analsys sciences - introduction to hplc

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AnalySys Scienceswww.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

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

sCK

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

0

0

Rt tK

t

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Relative retention (Selectivity / separation factor)

For baseline separation, a > 1.5

2

1

k

ka

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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 Rs

t tR

w w

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Peak Width (4s)

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Tailing factor (Asymmetry/ Skew factor)

BCAs

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)

CH 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

LH

t

LH

t

s

s

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Plate Count

2

2

164

255

R

R

t

t

s

s

2

5.542

R

LN

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 impliesbetter 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 sameconditions, 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

http://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

http://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)

0

% 100P

TP

10

1logA

T

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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

sin

sin

i

r

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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 thecathode.

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

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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


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


http://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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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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Rounded peaks

Sample overload

Reduce sample

concentration and/or

volume

Detector out of range

Adjust detector sensitivity


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Loss of peak height

Sample deterioration

Injector seal/System leak

Aging UV lamp

Wrong injection technique

Use total-loop technique


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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 strengthTroubleshooting Menu

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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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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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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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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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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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Dead volume

Excessive dead volume can

adversely affect results

Optimize tubing length and

diameter

Use correct detector flow cell

(10 to 20l for analytical HPLC)

Use Zero-Dead-Volume fittings


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Dead volume -2


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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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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


http://www.upchurch.com/

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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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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


http://www.rheodyne.com/support/product/troubleshooting/ts_injectors.htm

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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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Degassing - Sonication

The most common method of degassing solvents.

Sonicate solvents separately, since sonication causes mild heating.

Reasonably effective.

Inexpensive.


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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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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


216

Validation Basics

Guidelines from:Center for Drug Evaluation and Research (CDER), USFDAhttp://www.fda.gov/cder/

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http://www.analysciences.com/

http://www.fda.gov/cder/

217

Validation Basics

Method Validation

System Validation

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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. Table of contents.

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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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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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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 accuracy under the stated

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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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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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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 recommended.

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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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Reproducibility

The precision between laboratories as in collaborative studies.

Recommendations:

It is not normally expected if intermediate precision is accomplished.

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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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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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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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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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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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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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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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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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OQ

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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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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 system. Table of contents.

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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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 DriftSoftware 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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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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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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Method Development Primer

http://www.analysciences.com/

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. … trial and error.

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.

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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

ConditionsColumn: C18, 5cm x 4.6mm ID, 5µm particlesMobile Phase: acetonitrile:25mM KH2PO4, pH 2.3 with phosphoric acid (20:80)Flow Rate: 2 mL/minDet.: UV, 230nmInj.: 5µL mobile phase, analyte quantities shown

Analyte Data1. Dextromethorphan2. Acetylsalicylic acid

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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 UVRefractive 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.

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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.

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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.

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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.

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No work is complete…

… without paperwork!

Method validation Documentation Regulatory

compliance …till then, method

development is not complete!

258

Sample Preparation

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.

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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

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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

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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.

Source: www.supelco.com

http://www.supelco.com/

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SPE Hardware

Vacuum flask

Vacuum manifold

Automated SPE