introduction of lc

8/2/2019 Introduction of LC

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AT

TO


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What We Do!

Bio-Analytical Technologies (BAT) offers services toits global customer through its off-shoredevelopment centre and On-site consulting.

BAT is formed on the foundation of the domainknowledge in the field of Analytical andBiotechnology application.

BAT is focused on harnessing talents and multi skill

covering BT, Engineering, software technologies.


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About Us

BAT has successfully completedseveral projects for its customers

involving software development,software testing, integration testing,application scripting, methods

development.


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Our Activities in LCMS

Impurity profilingValidation of Method

Characterization of the compoundsRoutine analysisWater analysis/ Environmental analysis


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Agenda

IntroductionOverview of LCOverview of MSSample InletIonization TechniquesMass Analyzers

Quadrupole Operations


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Agenda

DetectorVacuumMethod Optimization for LCMS

Data InterpretationApplications Of LCMSSoftware- ANALYST

References


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Introduction

Mass Spectrometry is a powerful analytical techniquefor identifying the unknown compounds bydetermining their molecular weights, for qualitative& quantitative determination.

The basis in MS is the production of ions, that aresubsequently separated or filtered according to theirmass-to-charge ratio(m/z) and detected.

It is the most sensitive method of molecular analysis.


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Uses of Mass Spectroscopy

Identify compounds

Structural determination of new syntheticcompounds

Quantify compounds

Determine structural and chemical properties

Detection of minute quantities

Elicit drug detection


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Uses of Mass Spectroscopy

QA of pharmaceutical production

Determination of drugs and metabolite in body fluid

Determine structural formation of complexbiomolecules

Genomics and proteomics

Forensic analysis


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Glossary

IonsAMU

IsotopesHigh molecular weight ionsFragment ions


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Atom Ion

nucleus: neutrons andprotons

surrounded by equalnumber of electrons

charge 0

nucleus: neutrons and protons

surrounded by 1 or more or lesselectrons or protons

+ charge = number of surplusprotons

- charge = number of surpluselectrons

Atoms or molecules must be converted into ions in order to beanalyzed by mass spectroscopy


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Atomic Mass Unit (Dalton)

1 AMU is 1/12 the mass of a Carbon12 atom

Sum of the number of protons and the number of neutrons (inthe case of C12 it is 6 + 6)

Mass of electron is 1/2000 of a proton


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IsotopesNaturally occurringMore neutronsHigher amu but no changein charge

Atoms in the periodic tablewith higher numbers ofprotons have moreisotopes associated withthem

Lead has 82 protons and

either 122,124,125,or126 neutrons

N a t u r a l O c c u r a n c e o f L e a d ( P b )

0

1 0

2 0

3 0

4 0

5 0

6 0

2 0 4 2 0 5 2 0 6 2 0 7 2 0 8

A M U

%


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High Molecular Weight Ions

Multiple atoms join to form moleculesOrganic molecules have high weightsContain mainly C(12.00000), O(15.9491),

H(1.00783) and N(14.00307)These atoms have relatively few isotopesBy ionizing a molecule more than once is to increasethe effective mass range i.e. Isotopes reveal thedifference between 2700+ and 5400++

Increase the mass range by multiple charging


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Overview of

Liquid Chromatography


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Introduction to LC

Liquid chromatography (LC) is an analyticaltechnique of separation & identification of thesample which are dissolved in a solvent.

Discovered by Russian botanist Mikhail Tswett in

1903. He separated various plant pigments by

passing solutions through a glass column

containing calcium carbonate.

Chromameaning color + grapheinmeaning to

write


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Separation

Signal

Time, t

Packed

column

A+B

detector

A

B

A

B

t0 t1 t2

A

B

t3

A

Solvent

B

t4

B


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Chromatography

Chromatogram - Detectorsignal vs. retention time orvolume

time or volume

D

etectorSignal

1 2


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History of LC

British Chemists A.T.P.Martin and R.L.M.Synge developed partition chromatographyin 1942; ( Nobel prize in 1954)

Since 1940s, chemists used gravity-fed silicacolumns to purify organic materialsIn late 1960s, LC turned high performance

with small particle columns that requirehigh pressure pumps

Development of online detectors allow HPLCto become sensitive and quantitativetechnique for diverse applications


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Types of Chromatography


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Modes of Liquid Chromatography

1 Adsorption Chromatography: 1.1 Normal Phase1.2 Reverse Phase.

2 Partit ion Chromatography: 2.1 Liquid-liquid chromatography2.2 Bonded-phase chromatography


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Adsorption Chromatography

Normal Phase or Liquid-solidChromatography (NP,LSC)

-Separation based on adsorption/desorption of the

analyte onto a polar surface

Reversed Phase Chromatography (RPC)Separation based on analytes partition coefficients

between the mobile phase and the bondedstationary phase.


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Normal Phase Chromatography

Uses Highly Polar Stationary Phase :-Silica, Alumina , Amino-, Cyano- or Phenylbonded phase.-Polar analytes has longer retention times.

Relatively Non-Polar Solvent System : Hexane ,Diethyl chloride modified with IPA.Elution order Polar solutes elute later than non-

polar lypophilic ones.Excellent for non-polar analytes, isomers, groups

separations and for sample clean up.


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Reversed Phase Chromatography

Separation is based on Partitioning of the analyteinto Hydrophobic Stationary Phase bonded onSilica support.

e.g. C18, C8, etc.Uses polar mobile phase such as Methanol/Acetonitrile and Water; Polar analyte elute firstthan non-polar.

Most common HPLC mode used in > 60% all LCseparations

For polar (water soluble) , medium polarity andsome non-polar analyte.Ionic analytes can be separated using ion-pairing

reagents.


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Partition Chromatography

Separation is mainly dependent upon the relativesolubility of the compounds (Solutes) in the liquidattached to the support (Stationary phase) and inthe liquid flowing through the stationary phase

(Mobile phase)If the affinity of the solute for the stationary phase

is high, the solute does not elute.Ex: Fat dissolves in oil but not in water. If oil is the

stationary phase, a fatty molecule will only elutewith an oily mobile phase.

Therefore, one goes from a weak solvent (water) toa strong solvent (oil).


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Bonded Phase Chromatography

Stationary phase is chemically bonded to a backbone, suchas siloxane

Si OH + SiCl

R

R

R Si O Si

R

R

R

R = CH3

R = functional group, ex: CH3 chain, CN, phenol...


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Adsorption Chromatography

+

-

-

-

-

-

-

-

-

+

+

+

+

+

+

+

Flow+

-+

+--

(Polar Stationary Phase)

Solute interacts directly byadsorption with the stationaryphase.

Solute and solvent compete foradsorption sites.

Ex. stationary phases: Silica,Alumina, Charcoal, Cellulose

Non-polar molecules interactweakly with polar adsorbents.

Polar molecules interact stronglywith polar adsorbents.


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Ion Exchange Chromatography

Used to separate ionic or ionizable analytes.IEC relies on charge-charge interactions between the

charges in your sample and charges immobilized on the

resin of stationary phase.Stationary phase is bonded cationic or anionic groups onpolymeric materials or silica.

Uses mobile Phase consisting buffered Solutions ofdifferent pH and Ionic strength

Common application are analysis of ions, amino acids,

proteins/peptides, polynucleotides, etc.


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Size Exclusion Chromatography

Separation based on sievingaction of cross-linkedpolystyrene with controlledpore sizes

- Common mobile phase istoluene, Ethylene dichloride.

- Smallest molecules penetratethe smallest pores, retainedlongest

Larger molecules are excluded,so they elute first

Useful for molecular weightdetermination of polymers

and biomolecules


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Chromatography

HPLC is a physical separation technique in which a sampledissolved liquid is injected into a column packed withsmall particles and is separated into is constituentscomponents.

The most important and widely used analytical techniquefor the quantitative analysis of organics andbiomolecules.

Applicable to many sample types- Most useful for pharmaceuticals, biomolecules, and

labile organics.


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The Chromatographic Process

In LC, analytes to be separated is distributedbetween two phases-Mobile Phase

-Stationary PhaseComponents is separated by differential

interactions with porous supportAn on-line detector monitors the concentration of

each eluting component and generates a tracecalled the chromatogram.


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HPLC: Principle

High Performance Liquid Chromatography is a separationtechnique utilizing differences in distribution coefficient ofcompounds between two phases, stationary phase andmobile phase.

Stationary Phase: Typically Designates a thin layer created onsurface of fine silica particles.

Mobile Phase : Designates the liquid flowing over the particles.


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HPLC: Basic Terminology

Retention Time (tR)Peak width (Wb)Capacity Factor (K)

- Plate number (n)- Height Equivalent of Theoretical Plate ( HETP)

Selectivity () Resolution (RS)


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HPLC: Basic Terminology

Columna tube that contains packed sorbents

Retentionthe tendency of a solute to be retained by thestationary phase in the column.

Sample capacitythe maximum sample load for the column

Selectivitythe ratio of retention of 2 adjacent peaks

Resolutionthe degree of separation of 2 peaks

Efficiencya measure of the narrowness of peak widthsproduced by a column.


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Retention time (tR)

A peak is characterised by the retention time (selectivity) and its shape(efficiency)


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Capacity Factor (k)

Injection

t0 Solventpeak

Retention time tR

Solute A

t0

ttRR

K= (tR-t0)/ t0 = tR/ t0.

k is a measure of peak retention or how many times the peak is retainedvs an unretained peak (t0).


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Selectivity Parameters ()

Solute A Solute B

K1 K2

( Separation factor) = k2/ k1

is dependent on the column

and mobile phase.

is measure of differential

retention time of two analytes

and must be > 1.0 for

peak separation.


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Resolution

Resolution(measure of theseparation between

two consecutivepeaks) depends onthe systemsselectivity andefficiency

R = t/Wb2


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HPLC:Diagrammatic presentation


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Basic Components of an HPLCSystem

Pump System. Mobile phase pressures up to 6000 psi are necessary toachieve reasonable column elution times (~ minutes). Typical flow ratesare 0.1 to 10 mL/minute.

I njection System. Used to introduce small samples (0.1 to 500 L) intothe carrier stream under high pressure.

Reservoirs (Solvents). Multiple solvents are necessary for performinggradient elution's (i.e. changing the polarity of the mobile phase during arun).

Chromatographic Column. Typically 2-30 cm in length containing apacking of particles 2-10 m diameter. Many types of columns areavailable, depending on the type of liquid chromatography desired.

Detector. Many types are available including UV, IR, refractive index,fluorescence, conductivity, mass spectrometry, and electrochemical.Diode array detectors are used when wavelength scans are desired.


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HPLC Pumps: Requirements

Provide precise and pulse free delivery of solvents-Allows high pressure operation (up to 5000 psi)

Compatible with common eluents-Accommodate organic solvents, buffers, salts-Wettable parts made from inert materials e.g. stainless steel,

PEEK, Teflon, sapphire, etc.

Reliable Operation with long pump seal life-easy to maintain and service

Two types of pumpsPneumatic pump

Reciprocating pump


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.

Gradient are formed byusing 2 or more pumps (highpressure mixing) or solenoid-actuated proportioning valves(Low-pressure mixing)

Unlimited capacity - butpulsed flow.

Most HPLC pumps are reciprocating A motor driven cam drives the piston to deliver solvent through theoutlet check valve.

Reciprocating Pump


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Sample Injection System

Used to introduce small samples (0.001 to 0.5 mL) into the carrierstream under high pressure

The loop is filled from the syringe The loop is inserted between the

pumpand the mobile phase flows from & the column so that the mobilephase

pump to column. sweeps the sample onto the column.


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Usually stainless steel : 1 - 5 mm diameter, ~5 mm packing. Veryefficient but limited length

Packing - usually silica. More usual, to use similar chemical speciesactually bonded to the silica (longer column life), e.g. R can be non-polar (C8 or C18 hydrocarbon chain) or polar (amine, nitrile etc.).

Si O Si

Me

Me

R

Columns


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An HPLC detector is equipped with a small flow cell connected tothe column outlet and monitors the concentration of the analytecomponent.

Common detectors are:

- UV/Vis absorbance UV/Vis)fixed or variable wavelengthRapid scanning diode array detector

-Fluorescence (Fl)-Refractive index (RI)-Mass spectrometry (MS)

Other detectors:- Electrochemical (ECD), Conductivity and Infrared

Detectors for HPLC


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An ideal detector should be:

Universal

Sensitive

Linear Response: between intensity of response & amount of analyte

Structural Information: of the analyte.

Criteria for ideal Detectors


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Isocratic elution -Simple HPLC uses mobile phase of constant composition.

Gradient elution For more complex mixtures. Programm a changing (stepwise orcontinuous) mobile phase composition during the run. In HPLC thisis the usual solution to the general elution problem

Mobile Phase Composition


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Mobile Phase Characteristics

Desirable Physical propertiesHigh purity, low cost, UV transparency, non-corrosive, low

viscosity, non-flammable, sample solubility. StrengthRelated to polarity of solventSolvent strength under normal phase are characterized by

Hildbrands scale (E) SelectivityDepends on dipole moment, induced dipole, H-bonding, and

dispersive characteristics of the solvents.


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Advantage of HPLC

Fast techniqueHigh Resolution

Various Types of columns UsedWide range of Samples

VersatileNon destructive (detector dependent)Good for quantitation

Variety of separation mechanismsScalable from Entry LevelOperates at near ambient temperature


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Applications of LC

Pesticides, Herbicides, Phenols,PCBs

Pollutants

Bile Acids, Drug Metabolites, UrineExtracts, Estrogens

Clinical Medicine

Drugs, Poisons, Blood Alcohol,narcotics

ForensicChemistry

Condensed Aromatics, Surfactants,

Propellants, Dyes

I ndustrial

Chemicals

Artificial Sweeteners, Antioxidants,Preservatives

Food Products

Amino acids, Proteins,

Carbohydrates, Lipids

Biochemical

Antibiotics, Sedatives, Steroids,Analgesics

Pharmaceuticals

SeparationField ofApplication


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