Introduction to HPLC

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)