mass spectrometry (1)

MASS SPECTROMETRY

PRESENTED BY:

MOHSIN SHAD 2013-BC-005

JAHANZAIB AZHAR 2013-BC-017

ASMAT SHAHZAD 2013-BC-026

MUHAMMAD ZULQARNAIN 2013-BC-027

HAMZA ZAFAR 2013-BC-032

PRESENTED TO:

DR.MUHAMMAD TAYYAB 1

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MASS SPECTROMETRY

• TECHNIQUE INVOLVES :

• - CREATING GAS PHASE IONS FROM THE ANALYTE ATOMS OR MOLECULES

• - SEPARATING THE IONS ACCORDING TO THEIR MASS-TO-CHARGE RATIO (M/Z)

• - MEASURING THE ABUNDANCE OF THE IONS

• - QUALITATIVE AND QUANTITATIVE ANALYSIS

• - PROVIDING INFORMATION ABOUT THE MASS OF ATOMS AND MOLECULES

• - MOLECULAR STRUCTURE DETERMINATION (ORGANIC & INORGANIC

• - IDENTIFICATION AND CHARACTERIZATION OF MATERIALS

PRINCIPLE OF MASS SPECTROMETRY

• MASS SPECTRA IS ALSO CALLED POSITIVE ION SPECTRA.

• IN THIS ELECTRON BOMBARDMENT IS USED TO CONVERT A NEUTRAL MOLECULE IN TO POSITIVELY CHARGED ONE.

• OBTAINING MASS SPECTRA CONSISTS 2 STEPS

• CONVERSION OF NEUTRAL MOLECULE IN TO POSITIVELY CHARGE DONE.

• SEPARATION OF POSITIVELY CHARGED FRAGMENTS FORMED BASED UPON THEIR MASSES USING ELECTRICAL & MAGNETIC FIELD:

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• SEPARATES GAS PHASE IONIZED ATOMS, MOLECULES, AND FRAGMENTS OF MOLECULES

• SEPARATION IS BASED ON THE DIFFERENCE IN MASS-TO-CHARGE RATIO (M/Z)

• M = UNIFIED ATOMIC MASS UNITS (U)

• 1 DALTON (DA) = 1 U = 1.665402 X 10-27 KG

• Z = CHARGE ON THE ION (MAY BE POSITIVE OR NEGATIVE

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• ANALYTE MOLECULE CAN UNDERGO ELECTRON IONIZATION

• M + E- → M●+ + 2E-

• M●+ IS THE IONIZED ANALYTE MOLECULE CALLED MOLECULAR ION

• RADICAL CATION IS FORMED BY THE LOSS OF ONE ELECTRON

• PERMITS EASY DETERMINATION OF MOLECULAR WEIGHT OF ANALYTE

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BASIC COMPONENTS OF MASS SPECTROMETRY

1. SAMPLE INTRODUCTION SYSTEM :VOLATILIZES THE SAMPLE AND INTRODUCES IT TO THE IONIZATION CHAMBER UNDER HIGH VACUUM

2. ION SOURCE IONIZES THE SAMPLE (FRAGMENTATION MAY OCCUR) AND ACCELERATES THE PARTICLES INTO THE MASS ANALYZER

3. MASS ANALYZER (OR MASS SEPARATOR) SEPARATES IONIZED PARTICLES BASED ON THEIR MASS-TO-CHARGE RATIO (M/E-)

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BASIC COMPONENTS OF MASS SPECTROMETRY

4. DETECTOR - ION COLLECTOR

MONITORS THE NUMBER OF IONS REACHING DETECTOR PER UNIT TIME AS A CURRENT FLOW

5. SIGNAL PROCESSOR

AMPLIFIES THE CURRENT SIGNAL AND CONVERTS IT TO A DC VOLTAGE

6. VACUUM PUMP SYSTEM

A VERY HIGH VACUUM (10-4 TO 10-7 TORR) IS REQUIRED SO THAT THE GENERATED IONS ARE NOT DEFLECTED BY COLLISIONS WITH INTERNAL GASES

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INSTRUMENTATION

(1) SAMPLE INTRODUCTION SYSTEMS o BATCH INLET

o DIRECT PROBE

o CHROMATOGRAPHY INTERFACE (GC-MS)

o INDUCTIVELY COUPLED PLASMA (ICP)

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SAMPLE INTRODUCTION SYSTEMS

I. BATCH INLET • SAMPLE IS VOLATILIZED EXTERNALLY AND ALLOWED TO “LEAK” INTO THE ION SOURCE

GOOD FOR GAS AND LIQUID SAMPLES WITH BOILING POINTS < 500 °C

II. DIRECT PROBE • GOOD FOR NON-VOLATILE LIQUIDS, THERMALLY UNSTABLE COMPOUNDS AND SOLIDS

• SAMPLE IS HELD ON A GLASS CAPILLARY TUBE, FINE WIRE OR SMALL CUP SAMPLE INTRODUCTION SYSTEM

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SAMPLE INTRODUCTION SYSTEMS

III. CHROMATOGRAPHY INTERFACE (GC-MS) • THE MS IS USED BOTH QUANTITATIVELY AND QUALITATIVELY

• MAJOR INTERFACE PROBLEM – CARRIER GAS DILUTION

• JET SEPARATOR (SEPARATES ANALYTE FROM CARRIER GAS)

IV. INDUCTIVELY COUPLED PLASMA (ICP) • OPERATES SOMEWHAT LIKE A NEBULIZER IN AN AAS

• ALSO IONIZES THE SAMPLE IN ARGON STREAM (AT VERY HIGH TEMPERATURES, >6000 °C)

• ONLY A SMALL AMOUNT OF ANALYTE IS UTILIZED (< 1%)

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ION SOURCE TECHNOLOGIES

• ELECTRON IONIZATION

• CHEMICAL IONIZATION

• MATRIX-ASSISTED LASER DESORPTION/IONIZATION

• GLOW DISCHARGE

• FIELD DESORPTION (FD)

• FAST ATOM BOMBARDMENT (FAB)

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ION SOURCE TECHNOLOGIES

I. ELECTRON IONIZATION • ELECTRON IONIZATION (EI, FORMERLY KNOWN AS ELECTRON IMPACT)

• IT IS AN IONIZATION METHOD IN WHICH ENERGETIC ELECTRONS INTERACT WITH GAS PHASE ATOMS OR MOLECULES TO PRODUCE IONS.

• THIS TECHNIQUE IS WIDELY USED IN MASS SPECTROMETRY, PARTICULARLY FOR GASES AND VOLATILE ORGANIC MOLECULE

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ELECTRON IONIZATION

• THE FOLLOWING GAS PHASE REACTION DESCRIBES THE ELECTRON IONIZATION PROCESS :

M + E- → M●+ + 2E-

• WHERE M IS THE ANALYTE MOLECULE BEING IONIZED,

• E- IS THE ELECTRON AND M+• IS THE RESULTING ION.

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CHEMICAL IONIZATION (CI) ION SOURCE

• A MODIFIED FORM OF EI

• HIGHER GAS PRESSURE IN IONIZATION CAVITY (1 TORR)

• REAGENT GAS (1000 TO 10000-FOLD EXCESS) ADDED; USUAL CHOICE IS ETHANE, CH4

• REAGENT GAS IS DIRECTLY IONIZED INSTEAD OF ANALYTE

• GENTLE; LITTLE FRAGMENTATION; EVEN-ELECTRON IONS PRODUCED MORE STABLE THAN ODD-ELECTRON IONS PRODUCED IN EI

• EXCESS ENERGY OF EXCITED IONS REMOVED BY MANY ION-REAGENT GAS COLLISIONS

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FAST ATOM BOMBARDMENT

• ION SOURCE FOR BIOLOGICAL MOLECULES

• AR IONS PASSED THROUGH LOW PRESSURE

• AR GAS TO PRODUCE BEAM OF NEUTRAL IONS

• ATOM-SAMPLE COLLISIONS PRODUCE IONS AS LARGE AS 25,000 DALTONS

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GLOW DISCHARGE

• SPUTTERING OF THE CATHODE MATERIAL (THE SAMPLE) BY AN ARGON PLASMA.

• IONIZATION OF THE ELEMENTS OF THE SAMPLE IN THE PLASMA.

• EXTRACTION AND ACCELERATION OF IONS.

• IONS SEPARATION WITH A MAGNETIC SECTOR

• IONS DETECTION BY A FARADAY CUP OR AN ELECTRON MULTIPLIER

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MATRIX-ASSISTED LASER DESORPTION/IONIZATION (MALDI)

• ANALYTE MIXED WITH RADIATION-ABSORBING MATERIAL AND DRIED

• SAMPLE ABLATED WITH PULSED LASER

• OFTEN COUPLED TO TIME-OF-FLIGHT (TOF) DETECTOR

• EXCELLENT FOR LARGER MOLECULES, E.G. PEPTIDES, POLYMERS

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MASS ANALYZER TECHNOLOGIES

• TIME-OF-FLIGHT

• QUADRUPOLE

• QUADRUPOLE ION TRAP

• LINEAR QUADRUPOLE ION TRAP

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A. QUADRUPOLE ANALYZER • IONS FORCED TO WIGGLE BETWEEN FOUR RODS WHOSE POLARITY IS RAPIDLY

SWITCHED

• SMALL MASSES PASS THROUGH AT HIGH FREQUENCY OR LOW VOLTAGE; LARGE MASSES AT LOW FREQUENCY OR HIGH VOLTAGE

• VERY COMPACT, RAPID (10 MS)

• R = 700-800

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B. TOF TIME OF FLIGHT MASS ANALYZER • SEPARATES IONS BASED ON FLIGHT TIME IN DRIFT TUBE

• POSITIVE IONS ARE PRODUCED IN PULSES AND ACCELERATED IN AN ELECTRIC FIELD (AT THE SAME FREQUENCY)

• ALL PARTICLES HAVE THE SAME KINETIC ENERGY

• LIGHTER IONS REACH THE DETECTOR FIRST

• TYPICAL FLIGHT TIMES ARE 1-30 ΜSEC

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MASS ANALYZER TECHNOLOGIES B. TIME OF FLIGHT MASS ANALYZER

SEPARATION PRINCIPLE :

• ALL PARTICLES HAVE THE SAME KINETIC ENERGY

• IN TERMS OF MASS SEPARATION PRINCIPLES:

o VPARTICLE = HER/M

HOLD H,E, AND R CONSTANT

o VPARTICLE = 1/M (CONSTANT)

o VPARTICLE IS INVERSELY PROPORTIONAL TO MASS

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DETECTORS

• MONITORS THE NUMBER OF IONS REACHING DETECTOR PER UNIT TIME AS A CURRENT FLOW

• THE DETECTION AND RECORDING OF IONS CAN BE DONE EITHER BY PHOTOGRAPHIC PLATES OR ELECTRICAL METHOD.

• PHOTOGRAPHIC PLATES

• ELECTRICAL METHOD

DETECTORS(1) PHOTOGRAPHIC PLATES: • IN THIS METHOD A PHOTOGRAPHIC PLATE IS KEPT AT RIGHT ANGEL TO THE PATH OF IONS SO IONS OF

SUCCESSIVE M/E VALUES FORM AN IMAGE

(2) ELECTRICAL METHOD:DETECTOR IS USUALLY ELECTRON MULTIPLIER

• PRODUCE ELECTRICAL SIGNAL PROPORTIONAL TO NUMBER OF IONS,

• THESE SIGNALS ARE AMPLIFIED BY A SERIES OF DYNODES.

• THE RESULT OF THESE AMPLIFIED SIGNALS IS PRESENTED IN THE FORM OF GRAPH

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APPLICATIONS OF MASS SPECTROMETRY IN DIFFERENT FIELDS

• DRUG DISCOVERY

• CLINICAL TESTING

• GENOMICS

• ENVIRONMENT

• GEOLOGY

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MASS SPECTROMETRY ROLE IN BIOCHEMISTRY

• DETERMINE PROTEIN STRUCTURE, FUNCTION, FOLDING AND INTERACTIONS• IDENTIFY A PROTEIN FROM THE MASS OF ITS PEPTIDE

FRAGMENTS• QUANTITATE PROTEINS IN A GIVEN SAMPLE• MONITOR PROTEIN DIGESTION

mass spectrometry (1)

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