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CH437 ORGANIC STRUCTURE ANALYSIS CLASS 1: INTRODUCTION AND MASS SPECTROMETRY 1 Synopsis. Introduction to spectroscopic methods used in the determination of organic structures. Principles of mass spectrometry. Electron impact (EI) and chemical ionization (CI). Introduction Structure determination is important throughout chemistry and closely related subjects. When a new compound has been synthesized or discovered, it is crucial to have a reliable check on its identity: Is its structure the same as that of the target molecule? Is it the same structure as a known natural product or previously synthesized substance? What is the structure of this new compound that has been recently isolated from pine needles, or grape skins or marine sponges and which exhibits promising therapeutic properties? These are just some kinds of questions that the application of spectroscopic (and other) techniques can be used to answer. Nowadays, spectroscopic methods are overwhelmingly the most important structure determination techniques, although making derivatives and other chemical and physical methods can be useful in certain circumstances. These used to be the core of structure determination, before the development of spectroscopic methods. The major spectroscopic techniques in organic chemistry are summarized in the table below. Name of method Type of transition Comments or process/energy of radiation (electromagnetic spectrum) Mass spectrometry Ionization (fragmentation) Information obtained from spectrum Includes GC-MS Molar and HPLC-MS. molecular mass, 1 (MS) Nuclear magnetic resonance spectroscopy (NMR) Electron resonance (ESR)* The former needs volatile substances Nuclear spin (in 1D and 2D. presence of Multinuclear, but 1H and 13C most magnetic field)/radiofrequency important region spin Electron spin/microwave region Infrared (IR) spectroscopy and Raman spectroscopy* Ultravioletvisible (UV-VIS) spectrocopy Vibrational (rotational)/ region Electronic/UVvisible region formulas, structure (sometimes) Structural skeleton. Also dynamic (kinetic) and thermodynamic information For radical Structural (unpaired skeleton electron) determination Fourier transform Functional groups IR IR (FTIR) most common Including diode array type for rapid data acquisition Presence of conjugated or aromatic systems. Also quantitative and empirical methods Circular Electronic/UVUses polarized Absolute dichroism (CD) visible region, light, usually in configuration and optical usually UV-visible region rotatory dispersion (ORD) X-Ray Diffraction by atoms Needs good Molecular and crystallography* in crystal lattice crystals crystal structure, hydrogen bonds, absolute configuration *Not considered in this course Mass Spectrometry 1: Principles and Methods of Ionization Mass spectrometry is an outstanding technique in today’s analytical methods: it is a major tool for the determination of organic structures, although it is widely applied throughout science. There are many kinds of mass spectrometry, but all rely on the production of ions, followed by their selection (in the gas phase) by an analyzer and their subsequent detection. The essential features of a mass spectrometer are 2 shown below: the various types of spectrometer vary according to the type of ionization, the type of analyzer used and the type of detector. Vacuum or atmospheric pressure Inlet probe or flow from GC, HPLC or CZE instrument Vacuum (typically 10-5 or 10-6 torr) Mass analyzer where ions are ions "sorted", ions Ion source according (ionizer) to m/z. May be more than one analyzer (tandem MS) Detector signal Computer Data (spectrum) Ionization The major methods of ionization are: Electron ionization (EI) Chemical ionization (CI) Electrospray ionization (ESI) Atmospheric pressure chemical ionization (APCI) Matrix-assisted laser-desorption ionization (MALDI) ESI and APCI are especially popular with HPLC (HPLC-MS). The above ionization methods are reviewed in this course, but other methods include: Fast atom bombardment (FAB), liquid secondary ion mass spectrometry (LSIMS), thermospray ionization (TSI), field-desorption ionization (FDI), plasma 3 desorption ionization (PDI), particle beam ionization (with HPLC), and for inorganic and organometallic compounds, thermal cavity, spark source, glow discharge and inductively coupled plasma source ionization. Ionization can generally produce both positive and negative ions (they are called ions, but may be, in fact, radical ions – odd electron ions), as illustrated for electron ionization. Positive ion MS is more common. electron ejection M.+ + M : + e:M ._ electron absorption 2e- positive ion MS negative ion MS In many cases, especially EI, the production of ions occurs at energies high enough to cause dissociation (fragmentation) of some of the molecular ions: N loss of neutral fragment + m.+ odd electron ion M.+ molecular ion (odd electron) m+ + even electron ion R. loss of radical fragment m.+ and m+ are called fragment or daughter ions Fragmentation pathways depend on structure and hence much structural information can be obtained from their study. Multiply charged ions (all the above examples are of singly charged species) can also be found in mass spectrometry, especially when ESI and MALDI are the ionization methods (see later). Note that although ionization can be carried out at high vacuum (EI, MALDI: 10 -6 torr) or low vacuum (CI: 10-3 torr) or atmospheric pressure (ESI, APCI), ion 4 selection and detection must occur at high vacuum. The main reason for this is to prevent ion-molecule reactions before the ions, produced only by initial ionization and fragmentation, reach the detector. Ion Selection During the course of a mass spectrometric determination, many different ions are normally produced: ion selection is a kind of sorting of the ions according to certain physical characteristics, principally their m/z ratios. Selection occurs in the analyzer part of the instrument: there may be more than one analyzer, as in tandem MS. The major types of analyzers are: Dispersive Analyzers (these actually separate ions according to m/z) Magnetic Sector and Electric Sector (BE) Quadrupole (Q) Ion Trap (QIT) Time-of-Flight (TOF) Non-Dispersive Analyzers (these sort the ions, without separating them) Ion Cyclotron Resonance (ICR) (used in Fourier Transform MS, FTMS) Detectors Detectors convert the collection of ions into an electric current when ions impact the detector. Because the number of ions reaching the detector is normally small, various kinds of amplification systems are used. Detectors fall into two major categories: Point ion Collectors – these detect the arrival of ions sequentially at one point. Array Detectors – these detect arrival of ions simultaneously along a plane. These two categories may be further subdivided into Faraday Cup detectors and Electron (Photon) Multiplier detectors. Each type has its advantages and disadvantages, see “Detectors”. 5 Computer and Data System The computer and data system are very important in modern MS instruments. The computer not only processes the mass spectral output from the detector, but also controls each section of the instrument in both real experiments and in “tuning”. This is summarized below. See also “GC-MS and HPLC-MS” for further accounts of how the computer operates. set mass range (m/z) Digital input: keyboard, mouse Digital-toanalog converter (DAC) Electron multiplier scan 0 - U volts RF generator Ion abundances analysis of masses to voltages Analog scan voltage Analog-toComputer digital converter (ADC) Digitalized calibration scan voltages and ion abundances Ionization Techniques This is fundamental to all MS methods: the substance being studied needs to be ionized and its ions subsequently selected (analyzed) and detected. Because ion selection and detection is carried out at high vacuum (10 -5 – 10-6 torr), the ions must be ultimately in the gas phase, but ionization can occur in either condensed phases or the gas phase. Either the substance must be vaporized prior to its ionization (as in APCI, EI and CI) or ionized prior to its vaporization (as in ESI) or vaporized and ionized at the same time (as in MALDI). Electron Ionization (EI) This is the oldest ionization technique still in use with commercial mass spectrometers. Electrons are emitted from a hot filament and are accelerated through an electric field of potential difference typically 70 V, after which they have energies of around 70 eV. A typical EI source is shown below. 6 Mass spectrum %A m/z An energetic electron interacts with a sample molecule when it passes close by or travels through its electron cloud. The usual result is the ejection of an outer electron from the molecule, producing an odd-electron cation (radical cation), known simply as the molecular ion: M: + e- M.+ + 2e- For a limited number of substances, radical anions are produced by electron absorption, especially if the electron is less energetic (say, 10 eV) and the molecule contains groups that stabilize a negative charge: M + e- M.- This gives rise to the (less common) negative ion MS: most EIMS is concerned with positive ions. Although the energy of the ionizing electrons can be altered, the standard value is 70 eV – high enough to give some of the molecular ions so much excess energy that they dissociate (fragment), often by several different routes (called 7 fragmentation pathways). The fragment ions produced by dissociation of larger ions (or molecular ion) are called daughter ions: most fragmentation occurs in the ion source. A simple scheme is illustrated below. qM.+ (undissociated molecular ion) pM.+ rA+ + sB.+ + c. (radical) p = q + r + s, in abundances d (molecule) This gives rise to the following mass spectrum. E.g. 100% Ion abundance or relative ion abundance (%) E.g. 30% r E.g. 20% s q m/z Advantages of EI: Gives much structural information from fragmentation patterns (EI is called a “hard” form of ionization). Disadvantages: Sometimes fragmentation is so extensive that M .+ is of very low abundance or is undetected: this gives lack of formula mass information. The sample needs to be volatile and thermally stable, as ionization occurs at high vacuum. EI is comparatively inefficient (~0.1% of sample molecules ionized) and so is less sensitive than others (e.g. ESI) 8 Chemical Ionization (CI) This is essentially high energy EI (typically 200 eV), with a controlled amount of reagent gas R (typically CH4, NH3 or isobutane) in the ionization chamber, so that the pressure is typically 10-3 torr, but it can be higher. The typical CI process is shown below. R R.+ + e- + R.+ + 2e- (1) (2) RH RH+ + R. M MH+ + R RH+ + (3) The concentration of reagent R is much higher than that of the sample M, so that electron ionization of reagent molecules is the more likely (1). Reagent molecular ions undergo reaction with neutral reagent molecules to form RH + (2), which then protonates sample molecules and regenerates R (3). An example of (2) and (3) shows CI with CH4, a common reagent gas: . CH4 + + CH4 + CH5 + CH5 + + M CH4 + . CH3 MH+ The last step of CI (protonation of M) is much less energetic than EI and hence CI is called a “soft ionization” technique. The “molecular ion” (here, MH +) receives relatively excess energy and so fragmentation is less pronounced than in EI. Hence CI is especially useful when EI gives a mass spectrum with low or zero abundance molecular ion, as shown below. 9 Electrospray Ionization (ESI) This mode of ionization is most commonly used with HPLC (including capillary column HPLC) and CZE, using mixed aqueous organic solvents (e.g. H2O/MeCN) and (sometimes) added buffer salts. It is an example of atmospheric pressure ionization (API). The HPLC or CZE eluent containing the sample is converted to an aerosol at atmospheric pressure by the action of a high voltage (typically 4 kV for aqueous solutions) applied to the capillary nebulizer tube (see diagram overleaf). The strong electric field (~ 106 V/m) not only produces very small droplets but also induces a high charge accumulation (caused by ionizations) at the droplet surface. See K. Hiroaka, and I. Kudaka, Rapid Comm Mass Spectrom, 4, 519 (1990). 10 The aerosol is made up many tiny charged droplets, which under the influence of a strong electric field and a drying flow of warm gas, shrink in stages, losing both solvent molecules and ions. At some point, a droplet will be too small for the number of ions it contains: it then disintegrates and discharges its ions (called ion desorption), the remaining solvent molecules being pumped to vacuum. The curtain of warm gas helps break up ion clusters. This process is illustrated below for the positive ion mode. By reversing the polarity of the lens, negative ions, rather than positive ions may be collected. 11 Because ESI is a “soft ionization’ technique, ESI mass spectra posses relatively little fragmentation, as illustrated below. This is one of its advantages over EI. Multiple-charged ions are formed if there are many ionizable sites in the molecule, as in peptides and proteins, so that the formula masses of large molecules can be determined by ESI – another big advantage over EI. Most analyzers have limits on the size of m/z that can be measured with acceptable accuracy. For example, if a sample molecule has M = 10,000, it would be difficult to measure its m/z value if the ion was merely M.+ or MH+, but if it has 20 ionizable groups it can form [M + 20H]20+ in ESIMS, with a mass of 10, 020/20 = 501; much easier to measure. Finally, ESI is a more efficient ionization method than EI and hence ESIMS is more sensitive than EIMS. ESI can be made even more sensitive by the use of a corona needle in the electrospray: this boosts the production of ions. 12 Multiply Charged Ions One of the original uses of ESI was in mass spectrometric investigation of proteins, which have many ionizable groups and hence produce multiply charged ions of the type [M + nH]n+ and [M – nH]n-. The ESI mass spectra of such compounds usually correspond to the statistical distribution of consecutive peaks corresponding to the multiply charged ions (e.g. from [M + 12H]12+ to, say, [M + 20H]20+), as shown for lysozyme, below. There are usually very few peaks arising from fragmentation or decomposition. By application of suitable computer algorithms, the molecular mass of the protein can be determined by (computer) conversion of the multiply charged ion data in the spectrum to singly charged ion data. An example of how this can be done manually is discussed next. For any two ions of the type [M + nH]n+, separated by j – 1 peaks, the charge on the ion corresponding to the lower m/z value {(m/z)1} can be obtained from equation 1. 13 z1 = j[(m/z) 2 _ mp] [(m/z)2 _ (1) (m/z)1] mp = mass of H+; 1.0073 Da The molecular mass (M) of the protein (in Da) is then determined by equation 2: M = z1[(m/z)1 + mp] (2) Several calculations of this kind can be carried out on different pairs of peaks and thus an average value for the molecular mass can be obtained. Additionally, high resolution allows direct determination of the charge state of an ion corresponding to a particular peak in the ESI mass spectrum, because resolution of the peak shows several peaks (with 1/z observed distance between them) corresponding to isotope distribution (see class 5). 14