Basic LC Terminology

Adsorption chromatography
The stationary phase is an adsorbent (like silica gel or any
other silica-based packing)
The separation is based on repeated adsorption-desorption
steps.

Normal-phase chromatography
The stationary bed is strongly polar in nature (e.g., silica gel),
and the mobile phase is nonpolar (such as n-hexane or
tetrahydrofuran).
Polar samples are retained on the polar surface of the column
packing longer than less polar materials.

Reversed-phase chromatography
The stationary bed is nonpolar (hydrophobic) in nature,
The mobile phase is a polar liquid, such as mixtures of water
and methanol or acetonitrile.
The more nonpolar the material is, the longer it will be retained.

Basic LC Terminology
Size exclusion chromatography (SEC)
column filled with material having precisely controlled pore
sizes, and the sample is simply sieved or filtered according to
its solvated molecular size.
Larger molecules are rapidly washed through the column;
smaller molecules penetrate inside the pores of the packing
particles and elute later.
Also called gel permeation chromatography (GCP)
although the stationary phase is not restricted to a "gel"

Ion-exchange chromatography (IC)

the stationary bed has a charged surface of opposite charge
to the sample ions.
Used almost exclusively with ionic or ionizable samples.
The stronger the charge on the sample, the stronger it will be
attracted to the ionic surface and thus, the longer it will take
to elute
The mobile phase is an aqueous buffer, where both pH and
ionic strength are used to control elution time

Analytical Applications of LC

The branches of the LC family:

Note this means analyte polarity

Basic Mechanisms used in LC Separations

High Performance Liquid Chromatography (HPLC)

HPLC utilizes a high-pressure liquid mobile phase (ca.

100-300 bar) to separate the components of a mixture

These analytes are first dissolved in a solvent, and then

forced to flow through a packed small-particle
chromatographic column, where the mixture is resolved
into its components

HP = high pressure and high performance

Resolution depends upon the extent of interaction

between the solute components and the stationary
phase

Differences between HPLC and Classical LC

Small ID (2-5 mm), reusable stainless steel columns

Column packings with very small (3, 5 and 10 m)

particles and the continual development of new

substances to be used as stationary phases
Relatively high inlet pressures and controlled flow of the
mobile phase
Precise sample introduction without the need for large
samples
Special continuous flow detectors capable of handling
small flow rates and detecting very small amounts
Automated standardized instruments
Rapid analysis
High resolution
From now on, LC refers to HPLC

Advantages and Disadvantages of LC

Advantages:
Speed (minutes)
High resolution
Sensitivity
Reproducibility
Accuracy
Automation
Disadvantages:
Cost
Complexity
Low sensitivity for some compounds
Irreversibly adsorbed compounds not detected
Co-elution difficult to detect

More on Reversed-phase (RP) LC

RP is the most widely used mode of HPLC (75%?)

Separates molecules in solution on basis of their
hydrophobicity
Non-polar stationary phase
Polar mobile phase

In practice: non polar functional group bonded to silica

Stationary phase
functional group bonded to silica
this corresponds to a volume (Van deemter)
Alkyl groups ( C4, C8, C18)
retention increases exp. with chain length

Mobile Phases
Polar solvent (water) with addition of less polar solvent (acetonitrile
or methanol)

The Packed Column and the Stationary Phase

Packed LC columns, usually made of stainless steel and

carefully filled with material, are the heart of the LC
experiment

The stationary phase fills the column its properties are

critical to the separation

Review of Molecular Interactions

The basis of separations (and most of chemistry)

Name

Energy (kcal/mol)

Description

Covalent

100-300

Hold molecules together, orbital

overlap

Ionic

50-200

Electrostatic attraction

Polar
Hydrogen bonding
Dipole-dipole
-stacking

3-10

Vary from electrostatic-type

interactions (e.g. hydrogen
bonds) to much weaker

Non-Polar
Van der Waals
(dispersion)

1-5

Weak, induced dipole

Retention Mechanisms in LC
HPLC is a dynamic adsorption process. Analyte molecules, while
moving through the porous packing bead, tend to interact with the
surface adsorption sites. Depending on the HPLC mode, the different
types of the adsorption forces may be included in the retention process
Hydrophobic interactions are the main ones in reversed-phase
separations
Dipole-dipole (polar) interactions are dominant in normal phase mode.
Ionic interactions are responsible for the retention in ion-exchange
chromatography.
Retention in LC is competitive:
Analyte molecules compete with the eluent molecules for the
adsorption sites. So, the stronger analyte molecules interact with
the surface, and the weaker the eluent interaction, the longer
analyte will be retained on the surface.

Retention Mechanisms in LC

Remember the elution order!

Normal-phase vs. reversed-phase LC

Physical Properties of Stationary Phase Particles

HPLC separations are based on the surface interactions,

and depends on the types of the adsorption sites (surface

chemistry). Modern HPLC adsorbents are the small rigid
porous particles with high surface area.

Key parameters:
Particle size: 3 to 10 m
Particle size distribution: as narrow as possible, usually within 10%
of the mean
Pore size: 70 to 300
Surface area: 50 to 250 m2/g
Bonding phase density (number of adsorption sites per surface
unit): 1 to 5 per 1 nm2

The Most Popular Particle: Silica

Different morphology for different applications:

Macroporous spherical silica particle. [K.K.Unger,

Porous silica, Elsevier, 1979]

Electron microphotograph of spherical and irregular silica particles. [W.R.Melander, C.Horvath,

Reversed-Phase Chromatography, in HPLC Advances and Perspectives, V2, Academic Press, 1980]

Different chemistry:
H

Si OH

Si OH

Adsorbed Water

Si

OH

O O
Si

Si

O
H

Free Silanol

OH

Geminal Silanol
Dehydrated
Oxide Siloxane

Si O
O
H
Si O
H
Bound and
Reactive
Silanols

Chemical Modifications to Silica

Silica (or zirconia, or alumina) by itself cannot do the job needed by

modern LC users it must be functionalized and modified to suit the
analytical problem

Functionalize
d groups

Residua
l
silanols

Si
OH

O
Si

Si

Si

OH
O

Si

Si
O

Si

OH
O

OH
Si

Diagram from Crawford Scientific

Si

O
O

Si

Chemical Modifications to Silica

Groups are usually attached via reaction

of an organosilane (which can be prepolymerized in solution)
Besides attaching groups, it is also
possible to polymerize the silica (or the
attached group)
Purpose: stability at low pH, more
coverage
High-carbon load
Monomeric phases are more
reproducible (easier reactions to control)
Monomeric phases are also known
as sterically-protected
Endcapping: fully react the silica
surface, remove silanols and their
acidity, more coverage
Diagram from K. A. Lippa et al., Anal. Chem.2005, 77,7852-7861

Common LC Stationary Phases

Name

Structure

Silica

Si

Propyl

Si

C8

Si

OH

Description
Normal phase, for separating polar, non-ionic
organics

C3H7

Reversed-phase, for hydrophobic interaction

chromatography (proteins, peptides)

C8H17

Reversed-phase, like C18 but less retentive,

used for pharmaceuticals, steroids,
nucleotides
Reversed-phase, retains non-polar solutes
strongly. When bonded to 300A silica can be
used for large proteins and macromolecules

C18

Si

C18H37

Cyano

Si

CH2CH2CH2CN

Reversed-phase and normal-phase, more

polar than C18, unique selectivity

Amino

Si

CH2CH2CH2NH2

Reversed-phase, normal-phase, and weak

anion exchange. RP used to separate
carbohydrates

Common LC Stationary Phases

Name

Structure

Phenyl

Diol

Si

Description

CH2CH2CH2

Reversed-phase, retains aromatic

molecules. Also used for HIC
(proteins)

Both reversed-phase and normalphase utility. Used for RP SEC,

also used for NP separations as a
more robust alternative to silica
(not ruined by trace water)

Si

OH
OH

Nitro

Si

NO2

Normal-phase, separates aromatic

and alkene-containing molecules

Polar Stationary Phase Interactions

Interactions

Sorbents
O

CN

NH2

Si

Si

2OH

Dipole/ Dipole

Si

HydrogenBonding

O
O

O H
H
O

Source: Crawford Scientific.

HydrogenBonding

Ionic Stationary Phase Interactions

Source: Crawford Scientific.

Non-Polar Stationary Phase Interactions

Sorbents

Interactions

Si

van der Waals

C8
Si

PH

C2

van der Waals

Si

van der Waals

Source: Crawford Scientific.

A Good Choice of Stationary Phase Depends on

the Analyte
Functionality
Hydrophobic

H-Bonding

Analyte

Mechanism
N
NH
H22

N
NH
H22

Non-Polar

Polar

Ionic

+
N
NH
H33

Source: Crawford Scientific.

Ion-Exchange

More Subtle Effects

Shape selectivity (correlates with stationary phase order),

temperature, coverage (and the role of bonding chemistry):

Diagram from K. A. Lippa et al., Anal. Chem.2005, 77,7852-7861

More Subtle Effects

The effects of
temperature on the
order of the stationary
phase are often
surprising:

Diagram from K. A. Lippa et al., Anal. Chem.2005, 77,7852-7861

Chiral Stationary Phases

Interactions between chiral analytes (enantiomers and

molecules with more than 1 chiral center) and chiral
stationary phases are also possible
Normal-phase is most common because of binding modes

A. Berthod, Chiral Recognition Mechanisms, Anal. Chem. 78, 2093-2099 (2006).

Chiral Stationary Phases

Interactions between chiral analytes and chiral stationary

phases are also possible.
Common chiral stationary phases:
Name

Chiral Recognition Mechanism

Analyte and Mobile Phase

Requirements

Protein based

Hydrophobic and electrostatic

interactions

Analyte must ionize, helpful if it

contains an aromatic. RP only.

Cyclodextrin

Inclusion complexation, H-bonding

Polar and aromatic groups, RP

and NP.

Polymerbased
carbohydrates

Inclusion interactions, attractive

interactions

H-bonding donors/acceptors, steric

bulk at chiral center, RP and NP.

Pirkle

H-bonding, interactions, dipoledipole interactions

H-bonding donor/acceptors, mostly

NP.

Adapted from L. R. Snyder, J. J. Kirkland, and J. L. Glajch, Practical HPLC Method Development, 2nd Ed., Wiley, 1997. Pg 545.

A Chiral LC Separation

Example: separation of
naproxen enantiomers
Chiral AGP column
AGP = 1-acid glycoprotein
(orosomucoid), 181 amino
acid residues and 14 sialic
acid residues
Isocratic (no change in mobile
phase composition during
separation)
(S)

HO

O
O

(S)-naproxen

(R)

HO

O
O

(R)-naproxen
Adapted from L. R. Snyder, J. J. Kirkland, and J. L. Glajch, Practical HPLC Method Development, 2nd Ed., Wiley, 1997. Pg 545.

Ion Chromatography (IC)

Form of LC, also known as ion-exchange chromatography

Basic mechanism is electrostatic exchange:

Source: Rubinson and Rubinson, Contemporary Instrumental Analysis, Prentice Hall Publishing.

Typical IC Results

Example: an isocratic method for

monovalent cations in ammonium
nitrate based explosives
Detection limits 50-100 ppb, max
working range 40 ppm
Method:
Sample Loop Volume: 50 L
Columns: IonPac CS3 Analytical,
IonPac CG3 Guard
Eluent: 25 mM HCl, 0.1 mM DAPHCl,
4% Acetonitrile
Eluent Flow Rate: 1.0 mL/min
Suppressor: Cation MicroMembrane
Suppressor (CMMS)
Regenerant: 100 mM
Tetrabutylammonium Hydroxide
Detector: Conductivity, 30 S full scale
Injection Volume: 50 L

From Dionex Application Note 121R

Mobile Phases in LC

The type and composition of the mobile

phase (eluent) is one of the variables
influencing LC separations
Desirable properties:
Purity
Detector compatibility
Solubility of the sample
Low viscosity
Chemical inertness
Reasonable price
Mobile phases differ for each LC mode
Normal phase solvents are mainly nonpolar
Reversed-phase eluents are usually a mixture of water with some
polar organic solvent such as acetonitrile.
Size-exclusion LC has special requirements for mobile phases
Must dissolve polymers
Must also suppress all possible interactions of the sample molecule
with the surface of the packing material
Figure from Phenomenex technical literature

Control of Eluent Polarity

Isocratic elution: the eluent composition remains constant
as it is pumped through the column during the whole
analysis.
Gradient elution: the eluent composition (and strength) is
steadily changed during the run.

% mobile phase

N 1 1 k
Rs

4 k

time

N 1 1 k

Rs

*
4 k
where k* is the k at the midpoint of the column

LC Instrumentation
Pumps, Mixers
and Injectors

Column

Detector(s)

Computer

LC Instrumentation

The Agilent 1100, a typical modern LC system

Solvent reservoirs
Solvent degasser
Pump
Autosampler
Column oven
DAD

Review: The Purpose of Key LC Components

column

separation chemistry
tubing to detector flow cell

signal transduction
amplification/scaling
filtering

detector
analog output

data acquisition
digitization

A/D
digital output

chromatogram

digital processing
data analysis

The LC Pump(s)
Modern pumps have the following parameters:
Flow rate range: 0.01 to 10 ml/min
Pressure range from 1-5,000 psi
Pressure pulsations : less than1 %

Types of Pumps
Constant pressure pumps
Constant flow pumps

Reciprocating Piston Pump (90% of HPLCs)

small internal volume
pulsed flow

Syringe type pumps (Displacement Pumps)

limited solvent capacity

Pneumnatic Pumps (pressure)

Temperature Control in LC

Thermoelectric heating/cooling
the ability of a surface to
produce or absorb heat
when current is applied
across the junction of two
dissimilar conductors or
semicondeucted
The effect can be reversed (i.e.
heating turned to cooling) by
reversing the DC current
through the junction
Also known as the Peltier effect
after its 1834 discoverer, a
French watch maker

Overview of LC Detectors

Common HPLC detectors

Refractive Index
UV/Vis
Fixed Wavelength
Variable Wavelength
Diode Array
Fluorescence Detector

Less common:
Conductivity
Mass-spectrometric (LC/MS)
Evaporative light scattering (ELSD)

Desirable Features of an LC Detector

1. Low drift and noise level
2. High sensitivity (ability to discriminate between
small differences in analyte concentration)
3. Fast response
4. Wide linear dynamic range
5. Low dead volume
6. Cell design that eliminates remixing of separated
bands
7. Insensitivity to changes in types of solvent, flow
rate, temp
8. Operational simplicity and reliability
9. Non-destructive

Baseline Noise and Drift

Detector Response
The definition of detector response depends on whether
it is mass sensitive or concentration sensitive
Mass sensitive mV/mass/unit time
R = hw/sM
Concentration sensitive mV/mass/unit volume
R = hwF/sM
h = peak height mV
W = width at .607 of height
F = flow rate
M = mass of solute
s = chart speed

Cell Efficiency
Example:
column 15,000 plates
15 cm long
2 min tR
2 ml at 1 ml /min
peak width of 80uL
flow cell of 20 ul
only four measurements

Things to note:
parallel light beam
flow cell volume <1/10 of peak volume
optimization of cell geometry

Ultraviolet/Visible Spectroscopic Detectors

Any chemical compound could interact with the electromagnetic field. Beam of the
electromagnetic radiation passed through the detector flow-cell will experience some change
in its intensity due to this interaction. Measurement of this changes is the basis of the most
optical HPLC detectors.
infrared (IR)

2,500 - 50,000 nm

near infrared

800 - 2,500 nm

visible

400 - 800 nm

ultraviolet (UV)

190 - 400 nm

Name

Chromophore

Wavelength [nm]

Molar extinction,

acetylide

-C=C

175-180

6,000

Aldehyde

210

1,500

amine

-CHO
-NH2

195

2,800

azo

-N=N-

285-400

3-25

bromide

-Br

208

300

carboxyl

-COOH

200-210

50 - 70

disulphide

-S-S-

194

5,500

ester

-COOR

205

50

ether

-O-

185

1,000

ketone

195

1,000

nitrate

>C=O
-ONO2

270

12

nitrile

-C=N

160

nitrite

-ONO
-NO2

220 - 230

1000-2000

210

strong

nitro

Fixed / Variable Wavelength Detectors

mercury vapor lamp emit very intense light at 253.7

nm. By filtering out all other emitted wavelengths,
manufacturers have been able to utilize this 254 nm
line to provide stable, highly sensitive detectors
capable of measuring subnanogram quantities of
any components which contains aromatic ring. The
254 nm was chosen since the most intense line of
mercury lamp is 254 nm, and most of UV absorbing
compounds have some absorbance at 254 nm.

Diode Array Detectors

Diode array detectors can acquire all UV-Visible

wavelengths at once.

Advantages:
Sensitivity
(multiplex)
Speed
Disadvantages:
Resolution

Figure from Skoog, et al., Chapter 13

Other Detectors
Fluorescence Detector

Electrochemical Detector

Evaporative Light Scattering

Putting it All Together: LC Method Development

The importance without a good method:

Co-elution can be missed

Unable to detect/assay key components
Basic consequences of method changes:

Choosing an LC Approach

Goals of a separation:
Resolution (Rs) > 1.5
Short separation time (5-30 minutes)
Good quantitative precision/accuracy
Acceptable backpressure
Narrow peaks
Minimal solvent use

Overall Strategy

First select an

appropriate method
If LC is best, then
determine nature of
the sample
Exploratory RP
runs, i.e. fast simple
gradients with C18
phases, are usually
helpful in assessing
retention and polarity

Solid-phase Extraction (SPE)

What is SPE?
The separation of an analyte or analytes from a mixture of
compounds by selective partitioning of the compounds
between a solid phase (sorbent) and a liquid phase (solvent)

Comparison with conventional liquid-liquid extraction (e.g.

the organic sep funnel approach):
SPE: selective towards functional groups (better)
LLE: selective towards solubility
SPE: more choices because no miscibility (better)
LLE: must avoid miscible solvents
SPE: concentrates analytes (better)
LLE: can concentrate analyte after stripping

The Typical SPE Process

Conditioning:
Equilibration:

solvates the sorbent

removes excess conditioning solvent,
matches with analytical conditions (prevents shock)

Solid-phase Extraction

Conditioning the cartridge:

Not conditioned

Conditioned

SPE cartridges have a range of chemistries that are often

similar to those of LC stationary phases, but are optimized

for adsorption/desorption

Solid-phase Extraction

Automated SPE systems for sample cleanup the Spark

SymbiosisTM

Can be hyphenated

with LC, MS, NMR,

etc or used as a
stand-alone sample
pretreatment

Images from www.sparkholland.com

Homework and Further Reading

Homework problems (for study only):

28-2, 28-3, 28-11, 28-14

For a detailed discussion of method development in LC:

L. R. Snyder, J. J. Kirkland, and J. L. Glajch, Practical
HPLC Method Development, 2nd Ed., Wiley, 1997.

For recent advances in understanding gradient elution,

see:
P. Nikitas and A. Pappa-Louisi, Anal. Chem., 2005, 77,
5670-5677 (a new derivation of the equation of
reversed-phase HPLC gradient elution)