inexpensive expensive expensive
low efficiency efficient very efficient
Typically used columns

Stationary phases: C 18, C 8

Partical sizes: 3, 5 µ m
Internal diameter: 1 mm (average flow rate 50 µ l/min)
2.1 mm (average flow rate 200 µ 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

Organic Soluble Water soluble Organic Soluble Water 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)