QUADRUPOLE MASS ANALYZER

FOR BASIC UNDERSTANDING..

1. Quadrupole mass analyzer how it works triple quadrupole mass spectrometer MIDHUNA (P21SH202)

2. Mass Spectrometry A technique for measuring and analyzing molecules, that involves introducing enough energy into a (neutral) target molecule to cause its ionization and disintegration. The resulting primary ions and their fragments are then analyzed, based on their mass/ charge ratios, to produce a "molecular fingerprint ESI Quadrupole Electron Multiplier MALDI Time-Of-Flight Faraday Cup

3. QMA is one type of analyzer used in MS Quadrupole mass filter Four cylindrical rods are parallel to each other Opposing rod pair is connected together electrically Mass Analyzer: Quadrupole (Q)

4. PRINCIPLE By applying DC and AC, Potential generated will cause the movement of ions As they move positive ions will migrate – ve electrode and negative ions migrate to the + ve electrode, and they will never get mixed up Ions travel through the quadrupole, on the produced electric field, only ions of a particular m/z will be focused on the detector (Resonant ion), all the other ions will be deflected into the rods. ( non-resonant ion)

5. If the applied voltage is composed of a DC voltage U and a radiofrequency (RF) voltage V with the frequency ω the total potential Φ is given by, Φ= U + V cos ωt The path stability of a particular ion is defined by the magnitude of the RF voltage V and by the ratio U/V. Reducing U relative to V, ( RF>DC), smaller m/z ion bombard to wall and distance to the detector will increase and larger ion bombard less, moves faster so increasingly wider m/z range can be transmitted simultaneously. If RF<DC , smaller ion hit the detector first

6. Path stability of a particular ion is determined by- varying the amplitude of the voltages (AC/DC )

7. TRIPLE QUADRUPOLE MASS ANALYZER analyzer in tandem three sets of quadrupole analyzers are used in sequence

8. Q1 can scan across a range of m/z values or selectively filter ions of a selected m/z. Q2 -collision cell - fragment the selected ions from Q1. The product ions formed scanned through Q3, to obtain a mass spectrum or Q3 can be fixed in order to monitor a particular ion. Combination of fixed or scanning modes of Q1 and Q3 determine the type of scan performed

9. SCAN MODES

10. Product ion scans also known as daughter ion scans • Q1 is set to allow only the transmission of one m/z • The parent ion collides with Argon gas in Q2 to create fragment or product ions • Product ions are scanned through Q3 - Once the product ions are recorded, Q1 can then fix on a new m/z and the process repeated. Applications: Bottom-up proteomics approach, the sequence of many peptides eluting off a chromatographic column can be sequenced. Targeted Metabolomics

11. Precursor ion scan –Q3 is set to allow only a fragment ion of one m/z to pass Q1 is scanned across the entire m/z range of the analyzer. The precursor ions subsequently pass through Q2 for CID. Q3 is kept fixed such that only product ions of a specific m/z are filtered through the quadrupole. Precursor ion scanning allows one to determine the m/z of all precursor ions that have the same product ion. This is valuable in proteomics when one wants to identify all peptides that may have the same functional group.

12. Precursor ion scans are used for screening experiments where a group of compounds all gives the same fragment ion Eg 1: m/z 216 = signature immonium ion for phosphotyrosine Precursor ion scan allows identification of all peptides containing phosphotyrosine

13. Eg 2: identification of fragment of m/z=84 from different compounds

14. Neutral loss scan – Track ions before and after the loss of a neutral group. Both Q1 and Q3 are scanned simultaneously over the entire m/z range but with Q3 offset from the Q1 by an amount that corresponds to the loss of a neutral fragment from the ion . all precursors that undergo the loss of the same neutral fragment can be monitored. Application: PTM

15. Eg : Performing low energy CID on peptides that are phosphorylated will often result in the loss of phosphoric acid (H3PO4, m/z ¼ 98) from the parent ion, identification of peptides with phosphorylated serine or threonine

16. Selected reaction monitoring – also called  Multiple reaction monitoring , ( MRM ), is a method used in tandem mass spectrometry  in which an  ion  of a particular mass is selected in the first stage of a tandem  mass spectrometer  and an ion product of a fragmentation reaction of the  precursor ions  is selected in the second mass spectrometer stage for detection. Thus, specific signature fragment ions originating from a compound of known mass can be monitored.

17. Eg :- Orphenadrine is an anticholinergic drug of the ethanolamine antihistamine class. Q1 is set to allow only the transmission of Orphenadrine m/z 269.9 - Q3 is scanned only to detect m/z 181.1 +/- 0.3

18. SPECIFICATION Mass range 10-4000 dalton(amu) Resolution, typically 1000 Scan rate 5000dalton/min Accuracy .1-.2dalton

19. Applications Compositional analysis of gas and volatile liquids Mainly used in residual gas analysis Used in LC-MS and GC-MS Both qualitative and quantitative Advantage Compactible and reliable Law cost analytical tool Excellent stability over long periods of time Disadvantage Low resolution, high sensitivity Only one m/z ratio can be obtained at a time Broader m/z obtained only after long scanning period Not suited for pulsed ionization method

20. REFERENCES 1. Mass Analyzers and Mass Spectrometers Anthony M. Haag 2. Jürgen H. Gross Mass Spectrometry 3. Lovrić , Josip - Introducing proteomics _ from concepts to sample separation, mass spectrometry and data analysis-Wiley-Blackwell (2012)

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