Chromatography

High Performance Liquid

Chromatography (HPLC)

HPLC
 High Performance Liquid
Chromatography (HPLC)
• HPLC instrument
• Mobile phases
• HPLC columns
• Stationary phase
• Sample introduction
• Detectors for HPLC
• Quantitative applications

HPLC
 HPLC
• The mobile phase is usually a liquid and the
stationary phase is a solid or a liquid film
coated on a solid surface.
– Adsorption chromatography
• Solutes separate based on their ability to adsorb to a
solid stationary phase.
– Partition chromatography
• A thin liquid film coating a solid support serves as the
stationary phase.
• Separation is based on a difference in the equilibrium
partitioning of solutes between the liquid stationary
phase and the mobile phase.

HPLC
 HPLC

– Ion exchange chromatography:

• Stationary phases consisting of a solid support with
covalently attached anionic (e.g., SO3–) or cationic
(e.g., –N(CH3)3+) functional groups. Ionic solutes are
attracted to the stationary phase by electrostatic
forces.
– Size-exclusion chromatography:
• Porous gels are used as stationary phases. Separation
is due to differences in the size of the solutes

HPLC
 Normal Phase and Reverse Phase

• Normal Phase
– Polar Stationary Phase eg. Silica
– Non-polar Mobile Phase, eg. Hexane, pentane
– Neutral compounds elute first; Polar compounds elute
last
• Reverse Phase
– Non-Polar Stationary Phase, eg C18, C8
– Polar Mobile Phase, eg, H20, MeOH, CH3CH2CN
– Polar Compounds elute first; Neutral Compounds elute
last.

HPLC
 NP and RP
• Reverse Phase is method of choice
– Very broad scope, allows separation of wide
variety of compounds with differing polarity
– Inexpensive mobile phase (H20, methanol,
acetonitrile)
– Can be applied to ionic and ionisable compounds
(ion pairing)
– Generally experimentally easier with rapid
equilibration, faster and more robust
separations
– Organic solvents can be more dangerous to use
• Limitations
– Columns are generally stable within the pH
HPLC range of 3-8
 HPLC instrument

Schematic diagram of a high-performance

HPLC liquid chromatograph
 HPLC instrument

HPLC
HPLC
 HPLC column

HPLC
 HPLC Pumps

• Isocratic
– Pump delivers constant composition of mobile
phase
• Gradient
– Pump delivers variable mobile phase composition
or flow rate
– Gradients are used for complex samples

HPLC
 Properties of HPLC mobile
phases

HPLC
 Isocratic Elution

• Performed with a single solvent

• In a reverse phase separation, eluent
strength decreases as the solvent becomes
more polar.

HPLC
 Isocratic Elution - Peak Identity

1. benzyl alcohol
2. phenol
3. 3’,4’-dimethoxyacetophenone
4. benzoin
5. ethyl benzoate
6. toluene
7. 2,6-dimethoxytoluene
8. o-methoxybiphenyl

HPLC
 Isocratic Separation

HPLC A - aqueous buffer; B - acetonitrile

 Decreasing Eluent Strength

HPLC
 Decreasing Eluent Strength

HPLC
 Gradient Separation

HPLC
 HPLC stationary phases

– In liquid–liquid chromatography the stationary

phase is a liquid film coated on a packing
material consisting of 3–10 m porous silica
particles.
– Bonded stationary phases are attached by
reacting the silica particles with an
organochlorosilane of the general form
Si(CH3)2RCl, where R is an alkyl or substituted
alkyl group

HPLC
 HPLC stationary phases

– To prevent unwanted interactions between the

solutes and any unreacted –SiOH groups, the
silica frequently is “capped” by reacting it with
Si(CH3)3Cl (end-capped columns)

HPLC
 HPLC Silica Bonded Phases

Cyano
Amino SAX

Methyl Hexyl C8 C8

Butyl
Butyl
Phenyl C18
C18

SCX
HPLC
 Surface of Reversed Phase Packing

HPLC
 Mobile Phase Considerations

• Solid particles
• Dissolved gases
• Stabilisers / Antioxidants
• U.V absorbing impurities
• Viscosity
• Toxicity

HPLC
 Solvent Filtration
Removes particulate matter and dissolved gases
Must always be done without exception!

HPLC
 UV spectrum of Acetonitrile 99.9%

HPLC
 UV spectrum of Methanol

HPLC
 HPLC Columns
Silica Normal phase, gel permeation
Alumina Normal phase
Graphite Reversed phase
Poly(styrene-divinyl benzene) Reversed phase
Alkyl silane Reversed phase
Phenyl Reversed phase
Cyano Reversed phase
Amino Normal phase, ion exchange
Alkylated amino ion exchange
Sulfonate ion exchange, ion exclusion
Alkyl sulfonate ion exchange
Diol Normal Phase
Poly(methylmethacrylate) Ger permeation

HPLC
 Particle Size

• Larger Particles
– Lower backpressure
– More economical
– Higher Capacity

• Smaller Particles
– Higher efficiencies
– Better sensitivity
– Reduced run time

HPLC
 Particle Sizes

HPLC
 Sample Introduction

– The sample is introduced using a loop injector

Sampling loops are interchangeable, and
available with different volumes.

HPLC
 HPLC Injectors

HPLC
 Detectors

• Ultra-violet (UV) detection

• Photodiode array (PDA)
• Conductivity
• Electrochemical
• Refractive index
• Fluorescence
• Evaporative Light Scattering
• Radioactivity

HPLC
 Detector for HPLC

UV/VIS detector

HPLC
 Variable UV Detector
Light Censor

Flow Cell

Grating
Lamp

Slits
HPLC
 Photo Diode Array Detector

HPLC
 Solvent UV-Vis Cut-off
Wavelengths

HPLC
 Derivatisation in HPLC

• Why derivatise?
– Increased sensitivity
– Able to use different detectors
– Eliminate interfering Compounds
• Types
– Pre-column derivatisation
– Post-column derivatisation

HPLC
 Choice of HPLC Method

HPLC
 Choice of HPLC method

HPLC
HPLC
HPLC
HPLC
 Quantitative Calculations

– Quantitative analyses are often easier to

conduct with HPLC than GC because injections
are made with a fixed-volume injection loop
instead of a syringe
– Quantitative measurements can be made using
external standards

HPLC
 Quantitative Analyses - Example
– A 2.013-g sample of dried soil is extracted
with 20.00 mL of methylene chloride. After
filtering to remove the soil, a 1-mL portion of
the extract is removed and diluted to 10 mL
with acetonitrile. Injecting 5 mL of the diluted
extract into an HPLC gives a signal of 0.217
for the PAH fluoranthene. When 5 mL of a
20.0-ppm fluoranthene standard is analyzed
using the same conditions, a signal of 0.258 is
measured. Report the parts per million of
fluoranthene in the soil.
20  0.217 3 1000
10 10  20   1671 ppm
0.258 2.013
HPLC