Analsys Sciences - Introduction to HPLC

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AnalySys Sciences
www.analysciences.com 1
AnalySys Sciences
www.analysciences.com
Training
Method development
Chromatography
Mass Spectrometry

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High Performance Liquid Chromatography
Table of contents.
History
Chromatography – an introduction
Essential Theory
HPLC Hardware
Pumps
Detectors
UV-vis detectors
Fluorescence
Refractive Index
Diode array detection
Evaporative Light Scattering
Charged Aerosol detection
Electrochemical detection
Conductometric detectors
Amperometric detectors
Columns
Injectors
Mass spectrometry in HPLC
Troubleshooting HPLC systems
Validating HPLC systems
Sample Preparation in HPLC
Method development basics

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HPLC – The Basics
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100 years of chromatography
March 21, 1903
At the Warsaw Society of
Natural Scientists, Russian
botanist, Mikhail
Semenovich Tswett
presented the first lecture
on chromatographic
separation.
Kroma = color
graphein = writing

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Tswett’s separation
Tswett, MS (1906) Physico-chemical studies on
chlorophyll adsorptions.
Berichte der Deutschen botanischen Gesellschaft,
24, 316-23
Tswett, MS (1906) Adsorption analysis and
chromatographic method. Application to the
chemistry of chlorophyll.
Berichte der Deutschen botanischen Gesellschaft,
24, 385
http://www.life.uiuc.edu/govindjee/Part2/34_Krasnovsky.pdf
http://web.lemoyne.edu/~giunta/tswett.html

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 When a chlorophyll solution
in petrol ether is filtered
through the column of an
adsorbent …then the
pigments will be separated
from the top down in
individual colored
zones…the pigments which
are adsorbed stronger will
displace those which are
retained more weakly.
 Amongst the adsorption
means I can provisionally
recommend precipitated
CaCO3 which gives the
most beautiful
chromatograms.

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 "Like light rays in the spectrum, the different
components of a pigment mixture, obeying a law,
are separated on the calcium carbonate column and
can thus be qualitatively and quantitatively
determined.
 I call such a preparation a chromatogram and
the corresponding method the chromatographic
method."

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Chromatography is …
 “…a method in which the
components of a mixture are
separated on an adsorbent
column in a flowing system".
M.Tswett
 A separation involving two
phases and the sample. The
sample mixture undergoes a
series of interactions between
these two phases, resulting in
separation of its components.
 Sample components elute in
increasing order of interaction

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What interaction?
Some mechanisms…
Adsorption
…analyte in mobile liquid phase
adsorbed onto stationary solid
phase. Equilibration between the
mobile and stationary phase results
in separation

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Partition
…thin film of a liquid stationary
phase formed on a solid
support.

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Ion-exchange
IE resin is used to covalently
attach anions or cations
onto it. Solute ions of the
opposite charge are
attracted to the resin

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Affinity
specific interaction between a
solute molecule and a
molecule that is immobilized
on a stationary phase
eg. Protein / antibody

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Size Exclusion
a porous gel separates
molecules by size.

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Chromatography is …
…a “tug-of-war” between the mobile phase and the
stationary phase – each tries to hold on to the
sample as long as possible.
At the end of this war we get …

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One Chromatogram

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Some Equations

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Retention Volume
Volume of mobile phase required to elute a
particular analyte.
VR = tR x Fc
tR = Retention time
Fc = Flow rate

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Retention Time
Dead Time/volume
Retention time / retention volume
taken by an unretained solute to
elute from the system. Represents
the combined volume of tubings,
detector flow cell, injector loop,
column volume.
Relative (corrected) retention
time
0R Rt t t  

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Partition Co-efficient
(Distribution / Adsorption co-efficient)
M
sC
K
C


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Partition Ratio (Capacity Factor)
 Measure of the time spent
by a solute in the mobile
phase, with respect to the
stationary phase.
 For baseline separation,
K’ > 2

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Relative retention (Selectivity / separation factor)
For baseline separation, a > 1.5
2
1
k
k
a




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Selectivity
 Depends on
• Nature of the two phases
• Column temperature
higher temperature
will increase a

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Resolution
For baseline separation, Rs >2
2 1
1 2
2
R R
s
t t
R
w w


 
 
 

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Peak Width (4s)

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Tailing factor (Asymmetry/ Skew factor)
BC
As
CA


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Tailing factor - 2

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System Suitability Parameters USP
 Plate count > 2000 plates/meter
 Tailing factor < 2
 Resolution > 2
 Partition ratio > 2
 Relative retention > 1.5
 Precision / repeatability RSD </= 1% for n >/= 5

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Chromatography Theories
or… why a column will not do what it’s told..

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Plate theory Martin and Synge (1941)
 Nobel in Chemistry, 1952 for “their
invention of partition
chromatography”
 Column assumed to be similar to a
distillation column.
 Separation occurs across a series
of theoretical plates.
 Height Equivalent to a theoretical
plate. (HETP)
 Higher number of theoretical plates
(smaller HETP) improves column
performance.

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Rate theory Dr JJ Van Deemter (1956)
 Plate theory does not explain
band spreading and peak
broadening.
 Does not take into account
packing material, flow rate and
column geometry.
 Rate theory takes into account
various factors that cause peak
broadening.

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Van Deemter Equation
linear velocity ( flow rate)
C
H A B


  


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A term – Multipath effect
 Eddy diffusion
 Analyte molecules take
different paths thro‟ the
packing, leading to band
broadening
 Reduce particle size
 Backpressure will increase

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B term
 Longitudinal diffusion / wall
effect
 Distortion of the mobile phase
front, due to varying velocity
across the column, especially
at the column wall
 Increase flow rate
 Backpressure will increase.

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C term – mass transfer resistance
 Analytes remain trapped in
stagnant pockets in the
packing.
 Decrease flow rate

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Columns – Van Deemter plot

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HETP Height Equivalent to a theoretical plate
2
2
4
16
2
5.54
R
R
L
H
t
L
H
t
s
s
 
   
 
   

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Plate Count
2
2
16
4
25
5
R
R
t
t
s
s
 
   
 
   
2
5.54
2
R
L
N
H
t
s

 
   

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Plate count – what it means to the user.
 The plate count gives an idea of the efficiency
and separating power of a column.
 Higher plate count for a given column implies
better performance (but does not guarantee it !!)
 Plate count is affected by:
 Nature of sample
 Flow rate
 Detector flow cell volume
 Dead volume in the HPLC system
 Temperature
 Detector settings
 Data system settings.
 Injector reproducibility, etc…
 Be wary when comparing plate counts!!

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Quantitation in HPLC
 Area (height) under the
peak is proportional to the
injected amount.
 Proportionality constant is
the response factor.

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Peak Area
 Integration
 Data system sub-divides
peak into small rectangles,
calculates area of each, and
adds them up.

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Quantitation – External standards
 Known concentrations of the
analyte using reference standards.
 Analyse unknown under the same
conditions, in the same run
sequence.
 Start with lowest concentration.
 Use bracketing technique
 At least 5 injections per level

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Internal Standards
 Chemically similar to the
analyte
 Added to the sample and
external standards
 Same amount added to both
 Accounts for variations in
injection volume and other
system variables
 Provides better precision
 Not always possible to obtain
chemically similar internal
standard.

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HPLC - The System

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Pumps

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LC – Pump Considerations
 Pulse-free flow
 Flow rate precision / accuracy
 Backpressure capacity
 Piston volume
 Flow path contact materials

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Reciprocating Pump
 Single-piston reciprocating
pump
 Cam-drive
 Single-pistons have a
significant pulse.
Source: www.lcresources.com

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Pumps - Components
 Piston: Sapphire
 Check valves: Ruby
 Piston seals: HDPE

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Pump dampening methods
 Mechanical pulse dampeners
 Asymmetric gears / elliptical
cams
 Electronic pulse dampening
 Free-floating piston
 High refill speed (<100
milliseconds)
 Add one more piston

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Reciprocating pumps
 Dual piston reciprocating pump
 Cam-drive
 Two pistons in tandem
 There is still a small pulse
 Due to the crossover point
Source: www.lcresources.com

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Pumps - Elution
 Isocratic elution
Mobile phase composition remains constant
during the run
 Gradient elution
Mobile phase composition changes during the
run.

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Why gradients?
 To separate analytes of differing polarities
 multivitamin mixture
 amino acids
 impurity profiles
 To shorten run time
 To improve separation efficiency

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Gradients – high pressure mixing
 One pump for each solvent
 Solvents mixed under
pressure.
 Mixed in a mixing chamber
 Static mixer
 Dynamic mixer

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Gradients - low pressure mixing
 Single pump
 Proportioning valve before
the pump mixes different
solvents
 Solvents mixed in a mixing
chamber
 Solvents must be degassed
before use.

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Gradient Mixers
 Static mixers
Mixing tee joint
Low dead volume
Inexpensive
Non-reproducible mixing
 Dynamic mixers
Small stirrer bar inside a mixing
chamber
High dead volume
Expensive
Homogenous reproducible mixing

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Pumps - degassing
 Mobile phase must be
degassed to remove dissolved
air.
 Especially in gradient elution
and where water is used in the
mobile phase.
 Else, noisy baselines and
pressure fluctuations will
result.

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Degassing methods
 Helium sparging
 Best method, but expensive.
 Prolonged sparging will alter
composition.
 Degas solvents separately.
 Ultrasonication
 Good degassing method.
 May heat the mobile phase and
alter composition.
 Degas solvents separately.

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Degassing -2
 Membrane filtration
 Not too bad, not too good.
 Use compatible membrane
 0.45 m pore size
 On-line membrane degassers
 Mobile phase moves across a
semi-permeable membrane.
 Dissolved gases permeate out of
the mobile phase.

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Typical pumps
 Typical single-piston pump
 Piston-seal rinse
 “Free-floating” piston
 0.01 to 10 ml/min

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Agilent 1100
 Typical dual-piston pump
 Piston seal rinse
 Built-in prime/purge valve

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HPLC – Sample introduction
 The injector must introduce small volume of sample against
high backpressure.
 Typical injection volumes are 10 to 20 l.

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HPLC – Sample Introduction
 Stop-flow injection
 Stop the pump briefly, inject sample thro‟ septum, resume flow
 Flow-rate inaccuracies, distorted peak shapes
 Obsolete
 On-line sample injection
 Rotary valve injectors
 Valco, Rheodyne

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Rheodyne 7725i

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Columns in HPLC

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HPLC - Columns
 The column is the heart
of the system
 Usually made of SS
316L
 Packed with
microparticulate
packings, of various
chemistries

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Microparticulate packings
 Usually silica (silicic acid)
 Silica can be chemically modified
with different functional groups
 3 to 5 m particle size
 Irregular or spherical particles
 Porous, ~ 100 A pore size

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Silica phases – normal phase.
 Silicic acid is made of silanol
groups.
 (SiOH)x
 Silanols are polar in nature,
and cannot retain non-polar
analytes.
 Silica is water-soluble, and
does not permit water in the
mobile phase.
 For non-polar separations,
silica must be chemically
modified.

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Bonded phases

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Reverse Phases

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Bonded phases

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End-capping
Steric hindrance prevents
complete reaction with bonded
phases.
This leaves unreacted silanol
groups and polar sites.
Causes peak tailing and poor
separations.

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End-capping
 A smaller hydrocarbon group
(usually C3) is used to „cap‟
the unreacted silanols, after
the initial reaction with a C18
or C8 hydrocarbon.
 This technique is called end-
capping.
 Improves peak shape
 Reduces tailing
 Increases resolution and
selectivity

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Reverse phase retention

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RP Column evaluation parameters
 Carbon load ~15%
 End-capped? Yes.
 Particle size and shape ~ 5 m
 Pore size ~ 80 to100 Ǻ
 Dead volume < 0.5 ml
 Plate count > 10,000
 Silica purity Ultrapure, base
deactivated silica
 Silanol activity
 Hydrophobicity Toluene test
 Always check and replicate the test chromatogram.

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Column fittings
 Low dead volume fittings
 Compression fitting
 SS frit.
 5 pore size for regular
analytical columns.
 2 for microbore columns.

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Detectors

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Detector types
 Solute property detectors
 Detect a property specific to the analyte
 UV, fluorescence, IR, mass spectrum
 Bulk property
 Detect overall changes
 Refractive index, conductance.

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Important Parameters
 Limit of detection
 Lowest amount that can be
detected.
 S/N 2:1 or 3:1
 Limit of quantitation.
 Lowest amount that can be
quantitated with acceptable
precision. Usually S/N 10:1
 Linear Dynamic Range
 That range of concentrations
over which detector gives a
linear, proportional response.

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UV-Visible Detectors.

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UV Detection - basics
 Transmittance
 Absorbance
Expressed as absorbance units. (AU)

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Beer’s Law
(Beer-Lambert-Bouguer law)
A = ebc
A = absorbance
e = molar absorptivity (L mol-1 cm-1)
(extinction co-efficient)
b = path length of the sample (cm).
c = concentration of the analyte
(mol/L)
Pierre Bouguer (1698 –1758),
French mathematician and
astronomer.
The original discoverer of
Beer’s Law, circa 1729.

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UV detectors

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UV – visible sources
 Low pressure Hg lamp
Emits lines at 253.7 nm (very strong), 313 nm,
365 nm, 407 nm, 435.8 nm, 546.1 nm, 577
nm, 579.1 nm
 Deuterium lamp
Emits a continuum from 180 to 700 nm
 Xenon arc lamp
Intense continuum from 180 to 1100 nm
 Tungsten-halide lamp
Continuum from 280 nm to 1100 nm

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Dispersion devices
 Diffraction Gratings
 Reflecting or transparent
substrate surface with fine
parallel grooves or rulings.
 Diffractive and mutual
interference effects occur,
and light is reflected or
transmitted in discrete
directions, called orders.

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Monochromator configurations
Czerny-TurnerLittrow Mount

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Quartz Flow cells
 RI effects will distort baseline.
Flow cell geometry must be
optimised
 Flow cell volume affects peak
shape and LOD
10 l for analytical HPLC
 Backpressure limit 500 psi

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Photomultiplier tubes
 Glass vacuum tube with a photocathode, several dynodes, and an anode. Incident
photons strike the photocathode and produce electrons. (Photoelectric effect)
 On striking the first dynode, more low energy electrons are emitted and these, in
turn, are accelerated toward the second dynode.
 A cascade occurs with an ever-increasing number of electrons. Finally at the
anode, there is a sharp current pulse.

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PMT’s
 Very sensitive
 Take time to stabilise
 Finite response time
 Tracking error at high scan
speeds
 Tunable sensitivity and gain
 Dark current and baseline
noise at high gain.

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Photodiodes
p-n junction
 When a photon strikes a
semiconductor, it can promote
an electron from the valence
band (filled orbitals) to the
conduction band (unfilled
orbitals) creating an electron(-) -
hole(+) pair.
 The concentration of these
electron-hole pairs is dependent
on the amount of light striking
the semiconductor.
 Photovoltaic detectors contain a
p-n junction, that causes the
electron-hole pairs to produce a
voltage that can be measured.

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Photodiodes - 2
 Short warm-up time
 Rapid response
 Inexpensive
 Not as sensitive as PMT‟s
 Best used as diode arrays.

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Photoelectric effect
 Upon exposing a metallic surface to
electromagnetic radiation, the photons are
absorbed and current is produced.
 The energy of the photon is absorbed by
the electron and, if sufficient, the electron
can escape from the material with a finite
kinetic energy.
 A single photon can only eject a single
electron, as the energy of one photon may
only be absorbed by one electron. The
electrons that are emitted are termed
photoelectrons.

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Diode Array Detectors.

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Is this is a PURE peak?
Diode Array Detection

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The Co-elution problem

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Peak Purity – Absorbance Ratios
 Absorbance is measured at
two or more wavelengths.
 Ratios are calculated for two
selected wavelengths.
 If the compound under the
peak is pure, the ratio will be a
square wave function
(rectangle).
 If not, the peak is not pure.

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Spectral Index

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Peak Purity – Spectral Overlay

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How does one scan a peak?
 Stop-flow scanning
 Stop the pump at the peak of interest and scan rapidly using a
scanning detector.
 Peak and/or peak merging broadening occurs
 Disturbance in flow and loss of resolution
 Not reproducible
 Obsolete
 On-the-fly scanning
 Use a high-speed detector to rapidly scan peak as it passes
through the flow cell.
 Unreliable spectra obtained
 Tracking error

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Enter … Diode array
 An array of photodiodes,
instead of a single PMT or
dual-photodiode
 Usually around 512 to 1024
diodes
 Resolution depends on
number of photodiodes and
polychromator resolution.

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PDA Schematic

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Spectral angle

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Diode Array – ‘Benefits’
Simultaneous plots of absorbance,
time, and wavelength
Easier to detect hidden peaks and
co-eluants. For eg. Secondary
metabolites.
Easier to estimate lmax
No scanning, no tracking error.
Expensive.

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PDA detectors - parameters
 Resolution
 „Electronic‟ resolution
 Wavelength range / no. of
diodes
 Usually around 1.2 nm
 „Optical‟ resolution
 Function of grating efficiency
 Usually around 2 nm
 Moral: More diodes doesn‟t mean
higher resolution.

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PDA - Not a substitute for good chemistry!
You still got to separate them!

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Refractive Index Detectors

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Refractive Index
 Fermat's principle or
the principle of least
time
 the path taken between
two points by a ray of
light is the path that
can be traversed in the
least time.

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Snell’s Law

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Refractive Index
 Refractive Index
Dependent on:
 Wavelength of incident light
 Temperature
 Viscosity
 Expressed as RIU.
 (refractive index units)

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RI Detectors
 „Universal‟ detectors.
 Reasonably sensitive.
 Generally used for analytes
that do not have
chromophores.
 Carbohydrates / sugars.
 Polymers.
 Proteins.

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RI detectors - optics
 Deflection type
Differential refractometer
 monitors the deflection of a
light beam caused by the
difference in refractive index
between the sample cell
and the reference cell.

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RI detectors – optics 2
 Reflection type
Fresnel refractometer
 monitors the loss of
intensity of an incident light
beam, caused by the
difference in refractive index
between the sample cell
and the reference cell.

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RI Detectors - Limitations
 Very sensitive to changes in temperature.
Column thermostat is a must.
 Sensitive to changes in flow rate.
 Very sensitive to changes in mobile phase
composition. CANNOT use gradients.
 Sensitive to small air bubbles and
particulates.
 Take a long time to stabilise, especially if
baseline is disturbed by any of the reasons
above.
 Use is limited to fairly simple molecules like
carbohydrates, that can be separated using
isocratic conditions.

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Fluorescence Detectors

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Fluorescence
 Re-emission of previously
absorbed light
 Fluorescence detectors are
probably the most sensitive
HPLC detectors. It is possible
to detect even a single analyte
molecule in the flow cell.
 Fluorescence sensitivity is 10 -
1000 times higher than that of
the UV detector for strong UV
absorbing materials.
 Very specific detectors

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Luminescence
 Fluorescence
 Shorter life-times, typically micro to nanoseconds
 Phosphorescence
 Longer lifetimes, upto 10 secs.

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Fluorescence detectors - optics
 900 optics
 Filter-based
 Low-sensitivity
 No scanning

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Fluorescence – Scanning detectors

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Fluorescence detectors - optics
 900 optics
 Dual monochromator
 Xenon source
 PMT detector

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Fluorescence - applications
 Compounds with conjugated p electrons.
 Polyaromatic hydrocarbons (PAH‟s).
 Functional groups like carbonyls.
 Aliphatics that can be derivatised with fluorophores.
 OPA derivatives of amino acids
 FAME‟s (fatty acid methyl esters)

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Aflatoxins

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A typical application
Amino acids in serum
 Amino acids are UV-transparent
 Derivatisation necessary
 Orthophthaladehyde (fluorescent
derivatives)
 Ninhydrin (detection at 650 nm)
 Phenythiohydantoin (UV detection)
 Post-column derivatisation
 Ion-ex columns
 Pre-column derivatisation
 Reverse phase columns
Automated derivatisation with o-
phthalalydehyde for estimation of amino
acids in plasma using reversed-phase
high performance liquid chromatography.
Indian Journal of Biochemistry and
Biophysics, 41, 322-325, Dec 2004

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Light Scattering Detectors

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Light Scattering
 Why is the sky blue?
 Due to selective scattering
or Rayleigh scattering.
 Small particles are more
effective at scattering a
particular wavelength of
light. Air molecules, are
small in size and thus
more effective at
scattering shorter
wavelengths of light
(blue and violet).

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 Why are clouds white?
 Mie Scattering is
responsible for the white
appearance of clouds.
Cloud droplets with a
diameter of 20 μ or so are
large enough to scatter all
visible wavelengths equally.
Because all wavelengths
are scattered, clouds
appear white.

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Light scattering in HPLC
 Any analyte can, under the right
conditions, scatter an incident beam of
light.
 Amount of light scattered is directly
proportional to the molecular weight, size
and concentration of the analyte.
 Thus, light scattering detection can be
used for many analytes.

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ELSD – principles
 Nebulisation
 Eluent from the column is nebulised
into a fine mist using a heated inert
gas (usually nitrogen).
 Evaporation
 The mist (aerosol cloud) is propelled
through a heated drift tube in which
the solvent evaporates and only
sample particles remain.
 Detection
 Analyte particles emerging from the
evaporation tube enter the optical cell,
where they pass through a beam of
light. The particles scatter incident
light. The amount of light detected is
proportional to the solute
concentration and solute particle size
distribution.

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ELSD – pros and cons
Pros
 Universal detection.
 Rapid equilibration.
 No restriction on use of
gradients.
 Easy to use.
 Sensitive.
Cons
 Reproducibility not good.
 Difficult to validate.
 Nebuliser gets clogged and
requires regular cleaning.

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Charged Aerosol Detection

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Corona CAD

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CAD – Principle.
 HPLC column eluent is first nebulized
with nitrogen and the droplets are dried
to remove mobile phase, producing
analyte particles.
 A secondary stream of nitrogen
becomes positively charged as it
passes a high-voltage, platinum corona
wire. This charge transfers to the
opposing stream of analyte particles.
 The charge is transferred to a collector
where it is measured by a highly
sensitive electrometer, generating a
signal in direct proportion to the
quantity of analyte present.

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CAD – advantages.
 More sensitive than ELSD.
 Higher reproducibility, <2%.
 Can be validated.
 Large dynamic range.

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CAD – applications.
Virtually any non-volatile compound,
including:
 Drugs.
 Carbohydrates
 Lipids
 Steroids
 Peptides/ Proteins
 Polymers
In industries such as:
 Pharmaceutical
 Foods
 Consumer products
 Industrial chemicals
 Life science research

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Electrochemical Detection

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Electrochemical Detection
What is electrochemistry?
 Branch of chemistry that studies
reactions that occur at the interface
of an electron conductor (the
electrode) and an ionic conductor
(the electrolyte)
 These reactions involve electron
transfer between the electrode and
the electrolyte.
 Electron transfer can be caused by
an external voltage, or by an internal
chemical reaction.
 Reactions in which electrons are
transferred between atoms are
called oxidation/reduction (redox)
reactions.

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Ohm’s Law
V = iR
V = potential difference, volts
i = current, amperes
R = resistance, ohms.
Any of these three parameters can be
used for quantitative estimations of
electroactive compounds.
 Resistivity or Conductance
 Conductometric detectors.
 Current
 Amperometric detectors
 Coulometric detectors.
George Simon Ohm, 1789-1854

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Conductometric detectors
 Conductance
 The ease with which electric
current flows through a
substance.
 Inverse of resistivity.
G = 1/R
 Expressed as siemens or
ohms-1 or mhos.

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Conductometric detectors.
 Bulk property detectors.
 The flow cell is placed in one arm of a
Wheatstone bridge.
 Any ions in the eluent will alter the
conductance and create an out-of-
balance signal.
 This signal is rectified and presented
as a chromatogram (null-balance
principle).
 If buffers are used in the mobile phase,
there will be a large background signal,
that must be suppressed.

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Conductometric Detectors
 Can be used only for analytes
that are already ionised, like
inorganic acids, bases, salts.
 Some examples:
 Pollutants in drinking water.
 Electroplating solutions.
 Carbonates in beverages.
 Nitrates/nitrites in processed
foods.

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Electrochemical Detectors
 An electrochemical (redox)
reaction in the detector flow
cell is generated by an
externally applied voltage.
 Analyte undergoes reduction
or oxidation.
 Current is generated as a
result.
 That current is directly
proportional to the analyte
concentration, and can be
measured and quantified.

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Redox Reactions
LEO the Lion says GER
 Loss of Electron = Oxidation
 Gain of Electron = Reduction

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A typical redox reaction
 This reaction requires a certain
amount of energy.
 This energy is supplied by an
externally applied voltage.
 Electron transfer occurs during the
redox reaction.
 This results in a current, that can
be measured.
 The optimum voltage required is
specific to this reaction.
O
O
OH
OH
+ 2H+ + 2 e-
Hydroquinone Quinone
oxidation
reduction

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Electrochemical cells
 An electrochemical cell is a device
that produces electric current from
energy released by a redox
reaction, i.e. it converts chemical
energy to electrical energy.
 Electrochemical cells have two
electrodes – the anode and the
cathode.
 The anode is where oxidation
occurs and the cathode is the
electrode where the reduction takes
place.
 Electrodes come in various forms
including metal, gas and carbon.

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Electrodes
 an electrode is a conductor
through which electric
current is passed. It is used
to make contact with a
nonmetallic part of a circuit,
eg with an electrolyte, or
with a vacuum.

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Electrochemical cells.
 Electrochemical work within an
electrochemical cell is done by a
potentiostat.
 A potentiostat is an electronic
device that controls the voltage
difference between a working
electrode and a reference
electrode.
 The potentiostat implements this
control by injecting current into the
cell through an auxiliary
electrode.
 The potentiostat measures the
current flow between the working
and auxiliary electrodes.

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Electrochemical cells
Working Electrode:
Electrochemical reactions occur here.
It can be metal or coated.
Reference Electrode:
Used in measuring the working
electrode potential.
Has a constant potential, provided no
current flows through it.
Auxiliary Electrode:
Is a conductor that completes the cell
circuit.
Prevents current from flowing into the
reference electrode.
Usually an inert conductor like
platinum or graphite.

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Reference Electrodes
 Potential difference is always
measured with respect to an
electrode of known potential.
 The reference electrode has a
known, invariant potential, against
which the potential of the working
electrode can be measured.
 Typical reference electrodes:
 Standard Hydrogen electrode
 Potential = 0 by definition.
 Ag/AgCl electrode
 Potential = 0.224V with respect to
SHE.

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The Ag/AgCl electrode
 A silver wire that is coated with a
thin layer of silver chloride, either
by electroplating or by dipping the
wire in molten silver chloride.
 When the electrode is placed in a
saturated potassium chloride
solution it develops a potential
proportional to the chloride
concentration, and remains
constant as long as the chloride
concentration remains constant.
 Most reference electrodes use a
saturated KCl solution with an
excess of KCl crystals.

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Amperometric Flow Cells
 Analyte moves across the
surface of the working
electrode.
 Redox reaction occurs on the
working electrode surface.
 Glassy carbon is the most
commonly used working
electrode.

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Thin layer flow cell.
R O R O R O
Reference Electrode
Counter Electrode
Outlet
Working
Electrode
Inlet

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Amperometric flow cells
Limitations.
 Redox reaction does not proceed to completion. Usually not more
than 5% of the analyte is reduced/oxidised.
 Sensitivity is not very high.
 Electrodes foul up regularly, maintenance and polishing needed at
regular intervals.
 Tend to drift, require long warm-up time.

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Coulometric flow cells.
 Working electrode is porous,
usually porous graphite.
 Analyte moves through the
electrode, not across it.
 Therefore, much higher area is
available for the redox
reaction.
 Complete reaction of the
analyte is possible, thus
achieving higher sensitivity.
Counter and
Reference electrodes
High pressure
cell body
Electrode
5020 cell
(55-0417)

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Dual flow cells
 Two working electrodes or flow
cells in series.
 Enables detection of analytes
at different redox potentials or
enhanced detection of the
same analyte.
 Or can be used to reduce
interfering substances in the
mobile phase.
Counter and
Reference electrodes
Working
electrode #2
Working
electrode #1
5010 cell
(55-0411)

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Electrode Arrays
 An array of working electrodes is
used. Upto 80 electrodes in series
have been connected.
 A progressively greater potential is
applied sequentially to the
electrodes of each consecutive unit.
This results in all the analytes
migrating through the array until
each analyte reaches the unit that
has the required potential to permit
its oxidation or reduction.
 Sample analytes are totally reacted
and each analyte it will be detected
by that unit that has the required
potential and not be sensed by other
units.

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Electrode Array - Advantages.
 The electrode array detector gives
improved apparent chromatographic
resolution similar to a diode array
detector.
 Two peaks that have not been
chromatographically resolved and are
eluted together can still be shown as
two peaks that are resolved
electrochemically and can be
quantitatively estimated.
 Produces a characteristic pattern of
peaks for a particular analyte, that can
be used to confirm the purity and
identity of the analyte.
 Array detectors produce less
background noise and enhanced signal-
to-noise ratios.

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ELCD - Modes
 DC Mode
 A constant potential is applied to the working
electrode and the current produced is plotted
against time.
 Most common mode.
 Scan Mode
 Used to generate a voltammogram of the analyte
of interest.
 By passing a solution of the analyte through the
detector cell, a current-potential curve is
generated that can be used to optimise the
detection voltage for that analyte.
 Scan mode does not involve a chromatographic
separation.

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ELCD - Modes
Pulse mode
 Reaction products can clog the surface of the
electrode, badly affecting its performance.
 In pulsed mode, a cyclic series of potentials is
applied to the working electrode to clean the
electrode surface.
 A measuring potential is applied and after a
suitable equilibration time, a measurement
of the current is made.
 A large positive potential is applied to the
electrode, that oxidises any reaction
products on the electrode.
 A negative potential is applied to reduce the
electrode and bring it back to its base
metallic state.
 Usually this cycle lasts less than 1 second,
and is done continuously during the
analysis.
E1
Acquisition delay Measurement
T1
E2
Cleaning
E3
Regeneration
T2
T3

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Coulometric detectors – pros and cons.
High conversion efficiency.
Maintenance free – no
polishing needed.
Fast equilibration time.
Less sensitive to flow
fluctuations.
Multiple cell arrays
possible.
Can clog up over time.
Once clogged, must be
replaced.
Noise can be higher than
in amperometric cells.

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General precautions
 Mobile phase must be able to conduct
current, hence water is essential. Therefore,
non-aqueous separations not possible.
 Mobile phase must be free from dissolved
gases, especially O2, hence thorough
degassing is a must.
 Mobile phase must be free from metal ions
and microparticulates.
 ELCD‟s are sensitive to flow rate variations,
and a very good HPLC pump is needed.
 Temperature control is critical, and a good
column thermostat is needed.

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Conductometry v/s ELCD.
Conductometric
 Analyte is already ionised.
 Bulk property detector.
 Detects overall change in
conductance.
 Not specific to the analyte.
Electrochemical
 Analyte is ionisable. It is
ionised inside the detector
flow cell by applying a
suitable voltage.
 Solute property detector.
 Specific to the analyte.

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Glossary of electrochemical terms
Potential Difference
 The electrical potential difference between two points in a circuit results in a flow of current.
In electrochemistry we typically cannot measure "absolute" potentials, only the "difference"
of potential between two points. The measurement unit of the potential is the volt.
Resistivity (Resistance)
 The measure of a material's inability to carry electrical current. The measurement unit of the
resistivity (resistance) is the ohm.
Current
 The movement of electrical charges in a conductor; carried by electrons in a conductor.
Electrical current always flows from the positive potential end of the conductor toward the
negative potential end.
 Direct current is the unidirectional continuous flow of current, while alternating current is the
oscillating (back and forth) flow of current.
 The measurement unit of current is the ampere.

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Mass Spectrometry in HPLC

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Introduction
 Designed to separate gas
phase ions according to their
m/z (mass to charge ratio).
 A mass analyser separates
the gas phase ions, via
electrical or magnetic fields, or
combination of both, to move
the ions to a detector, where
they produce a signal which is
amplified.
 The analyser is under high
vacuum, so that the ions can
travel to the detector with a
sufficient yield.

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Mass spectrum

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MS Schematic

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Electron Impact ionisation
The most widely used of all
ionization methods
Sample is vaporized into the
mass spectrometer ion source,
where it is impacted by a beam of
electrons with sufficient energy to
ionize the molecule.
For most organic molecules, the
ion yield is a maximum at 70 eV
energy.

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Chemical Ionisation
“Soft” ionisation technique.
Used when no molecular ion is observed in EI mass spectrum, or when you want
to confirm the m/z of the molecular ion.
Same ion source device as in EI. Reagent gas (e.g. ammonia) is first subjected to
electron impact. Sample ions are formed by the interaction of reagent gas ions and
sample molecules.
Reagent gas molecules are present in the ratio of about 100:1 with respect to
sample molecules.
Positive ions and negative ions are formed in the CI process. Depending on the
setup of the instrument (source voltages, detector, etc...) only positive ions or only
negative ions are recorded.
Eg. Mass spec of trisilyl derivatives of amino acids.

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Electrospray Ionisation
Analyte is introduced to the source
at low flow rates. Passes through the
electrospray needle at high potential
difference.
This forces the spraying of charged
droplets from the needle.
Solvent evaporation occurs. The
droplet shrinks until the surface tension
can no longer sustain the charge (the
Rayleigh limit) at which point a
"Coulombic explosion" occurs.
This produces smaller droplets that
repeat the process, until complete
ionisation occurs. A very soft method of
ionisation.

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Atmospheric pressure (APCI)
Analogous ionisation method to chemical ionisation.
The significant difference is that APCI occurs at atmospheric pressure.
Cannot be used for thermo-labile compounds
Can be used at high flow rates (1 ml/min) unlike ESI.

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APCI - 2
Analyte solution is introduced into a pneumatic
nebulizer and desolvated in a heated quartz
tube before interacting with the corona
discharge creating ions.
The corona discharge replaces the electron
filament in CI and produces primary ions by
electron ionisation.
These primary ions collide with the vaporized
solvent molecules to form secondary reactant
gas ions.
These reactant gas ions then undergo
repeated collisions with the analyte resulting in
the formation of analyte ions.

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MALDI
Soft ionization technique.
The ionization is triggered by a
laser beam (normally a nitrogen-
laser). A matrix is used to protect
the analyte from the laser beam.
The matrix consists of crystallized
molecules.
The laser is fired at the crystals in
the MALDI spot. The spot absorbs
the laser energy and the matrix is
ionized. The matrix transfers part of
the charge to the analyte, thus
ionizing it.

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Magnetic Sector
It uses an electric and/or magnetic
field to affect the path and/or velocity
charged particles.
The ions enter a magnetic or electric
field which bends the ion paths
depending on their mass-to-charge
ratios (m/z), deflecting the more
charged and faster-moving, lighter ions
more.
The ions eventually reach the detector
and their relative abundances are
measured.
The analyzer can be used to select a
narrow range of m/z's or to scan
through a range of m/z's.

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A typical Mag sector MS
AMD Intectra M40 SF

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Quadrupole
 Two pairs of metallic rods. One
set at a positive electrical
potential, and the other one at a
negative potential.
 A combination of dc and rf
voltages is applied on each set.
Vrf/Vdc ratio determines the mass
resolution.
 For a given amplitude of the dc
and rf voltages, only the ions of a
given m/z will resonate, have a
stable trajectory to pass the
quadrupole and be detected.
 Other ions will be de-stabilized
and hit the rods.

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Q’pole - Modes
 SIM mode (single ion monitoring)
The (amplitude of the dc and rf voltages ) are set to observe only
a specific mass, or a selection of specific masses. Provides the
highest sensitivity for specific ions or fragments.
More time can be spent on each mass (dwell time).
 Scan mode
Amplitude of the dc and rf voltages are ramped (while keeping a
constant rf/dc ratio), to obtain a mass spectrum over the required
mass range.

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Ion Traps

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Ion Traps
Ring electrode and two end cap electrodes. The ions are
stabilized in the trap by applying a RF voltage on the ring
electrode.
He or N2 used as a damping gas to restrict ions to the
center of the trap.
By ramping the RF voltage, or by applying supplementary
voltages on the end cap electrodes, or by combination of
both, one can:
destabilise the ions, and eject them progressively from the
trap (Scan mode)
keep only one ion of a given m/z value in the trap, and then
eject it to observe it specifically (SIM mode)
keep only one ion in the trap, fragment it by inducing
vibrations, and observe the fragments. (MS/MS).

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Ion traps v/s Quads
 Quads
 Good resolution
 Stable, reproducible.
 Better suited for LC-MS
Need additional mass
analyser(s) for MS-MS
Cost more than Traps
Traps
Compact, bench-top.
Do not need additional
mass analysers for MS-
MS.
Better suited for GC-MS.
Reproducibility issues.
Very sensitive to
moisture.

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Time-of-Flight
Ions formed in an ion source are
extracted and accelerated to a high
velocity by an electric field into a drift
tube. The ions pass along the tube until
they reach a detector.
The velocity reached by an ion is
inversely proportional to the square root
of its m/z value.
Since the distance from the ion origin to
the detector is fixed, the time taken for
an ion to traverse the analyser in a
straight line is inversely proportional to
the square root of its m/z value.
Thus, each m/z value has its
characteristic time–of–flight from the
source to the detector.

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Detection systems
An electron multiplier (continuous
dynode electron multiplier) multiplies
charge.
Ions induce emission of electrons on
PbO coated metal.
If an electric potential is applied from
one metal plate to the other, the
emitted electrons will accelerate to
the next metal plate and induce
emission of more electrons.
12 stages of acceleration will usually
give a gain in current of 10 million.

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Tandem Mass Spec
 Tandem mass spectrometry employs two or more
stages of mass spectrometric analysis.
 Each mass spectrometer might scan, select one ion
or transmit all ions.
 Dramatic increase in S/N and selectivity.
 Structure confirmation and identification.

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Product ion MS (daughter ion)
 MS1 is used to select a parent
ion, that is fragmented again.
 Usually by CAD (collision-
activated dissociation) with
argon.
 MS2 scans the daughter ion to
provide a mass spectrum.

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MSMS – pesticide residues

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SRM(Single reaction monitoring)
 By fixing MS1 on the mass-to-
charge ratio of interest, the signal
at the detector is improved.
 To eliminate interference from
isobaric ions and the isotopic
contribution of lighter analytes,
one can select, after
fragmentation, a product ion
characteristic for the analyte of
interest using MS2.
 A single reaction is monitored,
yielding a highly selective
detection with high sensitivity
because of the removal of
chemical noise.

186
Troubleshooting HPLC systems
Backpressure
Peak shape
Baseline
Retention
Maintaining columns
Restoring clogged columns
Avoid the void
HPLC Syringes
Table of contents

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High backpressure
Column frit or solvent filter clogged
Check-valves clogged or stuck
Sonicate or replace
Injector in wrong position
Leave injector in inject position
during run
Tubing diameter too small
Mobile phase viscosity too high
Minimise water
 High backpressure increases
wear and maintenance costs
 Do not neglect high
backpressure

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Low backpressure
• Loose fittings
• Solvent in-line filter
• Prime valve
• Dynamic mixer/tee joint
• Column / guard column end fittings
• Worn out seals / check valves
• Pump seals / Check-valves
• Injector seals
• Chronic high backpressure
•
Do not over-tighten any fitting
Avoid the use of teflon tape on
fitting threads !
Troubleshooting Menu

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Drifting baseline
 Steep gradients ( Refractive index effect)
 Change composition gradually
 Temperature fluctuations
 Use column oven
 Mobile phase changeover
 Late peak from previous injection
 Wait
 Aging UV lamp
 Reverse phase bleed (rare)
 Change column

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Baseline noise
 Random noise
 Bubble in flow cell.
 Degas solvents before use.
 Dirty solvents.
 Aging UV lamp.
 Pulsating baseline
 Pulse dampener failure.
 Voltage fluctuation.

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Baseline noise
 Synchronous noise
 Pump failure
 Spikes in baseline
 Air bubble in flow cell
 Particulate contaminants
 Voltage fluctuation

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Baseline noise
 Contaminated buffer

If you use a pH meter, never,
put the pH electrode in the bulk
mobile phase.
 Transfer an aliquot of the
solution to a test tube or small
beaker, measure the pH, and
then discard the aliquot.
 Contamination from the pH
electrode can contribute to
baseline noise and/or garbage
peaks.
Source: http://www.lcresources.com/wiki/index.php?title=ChromFAQ:PHAdjust

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Split peaks
 Void at column head
 If all peaks split
 Memory effect
 From previous injection
 Flush injector before use
 Sample deterioration
 If one or two peaks split
 Injector seal leak

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Broad peaks
 Injection volume too large
 System leak
 Excessive dead volume
 Wrong flow rate
 Mobile phase pH or composition
Source : www.lcgcmag.com

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Ghost peaks
 Late peak from previous run
 Flush column and injector
 Increase run time
 Contaminated sample
solvent or mobile phase
 Confirm with blank run

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Missing peaks
Sample degradation
Overnight use of
autoinjectors, unstable
samples, derivatised
samples
Use cryogenic sample tray
Store samples below
ambient
Use amber vials
Prepare derivatives fresh Troubleshooting Menu

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AnalySys Sciences
Negative Peaks
 Absorbance or refractive index
of sample lower than mobile
phase
 Change wavelength
 Detector polarity reversed
 If all peaks negative
 Ion-pair reagent / solvent
interaction
 Change solvent
 Contamination of mobile phase

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AnalySys Sciences
Rounded peaks
 Sample overload
 Reduce sample
concentration and/or
volume
 Detector out of range
 Adjust detector sensitivity

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AnalySys Sciences
Loss of peak height
 Sample deterioration
 Injector seal/System leak
 Aging UV lamp
 Wrong injection technique
 Use total-loop technique

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AnalySys Sciences
Tailing peaks
Active sites on column
Use 0.1 % TEA in mobile phase
Sample ionisation
Adjust pH to suppress ionisation
K‟ too large
Increase mobile phase strength
Insufficient end-capping
Change column
Hidden peak on tail
Change detection wavelength
Change mobile phase strength

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AnalySys Sciences
Fronting peaks
 Sample overload
 Reduce sample concentration
 Reduce sample volume
 Unresolved peak on the front
 Change wavelength
 Change mobile phase
 Sample solvent incompatible
with mobile phase
 Dissolve sample in mobile
phase

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AnalySys Sciences
Retention Time Changes
 Flow rate variation
 Check pump
 Change in mobile phase
 Altered composition
 pH change
 Temperature change
 Use column oven
 System leak

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AnalySys Sciences
Analytical HPLC Tubings
 0.009” ID
 From injector to column
 From column to detector
 0.020” ID
 From pump to injector
 0.040” ID
 Detector outlet

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AnalySys Sciences
Restoring clogged frits - 1
 Disconnect column from detector
 Reverse the column and reconnect to
pump
 If using buffers, first flush with water @
0.5 ml/min, one hour.
 Flush with MeOH or CH3CN @ 0.5
ml/min for one hour
 Check backpressure. If normal,
reconnect column in normal direction
 Check backpressure again, at usual
flow rate, with mobile phase.
 Revalidate column with SOP

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AnalySys Sciences
Clogged frits - 2
 Badly clogged frits
 Remove column
 Unscrew column end-fitting
 Carefully slide the frit out.
 Sonicate frit in 50% aq nitric acid
for 30 mins, followed by HPLC
water for one hour.
 Do not sonicate in chromic acid
 Sonicate in mobile phase for 10
mins
 Restore frit
 OR
 Buy a new frit

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AnalySys Sciences
Dead volume
 Excessive dead volume can
adversely affect results
 Optimize tubing length and
 diameter
 Use correct detector flow cell
 (10 to 20l for analytical HPLC)
 Use Zero-Dead-Volume fittings

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AnalySys Sciences
Dead volume -2

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AnalySys Sciences
Avoid the void
 Change flow rate gradually
 Use ramping feature in the
software
 Use guard column
 Same packing as main column
 Use column in flow direction
only.
 It is a sin to reverse the column
 Mechanical shocks disturb
packing

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AnalySys Sciences
Column maintenance
If using buffers:
 Flush system with water @ 0.5
ml/min for 30 mins, followed by
CH3CN or MeOH for 30 mins
 Leave injector in inject position
while flushing.
 Rinse piston seals with 100 ml
water, via piston rinse port, if
available
… DAILY
Do not store columns in water
 Store RP columns IPA or
acetonitrile
 Store NP columns in hexane
USE GUARD COLUMNS
Guard column packing must be
identical to main column packing.
Source: www.upchurch.com

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AnalySys Sciences
Syringe Maintenance
 Inject smoothly – do not pause
 Use total-loop fill technique
 Do not separate plunger and
syringe – they are a matched pair
 Do not sonicate or soak cemented
needle syringes in solvents
 Use needle cleaner wire regularly
 Use Chaney adaptor or needle
guides, to prevent plunger bends

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AnalySys Sciences
Injection techniques
 With the handle on LOAD, insert the syringe into the needle port until it stops.
Dispense the sample; turn the handle rapidly to INJECT.
Remove the syringe.
Do not load a sample volume equal to the loop volume.
You will lose up to 20% of sample via the vent tube.
 Load <50% of the loop volume (partial-filling) or >200% (total loop fill)
A 20 µL sample loop does not contain 20 µL.
The size designations of loops are nominal.
 Complete-filling provides the best precision (reproducibility),.
Keep vent tubes and needle port at the same level.
Adjust the end of the vent tubes to the same height as the needle port so liquid
does not siphon out. Siphoning sucks air into the loop.
Use the proper syringe needle.
The needle should be #22 gauge 0.7 mm (5 cm, 2 in) OD, 5.1 cm (2 in) long,
with a 90° (square end) and no electrotaper
Source : http://www.rheodyne.com/support/product/troubleshooting/ts_injectors.htm

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AnalySys Sciences
Flushing the injector
 It is good practice to flush the needle port after every ten or twenty
injections.
 To flush, use from 0.1 to 1 mL of mobile phase. Do it while still in the
INJECT position so flow goes directly out vent tube #5 and
bypasses the loop that has already been flushed by the pump.
 Flush using the Needle Port Cleaner, not a needle.
Use the Needle Port Cleaner (a small Teflon part without a needle,
attached to a luer tip syringe). This flushes the entire length of the
port. A fully inserted needle flushes none of it.

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AnalySys Sciences
Degassing - Sonication
 The most common method
of degassing solvents.
 Sonicate solvents
separately, since sonication
causes mild heating.
 Reasonably effective.
 Inexpensive.

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AnalySys Sciences
Helium sparging
 Bubble helium @ 0.5 ml/min
using a sparger.
 Sparge each solvent
separately
 Sparging is the best
technique
 BUT – expensive

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AnalySys Sciences
Vacuum filtration
 Reasonable alternative to
sonication.
 Best used in conjunction with
helium sparging.
 Also good for solvent clarification
before HPLC.
 Use a compatible membrane, 0.45
m pore size, 47 to 50 mm dia.
 Use an oil-free vacuum pump,
preferably. Troubleshooting Menu

AnalySys Sciences
216
Validation Basics
Guidelines from:
Center for Drug Evaluation and Research (CDER), USFDA
http://www.fda.gov/cder/
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217
Validation Basics
 Method Validation
 System Validation
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218
Method Validation
 Validation of a method is the process by which a method is tested for
reliability, accuracy and preciseness of its intended purpose.
 Methods should be validated and designed to ensure ruggedness or
robustness. Methods should be reproducible when used by other analysts,
on other equivalent equipment, on other days or locations, and
throughout the life of the drug product.
 Data that are generated for acceptance will only be trustworthy if the
methods used to generate the data are reliable.
 Validation is an on-going process.
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219
Reference Standards
 A reference standard is a highly purified compound that is well
characterized.
 Chromatographic methods rely heavily on a reference standard to
provide accurate data. Therefore the quality and purity of the reference
standard is very important.
 Guideline:
 USP/NF reference standards do not need characterization
 Non-compendial standard (working standard) should be of the
highest purity that can be obtained by reasonable effort and should
be thoroughly characterized to assure its identity, strength, quality
and purity.
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220
Accuracy
 Accuracy is the measure of how close the experimental value is to
the true value.
 Accuracy studies for drug substance and drug product are
recommended to be performed at the 80, 100 and 120% levels of
label claim.
Recommendations:
 Recovery data, at least in triplicate, at each level (80, 100 and
120% of label claim).
 The mean is an estimate of accuracy and the RSD is an estimate of
sample analysis precision.
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221
LOD
 Limit of Detection
 The lowest concentration of
analyte in a sample that can
be detected, but not
necessarily quantitated, under
the stated conditions.
 Usually s/n 2:1 or 3:1
 Limit of Quantitation
 The lowest concentration of
analyte in a sample that can
be determined with
acceptable precision and
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222
Linearity
 That range of analyte concentrations over which the detector yields a linear
response.
 The working sample concentration and samples tested for accuracy should be in the
linear range.
Recommendations
 The linearity range for examination depends on the purpose of the test method. For
example, the recommended range for an assay method for content would be NLT ±
20% and the range for an assay/impurities combination method based on area %
(for impurities) would be +20% of target concentration down to the limit of
quantitation of the drug substance or impurity.
 Under most circumstances, regression coefficient (r) is 0.999. Intercept and slope
should be indicated.
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223
Precision
 Measure of how close the data values are to each other
for a number of measurements under the same
analytical conditions.
 Precision is defined by three components:
 Repeatability
 Intermediate precision
 Reproducibility
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224
Repeatability
 Injection repeatability
 Multiple injections of
the same sample in
the same conditions.
 Analysis repeatability
 Multiple
measurements of a
sample by the same
analyst under the
same analytical
conditions.
 Recommendation
 A minimum of 10
injections with an
RSD of 1% is
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225
Intermediate Precision
 Evaluates the reliability of the method in a different
environment other than that used during
development of the method.
 The objective is to ensure that the method will
provide the same results when similar samples are
analyzed once the method development phase is
over.
 Depending on time and resources, the method can
be tested on multiple days, analysts, instruments, etc.
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226
Reproducibility
The precision between
laboratories as in
collaborative studies.
Recommendations:
 It is not normally expected if
intermediate precision is
accomplished.
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227
Range and Recovery
Range
 The interval between the high and low levels of analyte studied.
Recommendation is usually +/- 20%.
Recovery
 The amount/weight of the compound of interest analyzed as a percentage
to the theoretical amount present in the medium.
 Full recovery should be obtained for the compound(s) of interest.
 Simpler sample preparation procedure will result in a lower variation of
recovery.
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228
Robustness
Measure of the method's capability to
remain unaffected by small, but
deliberate variations in method
parameters.
 Vary some or all conditions, e.g., age of
columns, column type, column
temperature, pH of buffer in mobile
phase, reagents, is normally performed.
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229
Sample Solution Stability
Sample Solution Stability
 Solution stability of the drug substance or drug product after preparation
according to the test method should be evaluated.
 Most laboratories use autosamplers with overnight runs and the sample
will be in solution for hours in the laboratory environment before the test
procedure is completed. This is of concern especially for drugs that can
undergo degradation by hydrolysis, photolysis or adhesion to glassware.
Recommendations
 Data to support the sample solution stability under normal laboratory
conditions for the duration of the test procedure, e.g., twenty-four hours,
should be generated.
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230
Specificity and Selectivity
 The analyte should have no interference from other
extraneous components and be well resolved from
them.
 A representative chromatogram should be generated and
submitted to show that extraneous peaks either by addition of
known compounds or samples from stress testing are baseline
resolved from the parent analyte.
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231
System Suitability Tests.
 The accuracy and precision of HPLC data begin with a
well-behaved chromatographic system.
 The system suitability specifications and tests are
parameters that help achieve this purpose.
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232
System Suitability Parameters
 Plate count > 2000 plates/meter
 Tailing factor < 2
 Resolution > 2
 Partition ratio > 2
 Relative retention > 1.5
 Precision / repeatability RSD </= 1% for n >/= 5
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233
General Points
 The sample and standard should be dissolved in the mobile phase. If that
is not possible, then avoid using too much organic solvent as compared to
the mobile phase.
 The sample and standard concentrations should be close if not the same.
 The samples should be bracketed by standards during the analytical
procedure.
 If the sample is filtered, adhesion of the analyte to the filter can happen.
This will be of importance especially for low level impurities. Data to
validate this aspect should be submitted.
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234
Hardware validation – IQ/OQ/PQ
 Installation Qualification
 Was the instrument installed as per vendor’s guidelines?
 Operational Qualification
 Is the system performing as per claimed specifications?
 Performance Qualification
 Is the analysis compliant for each sample?
 System Suitability Tests.
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236
Flow rate check
 The flow-rate accuracy of the pump can be evaluated
by calculating the time required to collect a
predetermined volume of mobile phase at different
flow-rate settings.
 For example, the flow-rate accuracy at 1mL/min. can be
verified by using a calibrated stopwatch to measure the time
it takes to collect 25 mL of eluent from the pump into a 25 mL
volumetric flask or specific gravity bottle.
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237
Gradient performance
 The accuracy and linearity of
the gradient solvent delivery
can be verified indirectly by
monitoring the absorbance
change as the binary
composition of the two
solvents changes from two
different channels.
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238
Pressure Hold Test
Plug the outlet of the pump using a dead-nut.
Set the pump shutdown pressure to 6,000 psi. Pressurize
the pump by pumping methanol at 1 mL/min.
The pressure inside the pump head increases quickly as the
outlet of the pump is blocked. As the pressure
increases to about 3,000 psi, the flow rate is reduced
to 0.1 mL/min.
The pressure will gradually rise to the shutdown pressure if
the check valves are able to hold the mobile phase in
the pump. If the check valve is not functioning
properly, the pressure will fluctuate at about 3,000
psi instead of reaching the shutdown pressure.
The pressure in the pump head decreases slowly over time
after the automatic shutdown.
A steep decrease in pressure over time implies poor check-
valve performance or leaks within the pumping
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239
Detector Tests
 Wavelength test
 Done by filling a flow cell with a solution of a compound with
a well-known UV absorption profile, and scanning the solution
for absorption maxima and minima.
 The lmax or lmin from the scan profile is then compared to
the known lmax or lmin of the compound to determine the
wavelength accuracy.
 Solutions of potassium dichromate in perchloric acid and
holmium oxide in perchloric acid, or aqueous caffeine solution.
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240
Detector tests
 Linearity of response
 Can be checked by injecting or by filling the flow cell with a series of standard
solutions of various concentrations. The concentration range typically should
generate responses from zero to at least 1.0 AU.
 From the plot of response versus the concentration of the solutions, the
correlation coefficient between sample concentration and response can be
calculated to determine the linearity.
 Noise and Drift
Software is capable of calculating the detector noise and drift. Typically,
methanol is passed through the flow cell at 1 mL/min.
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241
Injector Tests
 Repeatability
 Repeated injections of the same sample volume.
 Linearity
 Variable volume of sample will be drawn into a sample
injection loop by a syringe or other metering device. The
uniformity of the sample loop and the ability of the metering
device to draw different amounts of sample in proper
proportion will affect the linearity of the injection volume.
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242
Injector tests.
 Carryover
 Small amounts of analyte may get carried over from the
previous injection and contaminate the next sample to be
injected.
 Carryover be evaluated by injecting a blank after a sample
that contains a high concentration of analyte. The response of
the analyte found in the blank sample expressed as a
percentage of the response of the concentrated sample can be
used to determine the level of carryover.
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AnalySys Sciences
243
Method Development Primer

244
The basic steps
 Select separation mode
 Select column
 Select detection mode
 Sample prep
 Validation

245
Method development – Key Tips
Keep the sample in the stationary phase… as
long as is reasonably possible.
 Longer time in column = better chances of
separation.
The sample decides which column chemistry to
use.
 Polar sample = polar column
 Non-polar sample = non polar column
 Chiral sample = chiral column, etc.
There’s no in-silico substitute for
 … old-fashioned chemistry.
 … common sense.

246
The basic questions
 Molecular weight?
 Size exclusion… or not.
 What is it soluble in?
 Mobile phase to be used
 Ionic, ionisable or neutral?
 Column chemistry to be used.
 How will I detect it? At what sensitivity?
 Detection system. Limit of detection.
 What is the sample matrix?
 Sample prep method to be used.

247
Isocratic or gradient?
Number of analytes
 Less than 4 or 5, then isocratic.
 More than 5 analytes or multiple functionalities or solubilities,
then gradient.
Key analytes improperly resolved
Isocratic run resolves analytes, but takes too long.

248
If using a gradient…
 Is the sample completely soluble in the
mobile phase …
 … at the selected temperature?
 … across the gradient being used?
 Can my analyte (s) be detected across
the gradient?

250
Common HPLC methods – ion
suppression
 Ionisation of the analyte is suppressed using the appropriate pH
 Analyte remains neutral and can be separated on a C18 column.
Used for weak acids and weak bases
 Mobile phase
 Buffer phase, usually phosphate buffer
 Organic phase, CH3CN or MeOH

251
HPLC methods – ion pair LC
 An ion pairing agent is used
to create a neutral complex
with the analyte
 Quaternary amines for
anionic analytes
 Sulfonates for cationic
analytes

252
Analgesics – ion suppression
Conditions
Column: C18, 5cm x 4.6mm ID, 5µm particles
Mobile Phase: acetonitrile:25mM KH2PO4,
pH 2.3 with phosphoric acid (20:80)
Flow Rate: 2 mL/min
Det.: UV, 230nm
Inj.: 5µL mobile phase, analyte quantities shown
Analyte Data
1. Dextromethorphan
2. Acetylsalicylic acid

253
Examples – sucrose in cola
Mol wt of sucrose: 342.3. Solubility: Highly polar. Freely soluble in water
Which column?
Polar sample = polar column. C18 wrong choice.
Polar column needed. Bare silica column cannot be used, since silica is soluble in water.
Si-NH2 column preferable. Or HILIC column would be ideal.
Which detection method?
Chromophores: Nil. Does not absorb UV
Refractive index preferable. Or ELSD, if you can afford it. However, RI and ELSD are
both non-specific detectors.
Specific detection method: Sucrose is ionisable. So, amperometric or coulometric
detection can be used.
Key considerations: Cost per sample. Detection limit required. Presence of interfering
analytes (like fructose).
For a cola drink, sucrose is present in high amounts. Interfering substances unlikely. Low
cost per sample is important. Therefore, Si-NH2 or HILIC column with RI detection
preferred.

254
Sucrose in cola drinks - 2
 Column Si-NH2. Detection: RI
 Mobile phase?
 Water. 100% water will elute sucrose
too fast. So, add MeCN to increase
sucrose retention on column.
 Start with 10% MeCN, increase to
30% until acceptable resolution is
attained.
 Flow rate?
 Usually 1 ml/min will suffice for a 4.6
mm, 5 um column.
 Temperature?
 30 – 40 deg C preferred, for better
resolution. RI detection is sensitive to
temperature, so a column oven is
mandatory.
 Sample prep?
 Membrane filtration, hydrophilic
membrane, 0.45 um.

255
Example – caffeine in cola
 Mol wt: 194
 Solubility: Moderately water-soluble.
Freely soluble in MeOH.
 Which column? C18 preferred.
 Detection?
 Strongly absorbs UV. lmax 273 nm
 Mobile phase?
 Water:MeOH. Start with 20% MeOH,
and increase.
 Sample prep?
 SPE using C18 sorbent.
 LLE using CHCl3
 Membrane filtration
 Dilution, if necessary.

256
Example – Insulin injection
 Mol wt: ~ 5800 Da.
 Unstable in solution.
 Which column?
 SEC
 C18 currently used.
 300A pore size.
 Detection? UV.
 Mobile phase?
 Buffer used to stabilise analyte and
suppress its ionisation. pH < 3.
 0.1% TEA added to improve peak
shape
 MeCN used as organic modifier.
Start with 20% MeCN and increase.
 Sample prep? Critical.
 Membrane filtration, using
hydrophilic membrane.

257
No work is complete…
… without paperwork!
 Method validation
 Documentation
 Regulatory
compliance
 …till then, method
development is not
complete!

259
Sample prep basics
Why sample prep?
 Sample clarification
 Removal of interfering substances and
particulates
 Analyte extraction / enrichment
 Solid phase extraction
 Protect the column and HPLC components

260
Sample Clarification
Filtration
 Depth filters for particulate removal
 Membrane filters for sample clarification and
removal of sub-micron particles

261
Depth filters
 Depth filters use a porous
filtration medium to retain
particles throughout the
medium, rather that just on
the surface.
 used when the fluid to be
filtered contains a high load
of particles.
 Used as discs
 Glass fiber
 Polypropylene

262
Membrane filters
 Polymer films with
specific pore ratings.
 Retain particles and
microorganisms on the
surface of the
membrane.

263
Membrane filters
 Materials
 Hydrophilic
 Cellulose acetate or
nitrate
 Regenerated cellulose
 Hydrophobic
 PTFE
 PVDF
 Nylon
 Disc diameters
 4 mm
 13 mm
 25 mm
 47 / 50 mm (for solvent
clarification)
 Pore sizes
 0.45 / 0.5 
 0.2 

264
Membrane filters - tips
 Always check compatibility with sample and
sample solvent
 Use appropriate disc diameters
 < 2 ml, use 4 mm
 2-5 ml, use 13 mm
 5-25 ml, use 25 mm
 > 25 ml – 500 ml, use 47 mm
 Sample loss can occur due to non-specific
adsorption onto membrane or depth filter

265
Sample clarification - Centrifugation
In general, Microcentrifugation
is a better method of
sample clarification.
Used for analytes that adsorb
onto filter membranes.
Samples should be spun at not
less than 15,000 rpm.

266
Analyte extraction
Solid phase extraction
 Used to isolate
analytes of interest
from a wide variety of
matrices.
 Especially useful for
difficult matrices
 Uses much less solvent
than LLE
 Can be automated

267
SPE cartridges
 SPE cartridge
is a mini HPLC
column
 Same packing
material as
used in HPLC
 Eg. C18, C8,
Ion-ex.

269
SPE Hardware
 Vacuum flask
 Vacuum manifold
 Automated SPE

Analsys Sciences - Introduction to HPLC

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