1. Chem. 133 – 4/28 Lecture

2. Announcements Lab Report 2.3 due Today Pass back graded materials (lab reports 2.2, Q5, and AP3.1) Today’s Lecture Mass Spectrometry Ionization Methods (liquid samples?) Mass Analyzers Interpretation/Other Topics

3. Mass Spectrometry Ion Source Liquid Samples –Electrospray Ionization (ESI) Liquid is nebulized with sheath gas Nebulizer tip is at high voltage (+ or –), producing charged droplets As droplets evaporate, charge is concentrated until ions are expelled Efficient charging of polar/ionic compounds, including very large compounds Almost no fragmentation, but multiple charges possible For positive ionization, major peak is M+1 peak (most common); or for multiply charged compounds, peak is [M+n] n+ where n = charge on ion For negative ionization, M-1 peak is common Adduct formation also is possible e.g. [M+Na] + Liquid in Nebulizing gas High voltage ++ + ++ M+M+

4. Mass Spectrometry Ion Source ESI Example: –glycodendrimer core (courtesy of Grace Paragas) –C 30 H 60 N 14 O 12 (sorry, no structure) –Mass = 808.451 or for M+H+: 809.459 Our first “high resolution” ESI-MS sample – Full Spectrum M+H+ peak mass error = -2.6 ppm (+/- 5 ppm needed) Internal Standard: used for calibration

5. Mass Spectrometry Ion Source ESI Example: –So if ESI results in no fragmentation, what are the other peaks? –For most peaks, answer is “I don’t know”, but can give guesses for some M+H and isotope peaks M+41 = M+Na+H 2 O M+2H/2 peak = (808+2)/2 = 405 13C isotope peaks observed at +1/2 amu 425 peak = (M+H+Na+H 2 O)/2

6. Mass Spectrometry Ion Source DESI – Desorption Electrospray Ionization –Use of Electrospray focused onto sample to produce ionization –Commonly used for remote MS analysis of untreated surface –Tip with electrospray is pointed toward sample with vacuum pick up line near by –Collisions of electrspray charged drops end up charging surface molecules –Resulting ions are picked up to mass spectrometer entrance Sample plate (electrically conductive) sample Mass Analyzer Electrospray source vacuum line to mass analyzer M+M+

7. Mass Spectrometery Ion Sources Ion Sources –For Liquids (continued) Atmospheric Pressure Chemical Ionization –Liquid is sprayed as in ESI, but charging is from a corona needle nearby - More restricted to smaller sized molecules –For Solids (other than by DESI) Matrix Assisted Laser Desorption Ionization –Ionization from Laser –Samples normally doped with compound that absorbs light strongly (to cause intense heating/ionization)

8. Mass Spectrometery Ion Sources For Elemental Analysis –Inductively Coupled Plasma Produces ions as well as atoms used in ICP-AES Most sensitive method of elemental analysis skimmer cone to mass analyzer

9. Mass Spectrometry Questions 1.Which ionization method can be achieved on solid samples (without changing phase) 2.If one is using GC and concerned about detecting the “parent” ion of a compound that can fragment easily, which ionization method should be used? 3.For a large, polar non-volatile molecule being separated by HPLC, which ionization method should be used? 4.When analyzing a large isolated peptide by ESI-MS, multiple peaks are observed (at smaller than parent ion m/z numbers). What is a possible cause for this? 5.What ionization method should be used to analyze for lead in a sample?

10. Mass Spectrometery Instrumentation Analyzers –Separates ions based on mass to charge ratio –All operate at very low pressures (vacuums) to avoid many ion – ion or ion – molecule collisions –Analyzers for chromatographic systems must be fast. (If a peak is 5 s wide, there should be 4 scans/s) –Most common types (as chromatographic detectors): Quadrupole (most common) Ion Trap (smaller, MS-MS capability) Time of Flight (higher speed for fast separations and can be used for high resolution applications)

11. Mass Spectrometery Instrumentation Mass Spectrometer Resolution –R = M/ΔM where M = mass to charge ratio and is ΔM difference between neighboring peaks (so that valley is 10% or 50% of peak height – see text for exact defintion). –Standard resolution needed: To be able to tell apart ions of different integral weights (e.g. (CH 3 CH 2 ) 2 NH – MW = 73 vs. CH 3 CH 2 CO 2 H – MW = 74) More important to have higher resolution when analyzing larger compounds (e.g. a resolution of 1000 would be sufficient for GC-MS but not for LC-MS) –High Resolution MS: To be able to determine molecular formulas from “exact” mass example: CH 3 CH 2 CO 2 H vs. CHOCO 2 H; both nominal masses are 74 amu but CHOCO 2 H weighs slightly less (74.037 vs. 74.000 amu) because 16 O is lighter than 12 C + 4 1 H (Note: need to use main isotope masses to calculate these numbers – not average atomic weights). Needed resolution = 74/0.037 = 2000 Resolution > about 10 4 to 10 5 is normally needed.

12. Mass Spectrometry High Resolution Calculation of Exact Mass –Several compounds can have a molecular weight of 84 –Examples: C 6 H 12 C 5 H 8 O C 4 H 4 O 2 C 4 H 4 S CH 2 Cl 2 –Each example above will have slightly different mass (go over mass calculations on board)

13. Mass Spectrometry Isotope Effects It also may be possible to distinguish compounds based on isotopic composition Compounds in high resolution example will have different expected M+1/M and M+2/M ratios (which will NOT require high resolution to see) Calculations covered briefly for lab (will give you M+n/M ratios for single atom) Main difficulty is accurately determining ratios (plus effects of contaminants, variation in ratio, etc.)

14. Mass Spectrometry Other Topics – Multiple Charges in ESI In ESI analysis of large molecules, multiple charges are common due to extra (+) or missing (-) Hs (or e.g. Na + ) The number of charges can be determined by looking at distribution of big peaks For + ions m/z = (M+1.008n)/n (most common) For – ions m/z = (M–n)/n Ion current m/z  m/z I am only showing an “approximate” method for determining n – this usually will work when H + is causing the charging, but not if Na + causes charging (M+n)/n (M+n+1)/(n+1) Example: m/z peaks =711.2, 569.3, 474.8, 407.1

15. Mass Spectrometry Questions - #2 1.A modification is made in a peptide chain of molecular weight 1503 (native form), in which one threonine residue (NH 3 CH(CHCH 3 (OH))CO 2 ) is replaced with cysteine (NH 3 CH(CH 2 SH)CO 2 ). What resolution is needed to separate the native peptide from the modified peptide? 2.Predict the M+1/M, M+2/M, M+3/M, and M+4/M ratios for the ion CH 3 SO 3 -.