1. MS Intro

2. MS requires gas-phase ions, why? MS uses magnetic and electric fields to control the path of a compound based on mass to charge ratio (m/z) m/z = 137.11192m/z = 68.0553 Remember you calculate mass using the most abundant isotope pH 3pH 7 MW = 138.1278

3. MS Intro m/z = 136.1351 m/z = N/A MS requires gas-phase ions, why? MS uses magnetic and electric fields to control the path of a compound based on mass to charge ratio (m/z) pH 4 pH 1 pH 7 MW = 137.1438 m/z = 138.1525

4. MS Intro MS requires gas-phase ions MS uses magnetic and electric fields to control the path of a compound based on mass to charge ratio (m/z), how? Variety of ways: 1. Control which ions hit the detector - Quadrupole, magnetic sector, ion trap, accelerator MS 2. Determine how fast an ion hits a detector - Time of flight 3. Measure frequency of orbital motion of trapped ions - Fourier transform ion cyclotron resonance (FT-ICR)

5. MS Intro CharacteristicQuadrupoleIon trap Magnetic Sector Time of Flight FT-ICR Mass Range (Da) < 4000 15,000Unlimited>10 4 Resolving power 400010 3 -10 4 10 2 -10 5 15,000>10 6 Mass Accuracy (ppm) 10050-1001-55-501-5

6. MS Intro CharacteristicQuadrupoleIon trap Magnetic Sector Time of Flight FT-ICR Mass Range (Da) < 4000 15,000Unlimited>10 4 Resolving power 400010 3 -10 4 10 2 -10 5 15,000>10 6 Mass Accuracy (ppm) 10050-1001-55-501-5 Resolving power is the ability to separate two neighboring peaks. This is a measure of how ‘sharpness’ of a mass peak. Example…

7. RP = m (average mass)  m (mass difference) Resolving power (RP) MS Intro m mm

8. CharacteristicQuadrupoleIon trap Magnetic Sector Time of Flight FT-ICR Mass Range (Da) < 4000 15,000Unlimited>10 4 Resolving power 400010 3 -10 4 10 2 -10 5 15,000>10 6 Mass Accuracy (ppm) 10050-1001-55-501-5 Resolving power is the ability to separate two neighboring peaks. This is a measure of how ‘sharpness’ of a mass peak. Example…

9. MS Intro RP = mmmm Resolving power (RP) Need to determine the accurate mass of both compounds using the most abundant isotopes of each element How much resolving power do we need to distinguish between the following compounds: C = 12.00000 amu H = 1.007825 amu O = 15.994915 amu P = 30.973762 amu S = 31.972072 amu 3C + 7H + S + 3O = 123.011592 amu 3C + 8H + P+ 3O = 123.021107 amu RP = (123.021107 + 123.011592)/2 RP = 12900 (123.021107 - 123.011592)

10. MS Intro CharacteristicQuadrupoleIon trap Magnetic Sector Time of Flight FT-ICR Mass Range (Da) < 4000 15,000Unlimited>10 4 Resolving power 400010 3 -10 4 10 2 -10 5 15,000>10 6 Mass Accuracy (ppm) 10050-1001-55-501-5 Resolving power is the ability to separate two neighboring peaks. This is a measure of how ‘sharpness’ of a mass peak. Example…

11. MS Intro CharacteristicQuadrupoleIon trap Magnetic Sector Time of Flight FT-ICR Mass Range (Da) < 4000 15,000Unlimited>10 4 Resolving power 400010 3 -10 4 10 2 -10 5 15,000>10 6 Mass Accuracy (ppm) 10050-1001-55-501-5 Mass accuracy Comparison of the measured mass to the accurate mass Example…

12. MS Intro 5 ppm = 5 1,000,000 Mass Accuracy (ppm) Mass accuracy to 4 decimal places Lower the ppm value the more accurate the measurement Require < 10 ppm accuracy for ‘accurate mass’ measurement MW = 123.011592 amu MW = 123.021107 amu = 6 x 10 -4 ppm value? 1,000,000 = measured mass - actual mass actual mass Example, you have a mass for an unknown compound which you expect is one of the compounds below, which ones is it most likely? Measured mass: 123.0204

13. MS Intro MW = 123.011592 amu ppm = (123.0204 - 123.011592) 123.011592 Example, you have a mass for an unknown compound which you expect is one of the compounds below, which ones is it most likely? What is the accuracy (ppm) of this measurement as compared to the 2 actual masses ppm value? 1,000,000 = measured mass - actual mass actual mass MW = 123.021107 amu ppm = (123.0204 - 123.021107) 123.021107 x 1,000,000 = 5.7 x 1,000,000 = 72

14. MS Intro CharacteristicQuadrupoleIon trap Magnetic Sector Time of Flight FT-ICR Mass Range (Da) < 4000 15,000Unlimited>10 4 Resolving power 400010 3 -10 4 10 2 -10 5 15,000>10 6 Mass Accuracy (ppm) 10050-1001-55-501-5 Mass accuracy Measured error in m/q divided by the accurate mass Example…

15. Quadrupole MS

16. - - --+ ++ + Ion focusing -RF only -All ions pass Quadrupoles

17. Ion focusing -RF only -All ions pass Quadrupoles

18. - - + ++ + - A DC potential is applied to the poles, making 2 diagonal poles negative and 2 positive Quadrupole MS How do the quads work? - - - The RF potential is superimposed over the DC potential

19. + ++ + + - -- - + - positive ion will be attracted to a negative pole - during the RF pulse the charges of the poles reverse, and the ion will be repelled from this same pole - ion will be attracted to a pole that is negative - this process continues as the ion moves through the poles (into the slide) + Quadrupole MS RF pulse - Select for m/z 500 (positive) How do they select for an m/z?

20. + ++ + + -- + + - positively charged poles focus ions into centre plane - negatively charged poles attract ions out of the plane Quadrupole MS How do they select for an m/z? - Select for m/z 500 (positive)

21. + ++ + + -- + + + + - positively charged poles focus ions into centre plane - negatively charged poles attract ions out of the plane - during the RF pulse these charges reverse and attractive / repulsive forces reverse and the ions are focused in the other plane Quadrupole MS RF pulse - Select for m/z 500 (positive) How do they select for an m/z?

22. + ++ +-- + - positively charged poles focus ions into centre plane - negatively charged poles attract ions out of the plane - results in a complicated path with the ion moving towards and away from the poles as the potential changes, but constantly being refocused into the centre Quadrupole MS - during the RF pulse these charges reverse and attractive / repulsive forces reverse and the ions are focused in the other plane - Select for m/z 500 (positive) How do they select for an m/z?

23. Quadrupole MS + + + + + -- -Kinetic energy of an ion in an electrical field is proportional to its mass - Select for m/z 500 (positive) How do they select for an m/z? -Because of this, ions of lower m/z (m/z < 500, in this example) are accelerated more aggressively and eventually collide

24. Quadrupole MS + ++ +-- + + + -In contrast ions of higher m/z (m/z > 500, in this example) are slow to respond to the change in potential during the RF pulse -Kinetic energy of an ion in an electrical field is proportional to its mass - Select for m/z 500 (positive) How do they select for an m/z?

25. Quadrupole MS + + ++-- RF pulse -In contrast ions of higher m/z (m/z > 500, in this example) are slow to respond to the change in potential during the RF pulse -Kinetic energy of an ion in an electrical field is proportional to its mass - Select for m/z 500 (positive) How do they select for an m/z? -They are not refocused into the centre plane and are lost to the negative poles (DC potential)

26. Quadrupole MS + + -In contrast ions of higher m/z (m/z > 500, in this example) are slow to respond to the change in potential during the RF pulse -Kinetic energy of an ion in an electrical field is proportional to its mass - Select for m/z 500 (positive) How do they select for an m/z? -They are not refocused into the centre plane and are lost to the negative poles (DC potential) + ++ +--

27. + ++ +-- + Quadrupole MS -Only very specific m/z have their path appropriately corrected so that they pass safely through the quads -Resolution of a quad is usually unity -Can tell the difference between 499 and 500, but not between 499 and 499.5 How do they select for an m/z?

28. Triple Quadrupole MS Mass analyzer Q1 Mass analyzer Q3 Collision Cell Q2 Ion source Det Mass filter RF only All ions pass

29. Triple Quadrupole MS Collision Cell Q2 Positive ion in the collision cell - No mass analysis, all masses are allowed to pass - Collision cell is filled with an inert gas: Ar, He or N 2. -Analyte is accelerated through the collision cell -The faster this acceleration the more energetic the collision, and the more fragmentation + - + + -Analyte collides with the inert gas N2N2 5-50 eV + + + - Parent ion Collision Product ions -Process is called collisionally- induced dissociation

30. Triple Quadrupole MS Mass analyzer Q1 Mass analyzer Q3 Collision Cell Q2 Ion source Det + N2N2 + + + - Parent ion selection Collisionally-induced dissociation + + Product ion selection

31. Triple Quadrupole MS Mass analyzer Q1 Mass analyzer Q3 Collision Cell Q2 Ion source Det Parent ion selection Collisionally-induced dissociation Product ion selection PFOA, 499 m/z Fragments Product, 369 m/z

32. Triple Quadrupole MS -Cheap and small -Little downtime, workhorses What is the selling point of a quadrupole instrument -The choice of two characteristic ions is very selective -Selectivity lowers background noise (game of S:N) -Very good sensitivity! -Some structural information -Limited analyte information, usually only nominal mass analysis What is the downside of a quadrupole instrument What is the selling point of a triple quadrupole instrument -Can have some interferences and ‘false detections’ What is the downside of a triple quadrupole instrument