Rachel Beck, MS, DFTCB

Basics of Liquid Chromatography

Overview Introduction to Liquid chromatography

Theory

Practical Applications

Types of column

Advantages/Disadvantages

What is Liquid Chromatography?

Liquid chromatography (LC) is a separation

technique.

LC Classifications

Liquid-Liquid

Mobile phase – liquid

Stationary phase – liquid

Example: Liquid/Liquid extraction

Liquid-solid

Mobile phase – liquid

Stationary phase – solid

Example: High Pressure Liquid Chromatography

What is HPLC? A separation technique in

which a liquid sample is passed over a solid adsorbent material packed into a column (stationary phase) using a flow of liquid solvent (mobile phase) at high pressures.

http://en.wikipedia.org/wiki/High-performance_liquid_chromatography

How does LC work? A sample mixture is injected

onto the column. Each component partitions between the stationary phase (column packing material) and the mobile phase (solvent system). Molecules that have a greater affinity for the stationary phase take longer to reach the detector.

LC Schematic

To Mass Analyzer

Sample Mixture + Mobile Phase

Injection Time 2.0 min Time 3.5 min Time 5 min

t0 t5 t6

Dete

cto

r S

ignal

Time

Theory

Theory In order to understand liquid

chromatography, one must understand migration rates, resolution, and peak shape.

Migration rates – Retention Times

Resolution

Peak Shape – Van Deemter Equation

Migration Rates Migration rates are equilibrium constants

describing the distribution of the solutes between the mobile and stationary phases.

Mathematically, migration rates are related to retention times.

Retention time is the time difference between injection and analyte detection.

t0 t5 t6

Dete

cto

r S

ignal

Time t1 t2 t3 t4

Inje

ctio

n

Resolution Resolution is the column’s ability to differentiate

between two peaks.

An R value of 1.5 indicates baseline resolution

An R value of >1.0 is necessary for quantification purposes.

α is the selectivity factor

κ is the capacity factor

N is the column efficiency

Ra 1

a

k

K 1

N

4

Selectivity Factor A fraction consisting of the partition ratios of two

retained species.

Selectivity is the chemistry of the system

how does the analyte interact with the stationary phase

The selectivity is always equal to or greater than one. (α 1.0)

α is the selectivity factor

κ is the capacity factor

N is the column efficiency

Ra 1

a

k

K 1

N

4

α = KB

KA

k'B

k'A

= = (tR)B-t0

(tR)A-t0 http://www.chromedia.org/chromedia

Capacity Factor A dimensionless term used in conjunction with retention

time to describe chromatographic behavior (time an analyte spends in the stationary phase)

Calculated from the chromatogram

α is the selectivity factor

κ is the capacity factor

N is the column efficiency

Ra 1

a

k

K 1

N

4

k' = tR-t0

t0 http://www.chromedia.org/chromedia

Theoretical Plates A measure of the degree of broadening of a

chromatographic band; often expressed in terms of plate height (H) or the number of theoretical plates (N).

The more theoretical plates and the smaller the plate height the more efficient separation or increased resolution.

α is the separation factor

κ is the retention factor

N = number of theoretical plates

Ra 1

a

k

K 1

N

4

Time

Ideal

Broad

Fronting

Tailing

Doublet

McNair Basic Gas Chromatography 1998

Peak Broadening

a) Original chromatogram with overlapping peaks

b) Increased band separation

c) Decreased band width

Skoog, West, & Holler. Fundamantals of Analytical Chemistry.

Increasing column efficiency (HETP) increases resolution.

Factors Affecting Column Efficiency Column length

Particle size

Packing quality

Linear velocity (flow)

Instrument quality (dead volume)

Capacity factor

The factors influencing efficiency are described mathematically by the Van Deemter Equation.

Peak Shape Peak Shape is the actual Guassian character

of the analyte peak and is mathematically described by the Van Deemter Equation.

H is the plate height in centimeters

U is the linear velocity of the mobile phase in cm/sec

A = multiple flow paths

B = longitudinal diffusion

C = mass transfer between phases

CS mass transfer associated with stationary phase

CM mass transfer associated with mobile phase

H A B u CS C M

Van Deemter Schematic

http://www.chromacademy.com/resolver-september2010_High_Efficiency_HPLC_Separations.asp

Linear Velocity (Flow)

Magnitude of kinetic effects on column efficiency clearly depends upon the length of time the mobile phase is in contact with the stationary phase

This depends on the flow rate of the mobile phase

Efficiency studies typically determine plate height as a function of mobile phase velocity

Optimum flow: a certain velocity at which the plate number is highest (& the plate height is lowest)

Eddy Diffusion

Eddy diffusion or zone broadening arises from the multitude of pathways by which a molecule can find its way through a packed column.

H A B u CS C M

The more paths the

molecule can take the

broader the peak

Flow Direction

Longitudinal Diffusion

Peak broadening process in which solutes diffuse from the concentrated center of a zone to the more dilute regions.

Dependent upon mobile phase flow rates.

Contributes to peak broadening only at very low flow rates below the minimum (optimum) plate height

Because mobile phase velocity is much higher than the diffusion coefficient of the component in solution, the B term is hardly significant in liquid chromatography

H A B u CS C M

Mass Transfer

H A B u CS C M

A chromatographic system is constantly changing with the mobile phase

The time for restoration of the equilibrium (resistance to mass transfer) results in slight broadened concentration profiles.

Practical Applications

Terminology Mobile phase is a general term that refers to

the moving phase in chromatography.

Gas Chromatography (GC) the mobile phase is a gas and is termed CARRIER GAS

Liquid Chromatography (LC) the mobile phase is liquid and is termed MOBILE PHASE

Stationary Phase refers to the column packing material in chromatography.

Mobile phase of GC In gas chromatography, the moving phase

is a gaseous mixture of sample and carrier

gas.

The liquid sample is injected into the inlet where flash vaporization occurs.

The carrier gas then moves the gaseous sample through the column to the mass analyzer.

Carrier gas should be inert so the integrity of the sample is maintained. Common carrier gases include Helium and Hydrogen

In liquid chromatography, the moving phase is a mixture of liquid sample and liquid mobile phase.

The liquid sample is injected into a loop which is flushed with mobile phase.

The mobile phase moves the liquid sample through the column to the mass analyzer.

The mobile phase can be a single solvent; but typically consists of a mixture of aqueous

and organic solvents. Common solvents are methanol,

acetonitrile, and buffers.

Mobile phase of LC

Mobile Phase Composition There are two theories for elution of solutes from a

liquid chromatographic column. Isocratic elution

Gradient elution

Isocratic elution – The composition of the mobile phase (mixture of A and B) is

maintained throughout the acquisition, or

A single solvent is used.

Gradient elution – the composition of the mobile phase is changed throughout the acquisition.

Similar to temperature programming of GC

Mobile Phase Gradients

To aid in separation, decrease elution time, and optimize peak shape the composition of the mobile phase mixture is changed throughout the sample acquisition.

This change in composition disrupts the interactions occurring between analyte and column stationary phase.

Guard Column • Introduced in front of the analytical column to increase

the life of the analytical column

• Removes particulate matter & contaminants from solvents

• Composition of the guard column should be similar to that of the analytical column

• The particle size is usually larger to minimize pressure drop

http://www.phenomenex.com/guard-hplc-column

Types of LC Columns

LC Stationary Phase Categories Normal Phase - based on

polarity

Reverse Phase – based on polarity

Size Exclusion – based on size

Mixed Phase – utilizes a combination of stationary phases

Ion Exchange – based on charge

Affinity – based on affinity

Chiral – based on chirality

LC Column Application

Skoog, West, & Holler. Fundamantals of Analytical Chemistry.

Size Exclusion Most applicable to high molecular weight species

Packing consists of small silica or polymer particles containing a network of uniform pores

Molecules that are larger than the average pore size have no retention

Separation is based on size not chemical

or physical interactions between analyte

and stationary phase

Two types of packing Polymer beads – polystyrene-divinylbenzene

Silica based particles – greater rigidity; easier

packing; greater stability http://www.shimadzu.com/an/hplc/support/lib/lctalk/55/55intro.html

Ion Exchange Separation and ion determination based upon ion-exchange

resins. (sulfonic acid and quaternary amines)

Stationary phases can be porous beads, pellicular beads, or porous microparticles of silica Porous beads are styrene and

divinylbenzene based with

Anion/cation functional groups

bonded.

Pellicular beads are large

nonporous, spherical glass or

polymer beads coated with a

synthetic ion exchange resin.

Porous silica microparticles are coated with a thin film of exchanger http://www.waters.com/waters/en_US/HPLC-Separation-Modes/nav.htm?cid=10049076

Affinity Separation occurs based on ligand recognition Antigen to antibody

Enzyme to substrate

Receptor to ligand

Most selective chromatography

Utilizes specific interactions between one kind of solute molecule and a second molecule immobilized on a stationary phase.

Not frequently used in

Forensic laboratories.

http://www.rpi.edu/dept/chem-eng/Biotech-Environ/CHROMO/be_types.htm

Chiral Chiral compounds contain atleast one asymmetric carbon

and are often observed as enantiomers and diasteromers same molecular weight and formula; different spatial

arrangements

Separation achieved by the number and type spatially selective interactions

Common stationary phases

are polysaccharide, ligand

exchange, protein, helical

polymers, and macrocyclic.

Not common due to

production demands

http://www.phenomenex.com/chiral-hplc-column#

Normal Phase Contains a POLAR stationary phase

Theory – polar compounds will interact with stationary phase while non-polar compounds will elute early

Normal phase stationary phases typically include polar functional groups (silica, amino, and cyano).

LC gradient transitions from non-polar to polar Interrupts interaction between polar analytes and

column stationary phase

This is NOT commonly used in Forensic Labs

Reverse Phase Contains a NON-POLAR stationary phase.

Theory – non-polar analytes will interact with stationary phase while polar analytes will elute early

Reverse phase stationary phases include C18, C4, and C8

LC gradient composition transitions from polar to non-polar. Disrupts non-polar interaction between analyte and column

stationary phase

Most commonly used stationary phase

Mixed Phase Contains a mixture of stationary phases.

Theory – optimizes separation of complex mixtures through multiple column chemistries

ADFS employs a Synergi Fusion column

Utilizes both polar and non-polar stationary phase

Optimal for a broad range of drugs http://www.phenomenex.com/Products/HPLCDetail/Synergi/Fusion-RP?returnURL=/Products/Search/HPLC

Liquid Chromatography Schematic

Column Pump A

Pump B

6-Port Valve

Mass Analyzer

Sample

Waste

Sample Loop

How does it Work?

Effluent from pump A and B are constantly mixed according to the programmed gradient and are initially flowed through the multiport valve to column.

Sample is injected into the multiport valve where it will fill the sample loop.

The valve switches and the column effluent is diverted to the sample loop where it pushes the sample through to the column and eventually to the mass analyzer.

6-Port Valve Mechanism

http://faculty.uml.edu/david_ryan/84.314/Instrumental%20Lecture%2018.pdf

Advantages Shorter Run Time (<10 mins.) no loss of separation

Volatility issues resolved – liquid effluent vs. gas effluent

Venting of mass analyzer not necessary for column change

Use of guard column prevents constant column maintenance

Ease of troubleshooting

Liquid mobile phase

Leak detection sensors

Advantages Cooled autosampler minimizes sample

evaporation

LC is capable of switching between two columns (different stationary phases).

Easily interfaced with a variety of mass analyzers (TOF, QTRAP, MS/MS, UV, etc)

Disadvantages Costs associated with mobile phase solvents

Autosampler limitations can exist

Clogged lines/injection needle more prevalent

Liquid samples often contain undissolved solutes

Partial clog can cause retention time to be unstable

Air bubbles can damage column and cause RT shifting

Summary

LC is an effective tool in the analysis of drug analytes in a forensic laboratory

setting and should be utilized as a complement to the traditional GC/MS

instrumentation.

Questions and/or Comments?