High Performance Liquid

Chromatography
most popular, powerful and versatile
form of chromatography
Principle
Resolution Power of chromatographic column is determined by
many factors
• number of theoretical plates (N) in the column and hence plate
height (H)
• Value of N increases with column length but there are practical
limits to the length of a column owing to the problem of peak
broadening
Principle:
• Surface area of the stationary phase is directly proportional to the
number of theoretical plates

• Smaller the particle size of the stationary phase, greater will be the
surface area, hence greater the value of N

• N is inversely proportional to particle size.

Principle: Backpressure

• Unfortunately, smaller the particle size, greater is the resistance to

the flow of the mobile phase

• This resistance creates a backpressure in the column

• that is directly proportional to both the flow rate and the column
length

• inversely proportional to the square of the particle size

Principle: Backpressure
• This back-pressure may cause the structure of the matrix to
collapse, thereby further reducing eluent flow and impairing
resolution
Principle: Backpressure problem solution
• development of small particle size stationary phases (5-10 µm
diameter with a narrow range)

• which can withstand pressures up to 40 Mpa

HPLC Principle: Particle Size
• Larger particle size phases are the basis of low-pressure liquid
chromatography

• flow of the eluent through the column is either gravity-fed or

pumped by a low pressure pump, often a peristaltic pump

• LPLC is cheaper to run than HPLC but lacks the high resolution
that is the characteristic of HPLC
For gradient elution two reservoirs &
two pumps are used with liquid-
phase mixing before entry to the
sample injection loop

Components of an isocratic HPLC system

Columns:
Conventional columns Microbore or open tubular
columns
• made of stainless steel • internal diameter of 1–2mm
• can withstand pressures of up • generally 25-50 cm long
to 50MPa
• flow rates of 5-20mm3 min–1
• columns are 3–25 cm long
• approximately 4.6 mm internal
diameter
• flow rates of 1-3cm3 min–1
Columns
Microbore columns have three important advantages over
conventional columns:
• reduced eluent consumption due to the slower flow rates

• ideal for interfacing with a mass spectrometer due to the reduced

flow rate

• increased sensitivity due to the higher concentration of analytes

that can be used
Sample Loading: Injector loop

• common method of sample introduction is by use of a loop

injector

• consists of a metal loop, of fixed small volume, that can be filled

with the sample
https://www.youtube.com/watch?v=8-D-mMfDw7g
 Cont…
• eluent from the pump is then channelled through the loop
• by means of a valve switching system and
• sample flushed onto the column via the loop outlet
• without interruption of the flow of eluent to the column
Guard column
• Highly impure samples viz. sera, urine, plasma or whole
blood, which have preferably been deproteinated, may
cause the column to lose its resolving power

• So a guard column is often installed between the injector

and the column
 Cont…
• guard column = short (1-2 cm) column of the same internal
diameter
• packed with material similar to that present in the analytical
column.
• packing in the guard column preferentially retains
contaminating material and can be replaced at regular intervals
Mobile phases
• Choice of mobile phase depends on the type of separation to be
achieved

• Isocratic elution = single pump/single eluent or two or more

eluents premixed in fixed proportions

• Gradient elution = separate pumps to deliver two eluents in

proportions predetermined by a gradient programmer
 Cont…
• All eluents must be purified

• traces of impurities can affect the column

• interfere with the detection system

• But even with pure eluent a 1-5mm microfilter is introduced

into the system prior to the pump
What is mobile phase degassing?
• Henry’s law states that ‘mass of a gas which will dissolve into a
solution is directly proportional to the partial pressure of that gas
above the solution

• When the solvents are mixed, solubility of air is less than it is

in same proportion of pure solvents

• and excess air will tend to come out of solution (bubble out – c/d
outgassing)

• Rough surfaces in HPLC system produce nucleation sites for

bubble formation
Problems due to gassing

• Air bubbles modify the flow of mobile phase through the column

• Gassing can alter column resolution

• Interfere with continuous monitoring of the eluate (Air bubbles

passing through detectors lead to spurious peaks)

• Practically it is not necessary to remove the entire dissolved air

but only a fraction can be removed
Degassing methods
• Helium purging: helium is bubbled through the solvent and removes up to
80% of dissolved air

• Vacuum degassing: solvent is exposed to a vacuum and the reduced

pressure removes more than 60% of the dissolved air

• Sonication: ultrasonic baths removes up to 30% dissolved air

• warming

• stirring vigorously with a magnetic stirrer

Pumps
• Capable of outputs of at least 50 MPa

• ideally there must be no pulses (i.e. cyclical variations in pressure); may

affect the detector response

• Flow capability of at least 10 cm3 min-1 and up to 100 cm3 min–1 for
preparative separations

• Constant displacement pumps maintain a constant flow rate through

the column irrespective of changing conditions within the column.
 Cont…
• Reciprocating pump is the most commonly used form of constant
displacement pump
• Such pumps produce small pulses of flow and pulse dampeners
are usually incorporated into the system to minimise this pulsing
effect
• All constant displacement pumps have inbuilt safety cut-out
mechanisms
• so that if the pressure within the column changes from pre-set
limits the pump is inactivated automatically
Detectors
• Since the quantity of material applied to an HPLC column is
normally very small

• it is imperative that the sensitivity of the detector system is

sufficiently high and stable to respond to the low concentrations of
each analyte in the eluate
Detectors: commonly used detectors

• Variable wavelength detectors

• Scanning wavelength detectors
• Fluorescence detectors
• Electrochemical detectors
• Mass spectrometer detectors
• NMR spectrometer detectors
• Refractive index detectors
Variable wavelength detectors
• based upon ultraviolet–visible spectrophotometry
Scanning wavelength detectors
• record the complete absorption spectrum of each
analyte, thus aiding identification
Fluorescence detectors
• greater sensitivity (10–12 g cm–3) than UV detectors
• Limited by the fact that few analytes fluoresce
Electrochemical detectors
• These are selective for electroactive analytes and
are potentially highly sensitive
Mass spectrometer detectors
• These enable the analyte to be detected and its
structure determined simultaneously.
NMR spectrometer detectors
• These give structural information about the analyte
Refractive index detectors
• These rely on a change in the refractive index of the
eluate as analytes emerge from the column.
GAS CHROMATOGRAPHY
Principle

• exploits differences in the partition coefficients of the

volatilised analytes between a stationary liquid phase and a
mobile gas phase

• confined to analytes that are volatile but thermally stable

• most volatile elute first

 Cont…
• temperature of the column is raised to 50-300 oC to facilitate
analyte volatilisation

• stationary phase consists of a high-boiling-point liquid material

such as silicone grease or wax
Principle
• widely used for the qualitative and quantitative analysis of a large
number of low-polarity compounds

• because it has high sensitivity, reproducibility and speed of

resolution
Components
of a GC
system
Columns: two types
• Packed conventional columns
• Capillary (open tubular) columns
Packed conventional columns
• consist of a coiled glass or stainless steel column 1-3m long
• 2-4mm internal diameter
• packed with stationary phase coated on an inert silica
support.
 Packed conventional columns
Commonly used stationary phases include the
• polyethylene glycols (Carbowax)
• Or methylphenyl- and methylvinylsilicone gums
• Commonly used support is Celite (diatomaceous silica)
• Celite is often treated so that –OH groups gets modified (to prevent
support-sample interaction)
• Modification is done by silanisation of the support with
hexamethyldisilazane
Capillary (open tubular) columns:
• made of high-quality fused quartz
• are 10-100m long
• 0.1-1.0mm internal diameter.
• Two types: wall-coated open tubular (WCOT) and
support-coated open tubular (SCOT), also known as
porous layer open tubular (PLOT) columns
• WCOT = SP is thinly coated directly onto the walls of the
column

• SCOT = matrix is bonded to the walls of the column and SP is

coated onto the support
Application of sample
• majority of non- and low-polar compounds are directly amenable
to GC

• compounds possessing polar groups viz. -OH, -NH2 and -COOH

are generally retained on the column for excessive periods of time

• Poor resolution and peak tailing usually accompany this excessive

retention.
Application of sample: Derivetization
• Problem can be overcome by derivatisation of the polar groups

• This increases the volatility and effective distribution coefficients

of the compounds

• Methylation, silanisation and perfluoracylation are common

derivatisation methods for fatty acids, carbohydrates and amino
acids
Application of sample
• Test sample is dissolved in a suitable solvent such as acetone,
heptane or methanol

• For packed and SCOT columns, sample is injected onto the column
using a microsyringe through a septum in the injection port
Application of sample
• Normally 0.1 to 10mm3 of solution is injected

• As there is only a small amount of stationary phase present in WCOT

columns, only very small amounts of sample may be applied to the
column

• Splitter system has to be used at the sample injection port so that only
a small fraction of the injected sample reaches the column

• remainder of the sample is vented to waste

Application of sample
• maintain the injection region of the column at a slightly higher
temperature (+20 to 50 oC) than the column itself as this helps to
ensure rapid and complete volatilisation of the sample.
Mobile phase
• mobile phase consists of an inert gas such as nitrogen for packed
columns or helium or argon for capillary columns
• gas from a cylinder is pre-purified by passing through a variety of
molecular sieves to remove oxygen, hydrocarbons and water
vapour
• It is then passed through the chromatography column at a flow
rate of 40-80 cm3 min–1
• A gas-flow controller is used to ensure a constant flow
irrespective of the back-pressure and temperature of the column.
Detectors
• Flame ionisation detector (FID) – detect all organic compounds
• Electron capture detector (ECD) – For analyte which captures
electron e.g. halogen containing compounds. Used for
polychlorinated compounds such as pesticides, DDT, dieldrin,
aldrin
• Nitrogen–phosphorus detector (NPD)
• Flame photometric detector
• Rapid scanning Fourier transform infrared detector
• Mass spectrometer detector