Chem. 230 – 11/18 Lecture

1. Chem. 230 – 11/18 Lecture

2. Announcements I • Exam 3 Results • Lower Average (72%) • Distribution • New Homework Posted Online (long problems due next week)

3. Announcements II • Special Topics Presentations • Need to prepare reading material (link to journal or photocopies in folder) one week before presentations (due today for group going 11/25) • Besides presentation, will need Homework Problems (I request 4 per group) on day of presentation

4. Announcements III • Today’s Lecture • Quantification • Methods of Calibration • Mass Spectrometry • Applications • Instrumentation • Use as Chromatographic Detector • Interpretation • Other Topics

5. Quantitation in ChromatographyCalibration Methods • External Standard • most common method • standards run separately and calibration curve prepared • samples run, from peak areas, concentrations are determined • best results if unknown concentration comes out in calibration standard range • Internal Standard • Common for GC with manual injection (imprecisely known sample volume) • Useful if slow drift in detector response • Standard added to sample; calibration and sample determination based on peak area ratio • F = constant where A = area and C = conc. (X = analyte, S = internal standard) Area Concentration AX/AS Conc. X (constant conc. S)

6. Quantitation in ChromatographyCalibration Methods Standard Addition Used when sample matrix affects response to analytes Commonly needed for LC-MS with complicated samples Standard is added to sample (usually in multiple increments) Needed if slope is affected by matrix Concentration is determined by extrapolation (= |X-intercept|) Surrogate Standards Used when actual standard is not available Should use structurally similar compounds as standards Will work with some detector types (FID, RI, ABDs) standards in water Area Analyte Concentration Concentration Added

7. QuantitationAdditional (Recovery Standards + Questions) Recovery Standards Principle of use is similar to standard addition Standard (same as analyte or related compound) added to sample, then measured (in addition to direct measurement of sample) Useful for determining losses during extractions, derivatization, and with matrix effects

8. QuantitationSome Questions/Problems Does increasing the flow rate improve the sensitivity of a method? Does the use of standard addition make more sense when using a selective detector or a universal detector? Is a matrix effect more likely with a simple sample or a complex sample? Why is the internal standard calibration more common when using manual injection than injection with an autosampler?

9. QuantitationSome Questions/Problems 5. A scientist is using GC-FID to quantitate hydrocarbons. The FID is expected to generate equal peak areas for equal numbers of carbons (if substances are similar). Determine the concentrations of compounds X and Y based on the calibration standard (1-octanol). X = hydroxycyclohexane and Y = hydroxypentane.

10. QuantitationSome More Questions/Problems 6. A chemist is using HPLC with fluorescence detection. He wants to see if a compound co-eluting with a peak is quenching (decreasing) the fluorescence signal. A set of calibration standards gives a slope of 79 mL μg-1 and an intercept of 3. The unknown gives a signal of 193 when diluted 4 mL to 5 mL (using 1 mL of water). When 1.0 mL of a 5.0 μg mL-1 standard is added to 4.0 mL of the unknown, it gives a signal of 265. What is the concentration of the unknown compound and is a significant quenching (more than 10% drop in signal) occurring?

11. QuantitationSome More Questions/Problems 7. A chemist is testing an extraction process for removing DDT from fish fat. 8.0 g of fat is first dissolved in 50 mL of 25% methylene chloride in hexane. The 50 mL is divided into two 25 mL portions, one of which is spiked by adding 2.0 mL of 25.0 ng mL-1 DDT. Each portion is run through a phenyl type SPE cartridge and the trapped DDT is eluted with 5.0 mL 100% methylene chloride. The methylene chloride is evaporated off, and the sample is redissolved in 0.5 mL of hexane and injected onto a GC. The un-spiked sample gives a DDT conc. (in 0.5 mL of hexane) of 63 ng mL-1, while the spiked sample gives a DDT conc. of 148 ng mL-1. What is the % recovery? What was the original conc. of DDT in the fat in ppb?

12. Mass SpectrometryOverview Applications of Mass Spectrometry Mass Spectrometer Components GC-MS LC-MS Other Applications

13. Mass SpectrometryApplications Direct Analysis of Samples Most common with liquid or solid samples Reduces sample preparation Main problem: interfering analytes Off-line Analysis of Samples Samples can be separated through low or high efficiency separations More laborious Chromatographic Detectors generally most desired type since this allows resolution of overlapping peaks

14. Mass SpectrometryApplications Purposes of Mass Spectrometry Quantitative Analysis (essentially used as any other chromatographic detector) Advantages: selective detector (only compounds giving same ion fragments will overlap) overlapping peaks with same ion fragment can be resolved (through deconvolution methods) semi-universal detector (almost all gases and many solutes in liquid will ionize) very good sensitivity Disadvantages cost requires standards for quantification

15. Mass SpectrometryApplications Purposes of Mass Spectrometry - continued Qualitative Analysis/Confirmation of Identity With ionization method giving fragmentation, few compounds will produce the same fragmentation pattern Even for ionization methods that don’t cause fragmentation, the parent ion mass to charge data gives information about the compound identity. Some degree of elemental determination can be made based on isotopic abundances (e.g. determination of # of Cl atoms in small molecules) Additional information can be obtained from MS-MS (further fragmentation of ions) and from high resolution mass spectrometry (molecular formula) if those options are available. Isotopic Analysis Mass spectrometry allows analysis of the % of specific isotopes present in compounds (although this is normally done by dedicated instruments to get good enough precision for use as source tracers) An example of this use is in drug testing to determine if testosterone is naturally produced or synthetic

16. Mass SpectrometryInstrumentation Main Components: Ion source Analyzer Detector Data Processor

17. Mass SpectrometryInstrumentation Ion Sources For Gases Electron Impact (EI): electrons from heated element strike molecules M + e- => M+* + 2e- M+ is the parent ion Because M+* often has excess energy, it can fragment further, usually producing a smaller ion and a radical Fragmentation occurs at bonds, but electronegative elements tend to keep electrons + gas stream M e- e- CH3-Br+* CH3+ + Br∙ CH3∙+ Br+ Main fragment Minor or unobserved fragment

18. Mass SpectrometeryInstrumentation Ion Sources For Gases Chemical Ionization (CI): Can produce positive or negative ions First, a reagent gas reacts with a corona discharge to produce a reagent ion: CH4 => => CH5+ (more likely CH4∙H+) Then the reagent ion transfers its charge to a molecule: M + CH5+ => MH+ (one of largest peak has mass to charge ratio of MW + 1) Less fragmentation occurs, so more useful for identifying the parent ion

19. Mass SpectrometeryInstrumentation Ion Sources For Liquids Earlier Methods (particle beam and thermospray) suffered from poorer efficiency and ability to form ions from large molecules 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 often M+1 peak; or for multiply charged compounds, peak is [M+n]n+where n = charge on ion Nebulizing gas High voltage M+ + + + + Liquid in +

20. Mass SpectrometeryInstrumentation 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 Atmospheric Pressure Photoionization UV light causes photoionization of molecules

21. Mass SpectrometeryInstrumentation Ion Sources For Solids (common off-line method) Matrix Assisted Laser Desorption Ionization Sample plus strong absorber placed on substrate solvent removed laser focused on sample heat causes desorption and ionization of analytes M+

22. Mass Spectrometry 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)

23. Mass SpectrometryInstrumentation Mass Spectrometer Resolution R = M/ΔM where M = mass to charge ratio and is ΔM difference between neighboring peaks (so that valley is 10% of peak height). Standard resolution needed: To be able to tell apart ions of different integral weights (e.g. (CH3CH2)2NH – MW = 73 vs. CH3CH2CO2H – MW = 74) High Resolution MS: To be able to determine molecular formulas from “exact” mass example: CH3CH2CO2H vs. CHOCO2H; both nominal masses are 74 amu but CHOCO2H weighs slightly less (74.037 vs. 74.000 amu) because 16O is lighter than 12C + 41H (Note: need to use main isotope masses to calculate these numbers – not average atomic weights). Needed resolution = 74/0.037 = 2000 To separate similar ions requires very high resolution > 104 to 105 However, to obtain “accurate” mass (error in mass under 5 ppm) is not quite as hard in terms of resolution but requires internal standards and clean peaks

24. Mass Spectrometry Instrumentation Analyzers – how separation works Analyzers can act as filters (only passing a specific m/z at a time) – e.g. in quadrupoles and ion traps, can give full spectrum in a short time (time of flights), or can give full information over an acquisition (Fourier Transform ion cyclotron resonance) Control of ion throughput makes sense in ion traps or in quadrupoles but in time of flight full spectrum comes (whether desired or not)

25. Mass SpectrometryInstrumentation Detectors: Faraday Cup (simple, but not sensitive) Electron Multiplier (most common) Array Detector (Multichannel Analyzer) M+ I Detection Process: Ion strikes anode Electrons are ejected Ejected electrons hit dynodes causing a cascade of electron releases Current of electrons hitting cathode is measured Anode Dynodes Cathode M+ e- e- I

26. Mass SpectrometeryUse with GC MS matches well to capillary GC flow rates With EI gives good qualitative information CI used if compound fragments too much Total Ion and Selective Ion Modes: Total Ion Current (TIC) gives full mass spectra at every point (better for qualitative analysis) Selective Ion Monitoring (SIM) only determines signal at several ions (the fragments of interest) (better for quantitative analysis because of better sensitivity)

27. Mass SpectrometeryUse with GC - Example Example of examination of co-eluting peaks Synthetic diesel sample shows large number of peaks – mostly alkanes and alkenes C12s peak cluster = (mostly) same number carbons 2-Alkenes 1-Alkene Alkane mostly branched alkanes

28. Mass SpectrometeryUse with GC – Example – Cont. Analysis didn’t match manufacturer’s assessment of 4% alcohols However, alcohols are hard to determine by MS due to loss of H2O in fragmentation CH3(CH2)6OH → CH3(CH2)5CH·+ (MW = 98 – same as expected for alkene M peak) Linear Alcohols found to elute at time of branched C10 alkanes mass spectrum shows alkyl chains

29. Mass SpectrometeryUse with GC – Example – Cont. Careful examination of fragmentation shows differences between right and left sides of peak with right side close to that of C7 alcohol standard right shoulder 1-heptanol

30. Mass SpectrometeryUse with GC – Example – Cont. Ion Extraction allows separation of chromatographic peaks based on 70 vs 71 fragments Could improve by: using CI, using slight difference in column polarity Identification stronger due to water washing fuel 70 (alcohol) fragment 71 (branched alkane) fragment

31. Mass SpectrometeryUse with HPLC One disadvantage is the volume of gas developed as solvent evaporates For this reason, HPLC flows must be low (e.g. semi-microbore), or splitters are needed With most common ionization (ESI), little fragmentation occurs, making identification of unknown compounds harder Because of little fragmentation, MS-MS is more common In MS-MS, ions leaving mass analyzer are then fragmented (by collisions with molecules) before entering a second mass analyzer or re-entering the mass analyzer Also, some compounds are hard to ionize efficiently

32. Mass SpectrometeryInterpretation Fragmentation Analysis Focus on possible structure of fragments (low end of spectrum) or of fragments lost (high end of spectrum) Isotopic Analysis For elements with more than 1 isotope in abundance Average MW not useful, MW of specific isotopes determines charge Formation of M+1, M+2, M+3 ... peaks to predict elements present Determination of Charge Important for interpreting MALDI and ESI peaks where multiple charges are possible

33. Mass SpectrometryIsotope Effects It also may be possible to distinguish compounds based on isotopic composition Average MW is not useful (except for very large MW compounds), but abundance of each isotope gives each element a “fingerprint” 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) Go over calculations on board for CH3SSCH3 Main difficulty is accurately determining ratios (plus effects of contaminants, variation in ratio, etc.)

34. Mass SpectrometryOther 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+n)/n (most common) For – ions m/z = (M–n)/n (M+n)/n Dm/z Ion current m/z (M+n+1)/(n+1) Example: m/z peaks =711.2, 569.3, 474.8, 407.1 Dm/z = (M+n)/n – (M+n+1)/(n+1) = (M+n)(n+1)/[n(n+1)] – (Mn+n2+n)/[n(n+1)] = M/[n(n+1)] = 141.9, (94.5, 67.7) Do rest on board

35. Mass Spectrometry Other Topics – Multiple Charges in ESI Another way to find charge on ions is to examine the gap in m/z between isotope peaks (0 13C vs. 1 13C) The +1 mass difference will be ½ if charge is +2 or 1/3 if charge is +3 gap = 405.73 – 405.23 = 0.50 Glycodendrimer core Glycodendrimer core

36. Mass SpectrometryOther Topics - MS-MS In LC-ESI-MS, little fragmentation occurs making determination of unknowns difficult In LC-ESI-MS on complicated samples, peak overlap is common, with interferants with the same mass possible (e.g. PBDPs) In both of above samples, using MS-MS is useful This involves multiple passes through mass analyzers (either separate MSs or reinjection in ion-trap MS) and is termed MS-MS Between travels through MS, ions are collided with reagent gas to cause fragmentation

37. Mass SpectrometeryQuestions I Which ionization method can be achieved on solid samples (without changing phase) 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? For a large, polar non-volatile molecule being separated by HPLC, which ionization method should be used?

38. Mass SpectrometeryInterpretation Questions • Determine the identity of the compound giving the following distribution:

39. Mass SpectrometeryInterpretation Questions 2. Determine the identity of the compound giving the following distribution:

40. Mass SpectrometeryInterpretation Questions 3. From the following M, M+n ions, determine the number of Cs, Brs and Cls: