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Quadrupole and Ion Trap Mass Analysers and an Introduction to Resolution a Simple Definition of a Mass Spectrometer

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Quadrupole and Ion Trap Mass Analysers and an Introduction to Resolution a Simple Definition of a Mass Spectrometer Quadrupole and Ion Trap Mass Analysers and an introduction to Resolution A simple definition of a Mass Spectrometer ◗ A Mass Spectrometer is an analytical instrument that can separate charged molecules according to their mass–to–charge ratio. The mass spectrometer can answer the questions “what is in the sample” (qualitative structural information) and “how much is present” (quantitative determination) for a very wide range of samples at high sensitivity Components of a Mass Spectrometer Atmosphere Vacuum System Sample Ionisation Mass Data Detector Inlet Method Analyser System How are mass spectra produced ? ◗ Ions are produced in the source and are transferred into the mass analyser ◗ They are separated according to their mass/charge ratio in the mass analyser (e.g. Quadrupole, Ion Trap) ◗ Ions of the various m/z values exit the analyser and are ‘counted’ by the detector What is a Mass Spectrum ? ◗ A mass spectrum is the relative abundance of ions of different m/z produced in an ion source -a “chemical fingerprint” ◗ It contains – Molecular weight information (generally) – Structural Information (mostly) – Quantitative information What does a mass spectrum look like ? ◗ Electrospray mass spectrum of salbutamol + 100 MH 240 OH NH tBu HO Base peak HO % at m/z 240 241 0 60 80 100 120 140 160 180 200 220 240 260 280 300 What information do you need from the analysis ? ◗ Low or High Mass range ◗ Average or Monoisotopic mass (empirical) ◗ Accurate Mass ◗ Quantitation - precision, accuracy, selectivity ◗ Identification ◗ Structural Information ◗ Isotope Ratios Mass Analyzers Types of Mass Spectrometer –Magnetic Sector –Quadrupole –Ion Trap –Time-of-flight –Hybrid- Sector/trap, Quad/TOF etc Mass Spectrometers separate ions according to their mass-to-charge (m/z) ratios A Triple Stage Quadrupole Mass Analyzer Finnigan TSQ Q2 is Non-Linear Collision Cell Q0 Q1 Q2 Q3 ESI Probe Square Rod Ion Transmission Hyperbolic, high Electron to Analytical Quads precision Multiplier, quadrupoles Detection System Hyperbolic Quadrupoles How does the quadrupole work ? The quadrupole consists of four parallel rods. The opposing rods have the same polarity whilst adjacent rods have opposite polarity. Each rod is applied with a DC and an RF voltage.Ions are scanned by varying the DC/Rf quadrupole voltages. Only ions with the selected mass to charge ratio will have the correct oscillatory pathway in the Rf field. -ve +ve Quadrupole Mass Analyser The ion is transmitted along the quadrupole in a stable trajectory Rf field. The ion does not have a stable trajectory and is ejected from the quadrupole. Non-linear collision cell in the TSQ A non-linear Q2 ensures that neutral species are ejected from the mass analyser pathway Non-linear collision cell in TSQ Non-linear collision cell removes the line of sight to the detector for neutral species - reducing the noise at the detector. A 90 Degree Square Q2 in the TSQ Quantum Dynode Q3 Q2 API Q1 Q0 Ion Trap ◗ Consists of ring electrode and two end caps ◗ Principle very similar to quadrupole ◗ Ions stored by RF & DC fields ◗ Scanning field can eject ions of specific m/z ◗ Advantages -MS/MS/MS….. - High sensitivity full scan MS/MS Resolving Power of a quadrupole TheThe abilityability of of a a quadrupole quadrupole to to resolveresolve masses masses isis proportionalproportional toto frequency frequency of of AC AC and and lengthlength ofof quadrupolequadrupole Resolving power of round rods Round rods can only approximate the field generated by hyperbolic rods - most ideal when Rrod/R0 = 1.16 Rrod Rrod = Radius of rod R0 R0 = Position of rod relative to axis of instrument Resolving power of round rods Manufacturing rods such that Rrod/R0 = 1.16 over the full length of quadrupole is non-trivial. Variability would assure that one M+ is selected at one position and another where the ratio is different - loss of specificity and sensitivity. Thermal expansion coefficients must be very low – expansion / contraction within the length of the rod changes Rrod/R0 Hyperbolic rods give excellent transmission across the full range of resolution - providing optimal analytical specificity. Compare sensitivity at unit resolution versus open resolution. At tighter resolution round rods systems tend to lose a greater proportion of ions through variation in Rrod/R0. Manufacture of round rods Round rods manufacture typically requires several steps - first, machine rods. Second, machine ‘holder’ by drilling four holes into a cylinder. Third, machine ‘dropout’ to create ion path. Insert rods. Error in Rrod/R0 grows by number of processes. Rrod R0 Resolution Basics MagneticMagnetic Sector Sector Instruments Instruments ConstantConstant Resolution Resolution with with mass mass 10%10% Valley Valley Definition Definition Resolution = m / ∆ m Resolution Basics QuadrupolesQuadrupoles IonIon traps traps andand TOF’s TOF’s ConstantConstant peak peak widths widths VariableVariable Resolution Resolution FWHMFWHM Definition Definition Resolution = m / ∆ m m/z 500/1, R = 500 m/z 500/0.1, R = 5000 What is the advantage of using high resolution mass spectrometry? The advantage of high resolution measurement is to eliminate chemical background of the same nominal mass but different accurate mass and, therefore to increase the signal to noise ratio and the sensitivity respectively. Better selectivity is obtained by applying high resolution in case of isobaric compounds, i.e. two compounds of same nominal mass but different accurate mass. With low resolution only a combined Spectral result is obtained under Product ion conditions . With high resolution separate detection and therefore separate isolation and MS/MS spectra are obtained. Example: R= 1000 R= 5000 Compound mass1: 372.351 Da OVERLAPPING SEPARATION Compound mass2: 372.421 Da Effect of changing resolution on peak shape 0.5 FWHM 1.0 FWHM 0.7 FWHM 0.3 FWHM 0.2 FWHM 0.1 FWHM Effect of Peak Width On Transmission QUADRUPOLE ROUND RODS 100 T * Resolution at m/z 500 r 80 a Unit n 1000 * s 60 % m 2500 * i 40 5000 * s s i 20 o n 0 2 1.5 0.7 0.5 0.2 0.1 Peak Width FWHM Resolving Target Compounds in the Presence of Interferences ◗ Examples: Mometasone and Alprazolam were spiked into PPG ◗ >5000 (FWHM) parent ion resolution allows mass selection of these compounds differing by less than 0.2 Da from PPG ◗ Increasing the resolution to exclude the PPG interference increases the selectivity and specificity for the quantitative assay Mometasone resolution in the presence of PPGs (Mometasone [M+H]+ = 521.2, PPG (n=8) [M+Na]+ = 521.3) mometasone 35Cl 521.2 mometasone 37Cl resolved Q1 mass spectrum 523.2 522.2 521.3 522.9 524.1 522.3 523.4 521.2 transmitted parent ions PPG Q1 at unit resolution 521.3 521.2 Q1 selecting only mometasone 35Cl mometasone 35Cl 523.2 Relative Abundance 37 transmitted parent ions mometasone Cl PPG Q1 at unit resolution PPG 522.9 523.4 522.8 523.2 Q1 selecting only mometasone 37Cl mometasone 37Cl 518.0 518.5 519.0 519.5 520.0 520.5 521.0 521.5 522.0 522.5 523.0 523.5 524.0 524.5 525.0 m/z Mometasone resolution in the presence of PPGs product ion spectra of 37Cl mometasone at various precursor ion resolutions Q1 resolution unit * = PPG - related peaks Q1 resolution 0.08 FWHM passing only mometasone 37Cl The increased resolution on product ion scan of 37Cl mometasone can selectively exclude PPG interferents with an actual mass difference of less than 0.2 Da. Alprazolam resolution in the presence of PPGs (alprazolam [M+H]+ = 309.0907, PPG (n=5) [M+H]+ = 309.2277) 250 pg/uL alprazolam in 20 uM PPGs 309.3 PPG Transmitted precursor Unit resolution on Q1 ions 309→309 alprazolam 309.1 309.3 0.08 FWHM resolution on Q1 PPG Set to pass PPG ions Relative Abundance 309.1 0.08 FWHM resolution on Q1 alprazolam Set to pass alprazolam ions 0.08 Da FWHM 309.3 306.0 306.5 307.0 307.5 308.0 308.5 309.0 309.5 310.0 310.5 311.0 311.5 312.0 m/z Alprazolam resolution in the presence of PPGs Product ion spectra at various precursor ion resolutions Q1 set at unit resolution, spectrum is a composite of alprazolam and interfering PPG ions Q1 set to pass only PPG ions Q1 set to pass only alprazolam ions The increased resolution on product ion scan of alprazolam can selectively exclude PPG interferents with an actual mass difference of less than 0.2 Da. Comparison of Quads and Traps Ion Traps Quadrupoles Mass Separation in time Mass Separation in space High sensitivity Full Scan Lower sensitivity Full Scan Lower sensitivity SRM High sensitivity SIM and SRM Offer mutiple stages of MSn No mutiple stages of MS Offers higher resolution Lower resolution Parent and neutral loss scans.
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