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What We Do!
Bio-Analytical Technologies (BAT) offers services to its global customer through its off-shore development centre and On-site consulting. BAT is formed on the foundation of the domain knowledge in the field of Analytical and Biotechnology application. BAT is focused on harnessing talents and multi skill covering BT, Engineering, software technologies.

About Us
BAT has successfully completed several projects for its customers involving software development, software testing, integration testing, application scripting, methods development.

Our Activities in LCMS

Impurity profiling Validation of Method Characterization of the compounds Routine analysis Water analysis/ Environmental analysis

Agenda
Introduction Overview of LC Overview of MS Sample Inlet Ionization Techniques Mass Analyzers Quadrupole Operations

Agenda
Detector Vacuum Method Optimization for LCMS Data Interpretation Applications Of LCMS Software- ANALYST References

Introduction
Mass Spectrometry is a powerful analytical technique for identifying the unknown compounds by determining their molecular weights, for qualitative & quantitative determination. The basis in MS is the production of ions, that are subsequently separated or filtered according to their mass-to-charge ratio(m/z) and detected. It is the most sensitive method of molecular analysis.

Uses of Mass Spectroscopy

Identify compounds Structural determination of new synthetic compounds Quantify compounds Determine structural and chemical properties Detection of minute quantities Elicit drug detection

Uses of Mass Spectroscopy

QA of pharmaceutical production Determination of drugs and metabolite in body fluid Determine structural formation of complex biomolecules Genomics and proteomics Forensic analysis

Glossary
Ions AMU Isotopes High molecular weight ions Fragment ions

Atom
nucleus: neutrons and protons surrounded by equal number of electrons charge 0

Ion
nucleus: neutrons and protons surrounded by 1 or more or less electrons or protons + charge = number of surplus protons - charge = number of surplus electrons

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

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 (in the case of C12 it is 6 + 6) Mass of electron is 1/2000 of a proton

Isotopes
Naturally occurring More neutrons Higher amu but no change in charge Atoms in the periodic table with higher numbers of protons have more isotopes associated with them Lead has 82 protons and either 122,124, 125,or126 neutrons

N a tu ra l O c c u ra n c e o f L e a d (P b )
60 50 40
%

30 20 10 0 204 205 206 AMU 207 208

High Molecular Weight Ions

Multiple atoms join to form molecules Organic molecules have high weights Contain mainly C(12.00000), O(15.9491), H(1.00783) and N(14.00307) These atoms have relatively few isotopes By ionizing a molecule more than once is to increase the effective mass range i.e. Isotopes reveal the difference between 2700+ and 5400++ Increase the mass range by multiple charging

Overview of Liquid Chromatography

Introduction to LC
Liquid chromatography (LC) is an analytical technique of separation & identification of the sample 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. Chroma meaning color + graphein meaning to write

Separation
Solvent
A+B

Packed column

A B A B

detector
t0 t1 t2

A t3

B t4

Signal

A Time, t

Chromatography
Chromatogram - Detector signal vs. retention time or volume
Detector Signal

time or volume

History of LC
British Chemists A.T.P.Martin and R.L.M. Synge developed partition chromatography in 1942; ( Nobel prize in 1954) Since 1940s, chemists used gravity-fed silica columns to purify organic materials In late 1960s, LC turned high performance with small particle columns that require high pressure pumps Development of online detectors allow HPLC to become sensitive and quantitative technique for diverse applications

Types of Chromatography

Modes of Liquid Chromatography

1 Adsorption Chromatography: 1.1 Normal Phase 1.2 Reverse Phase. 2 Partition Chromatography: 2.1 Liquid-liquid chromatography 2.2 Bonded-phase chromatography

Adsorption Chromatography
Normal Phase or Liquid-solid Chromatography (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 bonded stationary phase.

Normal Phase Chromatography

Uses Highly Polar Stationary Phase : -Silica, Alumina , Amino-, Cyano- or Phenyl bonded 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 nonpolar lypophilic ones. Excellent for non-polar analytes, isomers, groups separations and for sample clean up.

Reversed Phase Chromatography

Separation is based on Partitioning of the analyte into Hydrophobic Stationary Phase bonded on Silica support. e.g. C18, C8, etc. Uses polar mobile phase such as Methanol/ Acetonitrile and Water; Polar analyte elute first than non-polar. Most common HPLC mode used in > 60% all LC separations For polar (water soluble) , medium polarity and some non-polar analyte. Ionic analytes can be separated using ion-pairing reagents.

Partition Chromatography
Separation is mainly dependent upon the relative solubility of the compounds (Solutes) in the liquid attached to the support (Stationary phase) and in the 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 elute with an oily mobile phase. Therefore, one goes from a weak solvent (water) to a strong solvent (oil).

Bonded Phase Chromatography

Stationary phase is chemically bonded to a backbone, such as siloxane
R R Si OH + Cl Si R Si O Si R

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

Adsorption Chromatography
Solute interacts directly by adsorption with the stationary phase. Solute and solvent compete for adsorption sites. Ex. stationary phases: Silica, Alumina, Charcoal, Cellulose Non-polar molecules interact weakly with polar adsorbents. Polar molecules interact strongly with polar adsorbents.
+++ - + + + + -

Flow

- + + (Polar Stationary Phase)

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 on polymeric materials or silica. Uses mobile Phase consisting buffered Solutions of different pH and Ionic strength Common application are analysis of ions, amino acids, proteins/peptides, polynucleotides, etc.

Size Exclusion Chromatography

Separation based on sieving action of cross-linked polystyrene with controlled pore sizes - Common mobile phase is toluene, Ethylene dichloride. - Smallest molecules penetrate the smallest pores, retained longest Larger molecules are excluded, so they elute first Useful for molecular weight determination of polymers and biomolecules

Chromatography
HPLC is a physical separation technique in which a sample dissolved liquid is injected into a column packed with small particles and is separated into is constituents components. The most important and widely used analytical technique for the quantitative analysis of organics and biomolecules. Applicable to many sample types - Most useful for pharmaceuticals, biomolecules, and labile organics.

The Chromatographic Process

In LC, analytes to be separated is distributed between two phases -Mobile Phase -Stationary Phase Components is separated by differential interactions with porous support An on-line detector monitors the concentration of each eluting component and generates a trace called the chromatogram.

HPLC: Principle
High Performance Liquid Chromatography is a separation technique utilizing differences in distribution coefficient of compounds between two phases, stationary phase and mobile phase. Stationary Phase: Typically Designates a thin layer created on surface of fine silica particles. Mobile Phase : Designates the liquid flowing over the particles.

HPLC: Basic Terminology

Retention Time (tR) Peak width (Wb) Capacity Factor (K) - Plate number (n) - Height Equivalent of Theoretical Plate ( HETP) Selectivity () Resolution (RS)

HPLC: Basic Terminology

Column a tube that contains packed sorbents Retention the tendency of a solute to be retained by the stationary phase in the column. Sample capacity the maximum sample load for the column Selectivity the ratio of retention of 2 adjacent peaks Resolution the degree of separation of 2 peaks Efficiency a measure of the narrowness of peak widths produced by a column.

Retention time (tR)

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

Capacity Factor (k)

k is a measure of peak retention or how many times the peak is retained vs an unretained peak (t0).
tR Retention time tR t0

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

Injection

t0

Solvent peak

Solute A

Selectivity Parameters ()

( Separation factor) = k2/ k1 is dependent on the column and mobile phase.

K1

K2

is measure of differential retention time of two analytes and must be > 1.0 for

peak separation.

Solute A

Solute B

Resolution
Resolution (measure of the separation between two consecutive peaks) depends on the systems selectivity and efficiency

R = t/Wb2

HPLC:Diagrammatic presentation

Basic Components of an HPLC System

Pump System. Mobile phase pressures up to 6000 psi are necessary to achieve reasonable column elution times (~ minutes). Typical flow rates are 0.1 to 10 mL/minute. Injection System. Used to introduce small samples (0.1 to 500 L) into the carrier stream under high pressure. Reservoirs (Solvents). Multiple solvents are necessary for performing gradient elution's (i.e. changing the polarity of the mobile phase during a run). Chromatographic Column. Typically 2-30 cm in length containing a packing of particles 2-10 m diameter. Many types of columns are available, 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.

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 pumps Pneumatic pump Reciprocating pump

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

Gradient are formed by

using 2 or more pumps (high pressure mixing) or solenoidactuated proportioning valves (Low-pressure mixing)
Unlimited capacity - but

pulsed flow.

Sample Injection System

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

The loop is filled from the syringe The loop is inserted between the pump and the mobile phase flows from & the column so that the mobile phase pump to column. sweeps the sample onto the column.

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

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

Me Si O Si R Me

Detectors for HPLC

An HPLC detector is equipped with a small flow cell connected to the column outlet and monitors the concentration of the analyte component. Common detectors are: - UV/Vis absorbance UV/Vis) fixed or variable wavelength Rapid scanning diode array detector -Fluorescence (Fl) -Refractive index (RI) -Mass spectrometry (MS) Other detectors: - Electrochemical (ECD), Conductivity and Infrared

Criteria for ideal Detectors

An ideal detector should be: Universal Sensitive Linear Response: between intensity of response & amount of analyte Structural Information: of the analyte.

Mobile Phase Composition

Isocratic elution Simple HPLC uses mobile phase of constant composition. Gradient elution For more complex mixtures. Programm a changing (stepwise or continuous) mobile phase composition during the run. In HPLC this is the usual solution to the general elution problem

Mobile Phase Characteristics

Desirable Physical properties High purity, low cost, UV transparency, non-corrosive, low viscosity, non-flammable, sample solubility. Strength Related to polarity of solvent Solvent strength under normal phase are characterized by Hildbrands scale (E) Selectivity Depends on dipole moment, induced dipole, H-bonding, and dispersive characteristics of the solvents.

Advantage of HPLC
Fast technique High Resolution Various Types of columns Used Wide range of Samples Versatile Non destructive (detector dependent) Good for quantitation Variety of separation mechanisms Scalable from Entry Level Operates at near ambient temperature

Applications of LC
Field of Application Separation
Antibiotics, Sedatives, Steroids, Analgesics Amino acids, Proteins, Carbohydrates, Lipids Artificial Sweeteners, Antioxidants, Preservatives Condensed Aromatics, Surfactants, Propellants, Dyes Drugs, Poisons, Blood Alcohol, narcotics Bile Acids, Drug Metabolites, Urine Extracts, Estrogens Pesticides, Herbicides, Phenols, PCBs Pharmaceuticals Biochemical Food Products Industrial Chemicals Forensic Chemistry Clinical Medicine Pollutants