molecule IR tells about the types of the bonds (functional groups) in a molecule. UV tells us about the conjugation (chromophores) What shape does a molecule have? X-ray answers this question better than any other method X-ray gives bond lengths and angles Why bother about spectroscopy when X ray is there? y Co-enzyme of a methanotropic bacteria Structure solved in 1979 1. You need a crystalline solid 2 It d ifi kill C t ll h ! Structure solved in 1979 2. It needs specific skills Crystallographer! 3. long time for collection and interpretation of data. Why do we need to know about mass spectrometry? Mass spectrometry Mass spectrometry How is it different from IR, UV or NMR spectroscopy What we measure is not energy but mass of a molecule or its fragment It is a plot of abundance Vs mass to charge (m/z). What is it used for? Mass spectrometry weighs the molecule. d f It is used for 1. determining masses of particles 2. Identifying a known compound by comparing with i ti t l d t existing spectral data 3. determining isotopic elemental composition of a sample, 4. Identification & structure elucidation of a new compound by observing its molecular ion & fragmentation pattern by observing its molecular ion & fragmentation pattern 5. quantifying the amount of a compound in a sample Mass Spectrometry Mass Spectrometry Molecular weight can be obtained from ll l a very small sample. A beam of high-energy electrons b k h l l breaks the molecule apart. Destructive technique, the sample b d cannot be recovered. The masses of the fragments and their l b d l f relative abundance reveal information about the structure of the molecule. 4 How does it work Mass spectrometer has three essential components 1 Something to volatalize and Magnetic sector mass spec. 1. Something to volatalize and ionize the compound into beam of charged particles Electron impact or chemical Electron impact or chemical ionization an ion source 2. Something that separates th s i ns b m ss/ch r these ions by mass/charge ratio - analyzer 3. Something that could d t t th i detect these ions. - detector Main principle: Lighter ions get deflected by the Main principle: Lighter ions get deflected by the magnetic force more than heavier ions (based on Newtons second law of motion, F = ma). Also ions with more charges gets deflected more Mass Spectrometer Principle : Ions can be deflected by magnetic field.F = ma 1.Ionization M ti fi ld b 2.Acceleration Magnetic field can be varied to allow all the streams to pass through to detector 3.Deflection 4.Detection http://www.chemguide.co.uk/analysis/masspec/howitworks.html#top,Dr.JimClark Stages Sample is ionized (ionization methods, will be discussed) Sample is ionized (ionization methods, will be discussed) The ions are accelerated so that they all have same kinetic energy Th i th d fl t d b ti fi ld di t The ions are then deflected by a magnetic field according to their masses. The lighter they are, the more they are deflected. The amount of deflection also depends on the number of positive h h Th h h d h charges on the ion. The more the ion is charged, the more it gets deflected. Analyzer separates them on the basis of their mass to charge ratio (ion separation methods, will be discussed) The detector detects the beam of ions passing though. The magnetic field can be varied to cover the entire mass range. The mass of each ion being detected is related to the size of the The mass of each ion being detected is related to the size of the magnetic field used to bring it on to the detector. Mass spectrum is recorded as a stick diagram showing the l ti t d d b i s f i m ss/ h ti relative current produced by ions of varying mass/charge ratio. i.e. relative abundance Vs. m/z Radical Cation Formation Radical Cation Formation When a molecule loses one electron, it then has a positive charge and one unpaired has a positive charge and one unpaired electron. This ion is therefore called a radical cation. 9 Electron Impact Ionization Electron Impact Ionization H C H C H H H C H C H H H H H C H C H + H H H H C H H C H H H + Other fragments can be formed when CC or CH bonds are broken during ionization. Only the positive fragments can be detected in MS. 10 The Mass Spectrum In the spectrum, the tallest peak is called the base peak and it is assigned an abundance of 100% The % abundance of all other peaks are 100%. The % abundance of all other peaks are given relative to the base peak. The molecular ion (M + ) corresponds to the mass of the original molecule the original molecule. Example Resolution In order to separate two adjacent peaks of 1 mass unit (Da) difference the valley between d fference the valley between them shouldnt be more than 10% of the highest peak. This degree of resolution is called unit resolution and can go unit resolution and can go upto 3000 Da on standard Instrument. = LRMS. R=3000/30002999=3000 High resolution Mass = 250 g R = 250.1933/ (250.1933-250.1807) = 19857 This is because atomic masses are non integers (except 12 C = 12.00000 by definition) (R can go upto 100000) Only HRMS can be used to determine atomic composition of an ion with accuracy Only HRMS can be used to determine atomic composition of an ion with accuracy Only HRMS can be used to determine Bee alarm pheromone Only HRMS can be used to determine atomic composition accurately MWCalcdObsError(ppm) C 6 H 10 O 2 114.068075114.1039358 6 10 2 C 6 H 14 N 2 114.115693114.1039118 C 7 H 14 O 114.104457114.10395 C 6 H 18 114.140844114.1039369 High Resolution MS High Resolution MS Masses measured to 1 part in million. R 20000-1 lac A molecule with mass of 44 could be C 3 H 8 C 2 H 4 O CO 2 A molecule with mass of 44 could be C 3 H 8 , C 2 H 4 O, CO 2 , or CN 2 H 4 . Using a mass with more significant figures would help identify the correct formula. identify the correct formula. For example, lets say the compound we are looking for has mass of 44.029, pick the correct structure from the table: 15 Gas ChromatographyMass Spectrometry (GCMS) The gas chromatograph column separates the mixture into its components. p The mass spectrometer scans mass spectra of the components as they leave the column. Typical mass spectrometer Typical mass spectrometer Lets talk about the various ionization methods first Ionization methods Gas phase ionization methods (MW < 1000) 1. Electron Impact ionization (EI) . Electron Impact on zat on (EI) 2. Chemical ionization (CI) Desorption ionization methods 1. Field desorption ionization (FD) 2 F t t b b d t (FAB) 2. Fast atom bombardment (FAB) 3. Plasma desorption ionization 4 Laser desorption ionization (MALDI) Evaporative ionization 1 Th m s m ss s t m t 4. Laser desorption ionization (MALDI) 1. Thermospray mass spectrometry 2. Electrospray mass spectrometry (ESI) Electron Impact Ionization (EI method) Widely used method. Vapor phase sample molecules are bombarded with high energy (70 eV) electrons, which eject an electron from sample to create a radical cation. Since IP of organic molecule = 15 eV, the excess ~50 eV energy g , gy is dissipated in breaking other bonds (3-10 eV). The breaking of bonds is highly extensive and critically, highly reproducible and is characteristic of a particular compound reproducible and is characteristic of a particular compound fragmentation pattern. The fragmentation process is also predictable. Advantages : Major libraries of 390000 compounds searched by t l ith i EI D t t hi i li bl f computer algorithms are in EI. Data matching is reliable proof. Coupled with a GC it becomes a powerful tool for structure elucidation Disadvantage: Sometimes molecular ion peak is not seen. Reduce Voltage. Chemical Ionization (CI method) Soft ionization technique to enable molecular ion peak Sample molecules in vapor phase are not bombarded with high energy Sample molecules in vapor phase are not bombarded with high energy electrons Reagent gas (usually methane, isobutane and ammonia are used) is introduced into the source and ionized introduced into the source and ionized. Sample molecules collide with ionized gas (CH 5 + , C 4 H 9 + etc.) in the relatively high pressure CI source and undergo secondary ionization by proton transfer giving [M+1] + by electrophilic addition producing proton transfer giving [M+1] , by electrophilic addition producing [M+15] + , [M+24] + , [M+43] + , or [M+18] + (with NH 4 ) + ions, or rarely by charge exchange giving M + ion. It sometime shows [M-1]+ by hydride abstraction. Less energy very less fragmentation, abstraction. Less energy very less fragmentation, Advantage: stable molecular ions enable molecular weight determination Disadvantage : Not useful for peak matching and structure elucidation EI Vs CI Desorption ionization methods Sample molecules are emitted directly from a condensed phase into vapor phase as ions U d f l l til i i d Used for large, non-volatile or ionic compounds Very less fragmentation Provides molecular weight or exact mass g Desorption ionization methods 1. Field desorption ionization (FD) sample is applied to metal i ( d ) d i hi h l di emitter (anode) and in a very high voltage gradient 2. Fast atom bombardment (FAB) Uses high energy Xenon or Argon atoms (6-10 Kev) to g gy g ( ) bombard samples in glycerol as a matrix controlled fragmentation- useful for proteins, carbohydrates etc. 1 Plasma desorption ionization 252 Cf used with TOF 1. Plasma desorption ionization 252 Cf used with TOF 2. Laser desorption ionization (MALDI)- a pulsed laser beam is used with nicotinic acid or sinapinic acid as a matrix Comparison Evaporative ionization Two methods in which the ions/ compounds in solution Two methods in which the ions/ compounds in solution have their solvent molecules stripped by evaporation with simultaneous ionization leaving behind the ions w th s multaneous on zat on leav ng beh nd the ons for analysis. Coupled with LC. 1. Thermospray mass spectrometry - outdated 2. Electrospray mass spectrometry (ESI or API) useful for proteins, peptides, multi charged species Comparison of various ionization methods p Now, lets talk about the various type of analyzers. Analyzers Analyzers Heart of the spectrometer p There are several types with different characteristics 1 M i S M S 1. Magnetic Sector Mass Spectrometers 2. Quadrupole Mass Spectrometers 3 I n T p M ss Sp t m t s 3. Ion Trap Mass Spectrometers 4. Time of Flight Mass Spectrometers 5 Fourier Transform Mass Spectrometers 5. Fourier Transform Mass Spectrometers Tandem Mass Spectrometry Tandem Mass Spectrometry Magnetic sector MS were the first commercially available instruments and remained important Separation is on the basis of momentum, each ion acquires same kinetic energy The magnetic field is scanned to bring the ions sequentially into focus as per m/z Single focusing Combined Electrostatic-Magnetic sector Very high resolution. Allows measurements of exact masses and molecular formulae Double focusing Quadrupole mass analyzers Much smaller and cheaper Consists of 4 cylindrical rods (10-20 Cms) A constant DC voltage modified by a Radio Frequency (RF) is applied g y q y ( ) pp As ions enter at one end only one m/z ion will pass through, at a certain combination of DC and modified RF voltage They are nothing but Tunable mass filters They are nothing but Tunable mass filters Thus entire mass range can be scanned in much < a second high sensitivity as no slits Less mass range 5000 And resolution Ion Trap Analyzer Unlike Quadrupole, it can TRAP ions for relatively long period The ions can be sequentially ejected to the detector to record spectrum mass-selective instability Consists of 3 electrodes a ring and two cap electrodes Ring electrode operates on a sinusoidal RF The endcap electrode operates either at ground potential/ DC / AC The endcap electrode operates either at ground potential/ DC / AC First, all the ions above certain cut-off are trapped then the RF voltage is raised leading to sequential controlled ejection of ions There are various Modes and Possibilities For designing specific experiments Using this Using this analyzer Tandem MS-MS Time of Flight (TOF) Concept Time of Flight (TOF) Concept A packet of stationary ions is A packet of stationary ions is accelerated to a defined kinetic energy and the time required to move through a q g fixed distance is measured First TOF design published in 1946 by W.E. Stephens Detector TOF The ions are accelerated through a potential and allowed to drift through a field-free region (a tube = 0.5 to several meters) to a detector. Assuming that all the ions arriving at the start of the drift tube have the same energy, the ions with different mass will have different velocities. The time ions arrive at the detector is measured and related to the m/z ratio. Unlimited mass range useful for very large biomolecules Unlimited mass range useful for very large biomolecules Instrument is not scanning, no tunneling, no trapping of ions No slits, filters, therefore excellent sensitivity Need to produce the ions at accurately known start time & position, so use of pulse ionization technique such as plasma or MALDI is a must Due to variation in energies of ions, the resolution <20000 Needs very complex and fast electronics for detection Fourier Transform (FT) Not very commonly used due to cost In a FTMS, ions are held in a cell with an electric In a FTMS, ions are held in a cell with an electric trapping potential within a strong magnetic field. An RF pulse is applied to generate the FT mass spectrum. Within the cell each ion orbits in a direction Within the cell, each ion orbits in a direction perpendicular to the magnetic field with a frequency proportional to ions m/z. An applied RF pulse brings all l d l f l l cycloidal frequencies into resonance simultaneously to yield an interferrogram, which is a time domain spectrum, which is Fourier transformed into a p frequency domain spectrum, which then yields a MS. Needs powerful magnet V t hi h s l ti n m ss n is Very accurate, high resolution, mass range is proportional to magnetic field strength Comparison of various analyzers Comparison of various analyzers Lowres.,fastelectronics Tandem Mass Spectrometry (MS-MS) Tandem Mass Spectrometry (MS MS) In MS-MS (MS squared), a parent ion from the initial fragmentation is selected and is induced to fragment further to fragmentation is selected and is induced to fragment further to give daughter ions In a complex mixture these daughter ions provide a unequivocal evidence for the presence of the known compound evidence for the presence of the known compound Very powerful screening tool No need for chromatographic purification of complex mixtures E.g. Urine analysis of humans or race horses to detect presence of drugs can be done accurately. In triple-quad,three quadrupoles are used in series (Tandem MS) p q , q p ( ) First quadrupole selects a specific ion of interest, second induces collisions using RF only, and third separates the product ions for recording spectrum of daughter ions product ions for recording spectrum of daughter ions. The three operations can be done simultaneously in an Ion Trap. Interpretation of mass spectra Interpretation of EI spectra Base Molecular ion 70eV Base peak [M+1] In EI spectra the molecular ion peak is usually the peak of highest mass number (except for the isotope peaks) but M+ could be the weakest peak as well. The Molecular Ion Peak The Molecular Ion Peak In EI spectra sometime the fragmentation is In EI spectra, sometime the fragmentation is so much that the molecular ion peak is missing How would you ascertain that it is a molecular y m ion peak? or an impurity? or a fragment? Simplest way is to record the spectrum again p y p g in CI mode. You will get an intense peak at [M+1] with very little and weak fragment k peaks Other way to rule out is - apply Nitrogen R l Rule The Nitrogen Rule - generalization The Nitrogen Rule generalization The molecule of even numbered molecular weight must contain either no nitrogen or an even number of must contain either no nitrogen or an even number of nitrogen atoms; an odd numbered molecular weight requires an odd number of nitrogen atoms. f d h DD f N h h If compound has ODD no. of Nitrogen then M+ has ODD nominal mass. If compound has EVEN no of Nitrogen then M+ has If compound has EVEN no. of Nitrogen then M+ has EVEN nominal mass. (Rare case, usually no N) Holds good for compounds containing C, H, N, O, S, X, P, B, Si, As & alkaline earths This rule results from nitrogen having a valence of three and an even atomic mass (which results in odd three and an even atomic mass (which results in odd molecular mass). Corollary: Fragmentation at a single bond gives an odd-numbered ion fragment from an even numbered l l molecular ion, and an even numbered ion fragment from an odd numbered l l h ll h ld molecular ion. For this corollary to hold, the ion fragment must contain all of the ( f ) f h l l nitrogen (if any) of the molecular ion. Exception: Inorganic compounds does p g p not follow this rule e.g. NO (m/z 30), NO 2 (m/z 46) 2 Some clues I t it f M d d th t bilit f l i Intensity of M+ depends on the stability of mol. ion 1. Aromatic> conjugated alkenes> cyclic > organic sulfides> normal alkanes> mercaptans. normal alkanes mercaptans. 2. ketones> amines > esters> ethers> carboxylic acids ~ aldehydes ~ amides ~ halides. [M-15] peak (loss of CH 3 ) or [M-18] peak (loss of H 2 O) Or [M-31] peak (loss of OCH 3 from methyl ester) is taken as confirmation of molecular ion peak confirmation of molecular ion peak. Peaks at M-1 (loss of hydride), M-2 (loss of hydrogen by thermolysis/fragmentation) or M-3 (from alcohols) are common. However peaks between M-3 to M-14 indicate impurity peaks or a fragment ion Peaks M-19 to M-25 are unlikely peaks or a fragment ion. Peaks M 19 to M 25 are unlikely except for fluorinated compounds F= 19, HF = 20. Unlikely masses 4-14, 21-25, 33, 37, 38 Isotope peaks [M+1,M+2.] Isotope peaks [M 1,M 2.] For example CO N CH N &C H HRMS CO, N 2 , CH 2 N &C 2 H 4 M. Wt. = 28 The sum of formula masses of the most abundant isotope gives HRMS For CO 1 12 000 1 15 9949 1x12.000 + 1x15.9949 = 27.9959 For N 2 14.0031 x 2 = 28.0062 For CH 2 N 1x12.0 + 2x1.00783 + 1x14.0031 = 28.0187 For C H For C 2 H 4 2x12.0+4x1.00783 = 28.0312. Atomic weight is however th f ll th the average of all the isotopes Index of Hydrogen Deficiency/ Sit (D ) f U t ti Sites (Degrees) of Unsaturation It is the no. of pairs of hydrogen atoms that must be It is the no. of pairs of hydrogen atoms that must be removed from the corresponding saturated formula to get the compound of interest. The index is, the sum of the number of rings, the h n s, th sum of th num r of r ngs, th number of double bonds and twice the number of triple bonds. (benzene =4, nitro = 1) Index for C n H m X x N y O z can be calculated from eqn. n m x y z q Index = (n) (m/2) (x/2) + (y/2) + 1 Calculate for C 7 H 7 NO 7 3 5 + 0 5 + 1 = 5 7 3.5 + 0.5 + 1 = 5 The index give some idea about the structure of molecular ion. Even-electron ions give an odd multiple of 0 5 while odd electron fragment ions give integer of 0.5, while odd electron fragment ions give integer values of the index. Determination of Formula Determination of Formula The Rule of 13 A C H d i l t (13 f 1) i Assumes C n H n and amu equivalent (13 for n=1) is present in all molecular fragment ions Step 1: Divide M+ mass by 13 this gives n Step 1: Divide M+ mass by 13, this gives n Step 2: Any remainder represents count of additional Hs example 1: for M+ = 78 mp f 78 13 = 6 n = 6 C 6 H 6 example 2: for M+ = 92 92 13 = 7.077 n = 7 7 x 13 = 91 1 extra H is present F l i C H C7H8 Formula is C 7 H 7+1 = C7H8 example 3: for M+ = 161 161 13 = 12.385 n = 12 12 x 13 = 156 5 extra Hs are present 12 x 13 = 156 5 extra Hs are present Formula is C 12 H 12+5 = C 12 H 17 Wh t b t h t t ? What about heteroatoms? Step 1: First derive formula as above Step 2: Next, modify using C n H m equivalents p , y g n m q Element C n H m equiv. Element C n H m equiv. 1 H12 C 31 P C 2 H 7 16 O CH 4 32 S C 2 H 8 14 N CH 2 16 O 32 S C 4 16 O 14 N C 2 H 6 35 Cl C 2 H 11 19 F CH 7 79 Br C 6 H 7 28 Si C 2 H 4 127 I C 10 H 7 example 4: for M+ = 108 108 13 = 8 308 n = 8 108 13 8.308 n 8 8 x 13 = 104 4 extra Hs are present Formula is C 8 H 8+4 = C 8 H 12 Possible candidates with heteroatoms C 8 H 12 CH 4 + O = C 7 H 8 O C 8 H 12 2(CH 4 ) + O 2 = C 6 H 4 O 2 C 8 H 12 CH 4 CH 2 + O + N = C 6 H 6 ON (even number?) Nitrogen rule? Says M+ cant be even numbered. So, C 8 H 12 2(CH 2 ) + N 2 = C 6 H 8 N 2 is possible M+ MS will only give you molecular weight data. One must use other spectral techniques to get p information about the presence of heteroatoms. CHN analysis, IR frequencies, NMR chemical shifts Problem The exact mass of a compound determined by HRMS is 212.0833. What is the molecular formula of the compound? H 1 00783 C 12 0 O 15 9949 N 14 0031 H 1.00783, C 12.0, O 15.9949, N 14.0031 212 (12%), 167 (4%), 105 (100%), 91 (45%), 77 (30%) 63 (10%) 51 (12%) St t ? (30%), 63 (10%), 51 (12%). Structure?