Separation techniques –

• Principles
• Instrumentation
• Applications

http://www.edusolns.com/hplc/
 Activity and Recap

• What is a stationary phase?

• What is a mobile phase?
• What are the different ways in which
separation is achieved in chromatography?
 Principle:
• Sample components are carried by a
mobile phase through a bed of stationary
phase.

• Individual species are retarded by the

stationary phase based on various
interactions:
Surface adsorption
Ion-exchange
Different Modes of HPLC

i) Normal phase
– Hydrophilic Stationary phase (bed) = polar (eg.
silica-unbonded).
– Hydrophobic Mobile phase = non-polar (eg. n-
hexane, tetrahydrofuran etc).
– Polar samples are retained on the stationary
phase (polar) longer. (Non-polar/hydrophobic
molecules elute earlier)
– Dipole-dipole (polar) interaction between
stationary phase and molecules
 Different Modes of HPLC
ii) Reverse phase
- Non-polar or Hydrophobic Stationary phase (bed)
= eg. C18- bonded
-Hydrophilic/Polar Mobile phase (e.g. mixture of
water and methanol or acetonitrile). Most common!
-Non-polar compounds retained longer
-Hydrophobic (non-specific) interaction between
stationary phase and molecules

iii) Ion exchange

 Basic HPLC equipment
• Solvent reservoir

• Pump

• Injector

• Column

• Detector

• Data system
The HPLC System
 Solvent (Mobile phase)
• Important qualities
– Purity
– Detector compatibility
– Solubility of the sample
– Low viscosity
– Chemical inertness
– Reasonable price

•Degassing of solvent
Reduce bubble formation in column or detector
by displacement with less soluble gass (helium
sparge)
by vacuum application
by heating
 Two elution types
• Isocratic elution
– Constant elution composition
– Better separation for particular component

Eg. ACN / MeOH / Water, 10 / 12 / 78, flow rate: 2.0ml/min

•Gradient elution
-Elution composition (and strength) is steadily
changed during the run
-Better separation for complex mixtures and
large variety samples.
Eg. ACN / 10% Methanol, Gradient: 40%-100%, 30min, flow
rate 2.0ml/min
 Isocratic vs Gradient Elution

http://www.hitachi-hightech.com/global/products/science/tech/ana/lc/basic/course3.html/
Isocratic vs Gradient Pumping

Gradient Elution is useful in separation /

resolution of a mixture of compounds with
different chemical properties.
 Sample Introduction
• By Injector port
or
•Autosampler
 Column
• Typical analytical columns
are 10, 15 and 25 cm in
length (longer usually better
resolution)
• fitted with extremely small
diameter (3 to10 m)
particles. The internal
diameter of the columns is
usually 4 or 4.6 mm
• 1.3 & 1.7 m particles
become more common
 Stationary Phase
• Packing material comes with column
• Type depending on application
• Reverse mode = non-polar stationary
phase, eg. C18 bonded spherical 4m
particles.
• Normal mode = polar stationary phase, eg.
silica spherical 5m partcles.
• http://www.mn-net.com/tabid/7118/default.aspx
 Detector
• Continuously monitor the column effluent
and detect the presence of solute.
• Highly sensitive
• The most commonly used detector is the
UV/VIS absorption detector (UV detector)
 Detectors
• Photometric (UV/Vis)
– Fixed wavelength, Variable wavelength,
Photo Diode Array)
• Fluorescence
• Refractive index
• Conductivity
• Mass-spectrometric (LC/MS)
• Evaporative light scattering
 Photometric Detector
• Measure the adsorption of ultraviolet or
visible radiation
•Lambert-Beer law, linearity over wide
concentration range
•Sensitivity: ~ ng
•Advantages: high sensitivity, small sample
volumes.
•nondestructive to sample

•suitable for gradient elution

Mass Spectrometric Detector
• Most sensitive
• Selective
• Universal
• Versatile
• Provide more structural
information on the analyte
• Most expensive
Typical HPLC chromatogram

Retention Time = tR
 Qualitative, Quantitative and
Preparative applications of HPLC

• Qualitative = to identify or determine the

presence of a component(s) in a sample.
• Quantitative = to determine the amount of
a known compound(s) in a sample.
• Preparative = to separate different
components in a sample and collect them
separately.
 Qualitative application
• For a given set of conditions, a compound has a fixed
retention time, tR.
Not highly specific, different compounds may have
the same tR.
To ensure that a peak represents the compound of interest,
spiking can be performed.

Is this Cpd X? Peak height /

area increases

Add Cpd X
Quantitative application
Measurement of Area - Integration

Area = ∫ Abs × dt

Modern HPLC
software have
integration
capabilities to aid in
the quantitation of
eluted compounds.
Standard curve using HPLC with
Photometric Detector
 Factors affecting retention
time
Depends on the
•affinity of the molecule in the sample for the
mobile phases versus the stationary phase
•pressure used (because that affects the flow
rate of the solvent)
•nature of the stationary phase (not only what
material it is made of, but also particle size)
•exact composition of the solvent
•the temperature of the column
 Internal Standards for Qualitative
& Quantitative applications
Retention time of a compound and amplitude of
detection can vary slightly each time due to:
1. Variation in operation conditions.
2. Variation in equipment function.
Introduce an Internal Standard as an internal control for
retention time as well as peak area.
Inject sample with
fixed amount of
internal standard
Internal each time.
Standard Take note of
retention time and
peak area of internal
standard.
Adjust for variation.
 Preparative applications
How can HPLC be used for preparative
purposes?

• can be used in pharmaceutical

development for troubleshooting
• purposes or as part of a systematic
scale-up process
Troubleshooting HPLC
(Credits: Sigma)
Troubleshooting HPLC
(Credits: Sigma)
Troubleshooting
HPLC
(Credits: Sigma)
Troubleshooting HPLC
(Credits: Sigma)
Troubleshooting HPLC
(Credits: Sigma)
Troubleshooting
HPLC
(Credits: Sigma)
Troubleshooting HPLC
(Credits: Pro04)
Troubleshooting HPLC
(Credits: Pro04)
Troubleshooting HPLC
(Credits: Pro04)
Troubleshooting HPLC
(Credits: Pro04)
 Recap
• Principles of HPLC
• Components of the HPLC
• Analysis of data from HPLC