BCMB 214 Principles of Biochemical Techniques PDF

BCMB 214 Principles of Biochemical Techniques 7/15/2024 1  SPECTROMETRY AND SPECTROSCOPY SPECTROMETRY is the measurement of the interactions between light and matter, and the reactions and measurements o...

BCMB 214 Principles of Biochemical Techniques 7/15/2024 1  SPECTROMETRY AND SPECTROSCOPY SPECTROMETRY is the measurement of the interactions between light and matter, and the reactions and measurements of radiation intensity and wavelength. - Spectrophotometry/Colorimetry - Spectro-fluorimetry - Atomic Absorption Spectrometry (AAS) - Mass Spectrometry (MS) 7/15/2024 2  SPECTROMETRY AND SPECTROSCOPY SPECTROSCOPY is the study of the absorption and emission of light and other radiation by matter. - Infrared-Spectroscopy - Nuclear Magnetic Resonance (NMR) - Electron Spin Resonance 7/15/2024 3  Electromagnetic Radiation and Spectra Electromagnetic Radiation is simply radiation that has both electric and magnetic fields and travels in waves through space. It includes radio waves, microwaves, infrared light, visible light, ultraviolet light, x-rays, and gamma rays Divided into various regions according to their wavelengths (λ) ultraviolet (UV)) region: 200-400 nm visible (VI) region: 400-700 nm infra-red (IR) region: 700 nm-500 µm radio-wave (RW) region: 1-5 m 7/15/2024 4  Electromagnetic Radiation and Spectra In the visible (VI) region, lights of different λs have different colors : – Violet and blue (low λ region) – Orange and red (high λ region) Wavelength (λ) of light: – Distance between adjacent peaks in a wave Defined by the equation: λ = c/v where c = speed of light v = frequency of light (waves/time) 5  Electromagnetic Radiation and Spectra When white light passes through or is reflected by a colored substance, - a characteristic portion of the mixed wavelengths is absorbed. - the remaining light will then assume the complementary color to the wavelength(s) absorbed. -For example, absorption of 420-430 nm light renders a substance yellow, -absorption of 500-520 nm light makes it red. Here, complementary colors are diametrically opposite each other. 6  Electromagnetic Radiation and Spectra When a substance in solution appears blue; – It absorbs reddish-orange light and transmits blue light. When a substance in solution appears red; – It absorbs bluish-green light and transmits red light A substance is said to have an absorption spectrum in the region it absorbs light. For example if a substance appears red, it means it absorbs bluish-green light and therefore the absorbance will be between 480-520nm 7  Colorimetry/Spectrophotometry They are analytical methods. Measure amount of light absorbed by a substance in solution Commonly used for: Quantitative determination of substances in solution. All substances in solution Absorb light of one wavelength (λ) and Transmit light of other λs. Absorbance is characteristic of a substance. May be related to amount of substance in solution 8 7/15/2024  Colorimetry/Spectrophotometry Colored substances only A colorimeter permits selection of wavelength of incident light by use of a colored filter. Color of filter employed depends on absorption maximum/color of substance. – Blue filter: red substance – Red filter: blue substance – Blue/green filter: yellow substance Used in VI region for routine analysis when a spectrophotometer is not required. 7/15/2024 9  Colorimetry/Spectrophotometry A substance not possessing significant color in VI region Can react quantitatively with other reagents to give a colored product. forms basis for assaying such substance Color (chromophore) produced under standard conditions from known quantities of substance. Extinction (absorbance) of the known quantities of substance is measured. Absorbance are plotted against conc. of substance producing color. Called a calibration curve 7/15/2024 10  Colorimetry/Spectrophotometry Unknown quantities of substance in a sample may be assayed by: – Producing color under same standard conditions – Measuring the extinctions/absorbances. Quantity of substance producing the extinction is determined from: – Calibration curve in a process called extrapolation 7/15/2024 11  Colorimetry/Spectrophotometry Points to consider in colorimetry Proportion of sample analyzed is not recoverable. Colorimetric assays most sensitive at the extinction peak of chromophore produced. Assays should be performed in duplicate and individual values NOT mean average plotted Allows for elements of duplicate points which do not fall within the calibration curve Best line through points should be drawn, NOT necessarily best line through origin/other points. 7/15/2024 12  Colorimetry/Spectrophotometry Points to consider in colorimetry New calibration curve should be prepared each time assay is performed because of: – Variation between baseline of reagents and standard. – Changes in environmental conditions – Sensitivity of instrument from day to day due to fluctuations in current Values should never be extrapolated beyond highest absorbance value. – Most accurate region of calibration curve is linear region – If absorbance of sample is beyond linear range it must be diluted or cuvettes of shorter path length used. 7/15/2024 13  UV and VI Spectrophotometry Principles Absorption spectra of a compound: Plot of light absorbed (extinction) against wavelength (λ) Plot may have one or more absorption max. (λmax ). Absorption spectra in UV ranges from (200-400 nm) and VI ranges (400-700 nm) regions. - Reflect energy transitions of: bonding (outer electrons of -C=C- ) non-bonding (lone electron pairs of N & O) 7/15/2024 14  UV and VI Spectrophotometry The λs of light absorbed depend on the actual transitions occurring; Produce specific absorption peaks Chromophore is defined as: Small part of a molecule which gives rise to distinct parts of an absorption spectrum, hence responsible for its color e.g. C=O or C=C groups. 7/15/2024 15  UV and VI Spectrophotometry Conjugation of double bonds: Lowers energy required for electron transitions causes increase in λ of chromophore absorption (BATHOCHROMIC SHIFT) For example, λmax of C=C in ethene is 170nm but λmax of beta- carotene which contains conjugated C=C is around 450 and 475 7/15/2024 16  UV and VI Spectrophotometry Protonation of a ring N decreases conjugation. - Increases energy required for electron transitions causes decrease in λ of chromophore absorption (HYPSOCHROMIC SHIFT) HYPERCHROMIC/HYPOCHROMIC effects refer to: - An increase/decrease in absorbance, respectively. 7/15/2024 17  The Beer-Lambert’s Law Quantitative Aspects of Light Absorption Amount of light passing through a substance is called transmittance (T) or % transmission (% T). Defined by the following equations: T = I/I₀ where I₀ = intensity of incidence light I = intensity of transmitted light % T = I/I₀ x 100 7/15/2024 18  The Beer-Lambert’s Law However, -log T = Absorbance (A). And the absorbance is directly proportional to the length of light path (L) It is described by the Lambert law and expressed as: - log T = - log I/I₀ = A = k₁ L where L = path length of cell; k₁ = a constant Also, - log T is proportional to c as described by Beer’s Law. - log T = - log I/I₀ = A = k₂ c where c = conc. of subst. ; k₂ = a constant 7/15/2024 19  The Beer-Lambert’s Law Combining the 2 laws as Beer-Lamberts’s Law - log T = - log I/I₀ = A = ɛcL where ɛ = extinction coefficient incorporating k₁ & k₂ c= concentration of substance L= path length (cm) ɛ is dependent on: i) wavelength of light ii) chemical nature of substance Abs is used instead of % Transmittance because: The former is linear with conc. and the latter is exponential 7/15/2024 20  Definition of Extinction Coefficient Absorption coefficient is expressed as molar absorption coefficient (ɛ): - Measures how strongly a substance absorbs light at a fixed λ Units of molar extinction coefficient depends on units of c and vice versa. - If units of c is expressed as molL-1, that of ɛ is Lmol-1cm-1 Because ɛ = A/cL = 1/mol L-1 x (cm) = mol-1 L cm-1 7/15/2024 21  UV or VI Spectrum of a substance and the λmax To use Beer-Lambert law to determine the conc. of a substance, - Light of a fixed λ must be chosen A spectrum of the pure substance - Absorbance (Abs) of the substance as a function of λ - Obtained by using a dual beam spectrophotometer automatically changes the λ of the incidence light and records Abs Dual beam permits corrections to be made for solvent/reagent blank or baseline as a function of λ. -Solvent blank/reagent blank is placed in reference beam path contains all of the reagents except the substance of interest -Absorbing sample is placed in sample beam path. 22  UV or VI Spectrum of a substance and the λmax A single beam may be used but is tedious because: - A blank is used to zero equipment at a particular λ before putting in sample. The λ that gives the maximum absorption of light of a substance is known as the λmax - Used to measure changes in Abs with conc. of substance. λmax for the substance whose spectra is shown will be 471nm 23  Determination of Extinction Coefficient and Conc. If ɛ of a substance at its λmax and path length (L) [1.0 cm] are known; - Conc. of substance can be determined from Abs value ɛ can be obtained from: -Literature -Measuring Abs of the substance at different concs. (calibration curve) plot of Abs vs Conc. should give a linear curve whose slope = ɛ when L = 1 cm. 24  Determination of Extinction Coefficient and Conc. If ɛ = 10,000 L mol-1cm-1 Abs (A) = 0.01 (minimum Abs detectable) L = 1.00 cm c = A = 0.01 = 1.0 x 10-6 M ɛL 10,000 (minimum conc. of substance that can be measured) If ɛ = 100,000 L mol-1cm-1 (10 x the above) c = 0.01 = 1.0 x 10-7 M 100,000 (minimum conc. of substance that can be measured) The higher the ɛ -the lower the conc. that can be measured (higher sensitivity). Substances with high ɛ values - give higher Abs values at similar concs. 25  Determination of Extinction Coefficient and Conc. When conc. of a substance is too high in a sample: - Abs values may be too high to fall in linear range curve. Hence experimental parameters must be modified by: - Dilution of sample or - Reduction of path length (use of a spacer – placed in the 1 cm cell containing the substance; glass or quartz of size (9.9-9.98 mm). thus path length becomes (10.0-9.98 mm/10.0-9.90 mm) = 0.02 mm/0.1 mm =0.002 cm/0.01 cm). If conc. of substance is too low or ɛ is too low then: - Absorbance will be too low to measure and hence: a cell of bigger path length can be used (5.0-10.0 cm) to obtain higher and more accurate absorbance. 26  Non-coloured substances (Spectrophotometry) Many organic substances are not colored and hence do not absorb in VI but UV region. - Proteins: λmax at 280 nm due to tyr and trp amino acids - Nucleic acids: Absorb around 255 – 260 nm due to presence of purine and pyrimidine constituents. - Coenzyme NADH: Absorbs at 340 nm due to presence of purines Chemical reactions can generate substance that has absorption maximum in VI/UV regions - So that substance can be quantified. This may involve: - Addition of an indicator, complexing or chelating agent - An oxidative or reduction reaction - A dehydration and condensation reaction 27  Instrumentation The amount of light that is absorbed by a substance may be measured by: Spectrophotometers or Colorimeters These have several parts which include: Light source, Monochromator or colored filter (to give selected λ), Variable slit, Sample holder, Photo-detector and Meter. 28  Instrumentation Light Source Diffraction light sources required for different light regions - VI region: tungsten lamp (400-700 nm) - UV region: deuterium lamp (200-400 nm) Collimator/Lens Narrows the beam of particles or waves. - cause the directions of motion to become more aligned in a specific direction 29  Instrumentation Monochromator/Filters Selects λs of light in VI or UV regions Made up of prisms or diffraction gratings - Gives light of a narrow band of λs Light emerging from monochromator consists of a group of λs known as spectral slit width/band differing in sizes 5-35 nm (simple) F 1,6 BP + ADP (PFK-1) F 1,6 BP ------------> G 3 P + DHAP (Aldolase) G 3 P + DHAP + NAD + + Pi ------------> 1,3 BPG + NADH (G3PD) Rate of NADH production and hence increase in Abs at 340 nm is a measure of PFK-1 activity. 37  Applications Of Spectrophotometry b)Substrate assays All substrate converted to product in enzymic reaction - Total change in parameter (e.g. UV absorption) recorded. change is used to compute amount of substrate originally present Sufficient enzyme is used to ensure reaction goes to completion in a reasonable time 38  Applications Of Spectrophotometry Difference Spectroscopy (DS) Absorption spectra of 2 samples of slightly different composition or physical state are compared. Differences between 2 absorption spectra can be obtained a) indirectly or b) directly Directly: using one compound in reference cuvette whilst measuring absorption spectrum of the other. Indirectly: subtraction of an absorption spectrum from another. Advantage of DS Enables detection of relatively small absorbance changes in a system with a large absorbance e.g. changes in oxidative state of components of the respiratory chain in intact mitochondria/chloroplasts. 39  Applications Of Spectrophotometry Characteristics of DS Both Absmax and Absmin are often displayed. Contains negative Abs values Points at zero extinction in difference spectrum are equivalent to: - Wavelengths where both reduced and oxidized forms of compound have identical extinctions (Abs). Uses of DS Substrate binding difference spectra may be used to study: - Extent of interaction between an enzyme and its substrate e.g. the binding of a drug (substrate) to liver microsomal monooxygenase (MFO) - causes a blue shift of the CYP450 component of enzyme from 420 nm to 390 nm. 40  Applications Of Spectrophotometry Can be used to study conformation of proteins and NAs in solution By examining them under different solvent conditions of: - pH, temperature, conc. of organic solvent etc Protein structural studies Change in solvent polarity Causes changes in Abs spectrum of a constituent amino acid chromophore of a protein without changing its conformation Accessibility of amino acid residue to solvent at surface of protein is called: SOLVENT PERTURBATION Effects of pH, temperature and ionic strength on 2o structure of a protein may be studied 41  BCMB 214 SPECTROSCOPIC AND RADIOISOTOPIC TECHNIQUES 7/15/2024 1  Spectrofluorimetry A conjugated system connected p-orbitals with delocalized electrons in compounds with alternating single and multiple bonds, lower the overall energy of the molecule and increase stability. Organic molecules containing large conjugated p-electron systems are intensely fluorescent e.g. most polycyclic aromatic hydrocarbons 2 3  Spectrofluorimetry Compounds that do not exhibit intense native fluorescence can be made fluorescent by: Derivatization - reaction with chemicals to convert them to fluorescent derivatives Attachment of a fluorescent tag/label Selectivity It is more selective than UV/VI absorption spectrometry for two reasons. Many molecules absorb strongly in the UV or VI range - but do not exhibit detectable fluorescence. Two λs (excitation and emission) are available in fluorometry - but only one λ is available in spectrophotometry. 4  Spectrofluorimetry Two sample constituents with similar excitation spectra but different wavelengths of fluorescence may be distinguished from one another by: appropriate choice of emission wavelength Two compounds that have similar fluorescence spectra but different wavelengths of excitation may be distinguished from each other by: proper choice of excitation wavelength (selective excitation). Limitations of fluorescence selectivity is due to: - Broad, featureless nature of the: absorption/excitation and fluorescence spectra of most molecules. 5  Physical and Chemical Principles o The initial step in a fluorescence measurement is: Excitation of a molecule via absorption of a photon o An excited molecule can decay to rid itself of energy imparted to it by absorption through: Fluorescence (the desired decay route) or Non-radiative decay processes, leading to: - release of energy in the form of heat rather than light o Other sample constituents may interact with an excited molecule to prevent it from fluorescing Such processes are called quenching 6  Physical and Chemical Principles o An excited molecule may undergo a chemical reaction Leading to photodecomposition o Thus fluorescence occurs when: A molecule is promoted to excited singlet state - and decays back to the ground singlet state by emission. 7  Information from Fluorescence Measurements o Two basic types of spectra can be produced by a fluorescence spectrometer. - A fluorescence/emission spectrum keeping the λexc constant and measuring the emission spectra - An excitation spectrum keeping emission λems fixed and measuring the excitation spectra o An excitation spectrum may be similar to its UV/VI absorption spectrum o There is an overlap between absorption and fluorescence spectra of a compound. Both spectra may exhibit wavelength shifts whenever the solvent is changed largest for polar solutes in polar/hydrogen-bonding solvent. 8  Information from Fluorescence Measurements o At low concentrations of fluorophore, the fluorescence intensity of a sample is essentially linearly proportional to concentration o However, as the concentration increases, a point is reached at which the intensity is progressively less linear, and the intensity eventually decreases as concentration increases further. 9  Information from Fluorescence Measurements o Stokes’ law states that the wavelength of fluorescent light is always greater than that of the exciting radiation. o Hence, for any fluorescent molecule, the wavelength of emission is always longer than the wavelength of absorption If λmax in fluorescence spectrum of a compound is > λmax of its absorption spectrum. o The difference between the excitation and emission wavelengths is called the Stokes shift Δλ between λmax Abs/Exc and λmax Fluorescence/Em is the Stokes shift ( often large (20-50 nm),especially for polar solutes in polar solvents) 10 Information from Fluorescence Measurements 11  Information from Fluorescence Measurements o Fluorescence rate of a molecule can be measured Changes in fluorescence spectra as a function of time (time- resolved spectra) is obtained. o Measurements of time-resolved spectra/decay times Aid in analytical applications of fluorimetry Provide unique fundamental information in the study of: - very fast chemical and physical phenomena Analytical Information: The Fluorescence Advantage o Main analytical application of molecular fluorescence spectrometry is: Detection and quantification of species present at very low concs. 12  Information from Fluorescence Measurements Analytical Information: The Fluorescence Advantage o Conditions to be satisfied for the fluorescence advantage are that: Analyte absorbs strongly at λexc Radiation source generates a large number of photons/time at that λexc Excited analyte molecules exhibit a high probability of decaying via fluorescence Detector exhibits high sensitivity at λems of analyte 13  Instrumentation General Parts of a spectrofluorometer o Light source (75 to 450 W high-pressure xenon arc lamp or lasers) o Excitation monochromator o Sample holder (quartz/optical glass/plastic cells) o Emission monochromator o Detector o Reference sample 14  Instrumentation Light Source o Must produce high optical power (a large number of photons per unit time). o An intense continuum source that emits in the UV, VI and near IR regions. Source used in most commercial fluorimeters is xenon arc lamp Wavelength Selectors o Portable/inexpensive fluorimeters use filters as λ selectors. Used to measure fluorescence intensity at single excitation and emission λs. o Most fluorimeters use grating monochromators as excitation and emission λ selectors. 15  Instrumentation Sample Illumination o Right-angle geometry is most common arrangement Fluorescence is viewed at a 90° angle - relative to the direction of exciting light beam Suitable for weakly absorbing solution samples Solution samples held in rectangular 1-cm glass/fused silica cuvettes with four optical windows o Front surface geometry is preferable for: Solutions that absorb strongly at the λexc Solids (samples adsorbed on solid surfaces, such as TLC plates). - fluorescence viewed from face of sample on which exciting radiation impinges 16  Instrumentation Sample Illumination o Flow/windowless cells have been designed for specialized applications. When very low limits of detection are required or To illuminate a very small Detectors o Key requirement for a detector is: Its ability to detect weak optical signals. o A photomultiplier tube (PMT) is used as the detector in most fluorescence spectrometers. 17  Instrumentation Detectors o PMTs operated at such temperatures to: Improve their signal-to-noise ratios Increase sensitivity o Main shortcoming of a PMT is that: It is a single-channel detector. o To obtain a spectrum, the appropriate monochromator is scanned across range of λ spectrum o Multi-channel instrument with an array of detectors Allows the entire spectrum to be viewed at once. The charge-coupled device (CCD) is a promising electronic array detector for fluorimetry 18 Instrumentation 19  Applications of a Spectrofluorometer Biochemistry o Used generally as a non-destructive way of tracking or analysis of biological molecules (e.g. proteins) o Used in the analysis of aromatic amino acids (phenylalanine, tyrosine, tryptophan) o Fingerprints can be visualized with fluorescent compounds such as ninhydrin Environmental Importance o Used to detect environmental pollutants such as polycyclic aromatic hydrocarbons (e.g. pyrene, benzopyrene, carbamate insecticides) 20  Applications of a Spectrofluorometer Analytical chemistry o To detect compounds from an HPLC flow Geology o Many types of calcite (carbonate mineral) and amber will fluoresce under shortwave UV. o Rubies, emeralds and hope diamond exhibit red fluorescence under shortwave UV light. Pharmacy o Direct or indirect analysis of drugs such as vitamins o Immunoassay procedures for detection of specific constituents in biological systems 21  Applications of a Spectrofluorometer o Environmental remote sensing (hydrologic, aquatic, and atmospheric). o In situ analyses in biological systems (such as single cells) and cell sorting (flow cytometry). o Use of fluorescent tags to detect non-fluorescent molecules has numerous applications (DNA sequencing). 22 Applications of a Spectrofluorometer 23  ATOMIC ABSORPTION SPECTROMETRY (AAS) A spectro-analytical procedure for: Qualitative/quantitative determination of elements Employs absorption of light by free atoms in gaseous state Used to determine conc of a particular element (the analyte) in a sample - Over 70 different elements in solution or in solid samples Principles oTechnique uses absorption spectrometry to assess: o Concentration of an analyte in a sample oRequires standards with known analyte content to establish a calibration curve. 24  Principles oTechnique uses absorption spectrometry to assess: Concentration of an analyte in a sample oRequires standards with known analyte content to establish a calibration curve. Relies, therefore, on Beer-Lambert Law. oElectrons of atoms in atomizer can be promoted to higher orbitals (excited state) for a short period by: Absorbing a defined quantum of energy (radiation of a given wavelength). - specific to particular electron transition in a particular element 25  Principles o In general, each wavelength corresponds to only one element. Gives technique its elemental selectivity o Radiation flux with/without sample in atomizer measured using a detector. Ratio between the two absorbance values is converted to analyte concentration/mass. 26  Instrumentation Atomizer/Nebulizer oAtomizers most commonly used are: Spectroscopic flames Electrothermal (graphite tube) oTo analyze a sample for its atomic constituents It has to be atomized. oSample solution is aspirated and transformed into an aerosol. oBurner head on top of a spray chamber produces a flame. 27  Instrumentation Atomizer oAerosol is introduced into spray chamber, mixes with flame gases to produce Finest aerosol droplets (< 10 μm) to enter flame oAtoms are irradiated by optical radiation from: An element-specific line radiation source or A continuum radiation source Continuum/Lamp Source oHigh-pressure xenon short arc lamp operating in a hot- spot mode 28 Instrumentation 29  Instrumentation Continuum Source oLamp emits radiation of intensity far above that of a Hollow Cathode Lamp (HCL) Over λ range 190 nm to 900 nm oRadiation beam passes thru flame at its longest axis Flame gas flow-rates may be adjusted to produce highest concentration of free atoms. Burner height may also be adjusted so radiation beam passes through zone of highest atom cloud density in flame. 30  Instrumentation Flame Processes in a flame include these stages: oDesolvation (drying): - Solvent evaporated whilst dry sample nano-particles remain oVaporization(transfer to the gaseous phase): - Solid particles converted into gaseous molecules oAtomization: - Molecules dissociated into free atoms o Ionization: - Atoms partly converted to gaseous ions depending on: ionization potential of analyte atoms energy available in a particular flame 31  Instrumentation Monochromator oSpectrometers use compact double monochromator with: A prism pre-monochromator and An echelle grating monochromator without an exit slit for high resolution oSeparates element-specific radiation from other radiations emitted by radiation source Detector oA linear charge coupled device (CCD) array with 200 pixels is used as detector. 32 Instrumentation 33  BCMB 214 SPECTROSCOPIC AND RADIOISOTOPIC TECHNIQUES 7/15/2024 1  Mass Spectrometry Principle Mass spectrometry is an analytical tool that separates ionized particles such as atoms, molecules, and clusters by measuring differences in the ratios of their charges to their respective masses (mass/charge; m/z), and can be used to determine the molecular weight of the particles as well as to elucidate the structure of compounds. 2  Mass Spectrometry Theory behind Principle o A moving ion may be deflected by a magnetic field o Degree of deflection is dependent on mass and velocity (momentum) o Deflections of ions of lower momentum (mv) > ones of larger momentum o A mixture of ions of different masses but constant velocity will be deflected in proportion to their mass. 3  Mass Spectrometry Theory behind Principle o Molecules of a compound are ionized either by: Ejection of an electron (cation) or Capture of an electron (anion). o Gives parent molecular ion the energy to cause: Fragmentation thus producing a series of fragmented ions. o Knowledge of mass of the molecular ion and its major fragment ions Enables structure of parent compound to be deduced 4  Mass Spectrometry Theory behind Principle o Majority of ions produced during initial ionization procedure have a single (+) charge One electron is removed from molecule or fragment mass to charge ratio (m/e) = mass (m) Ions produced differ only in their mass o Occasionally molecules lose more than one electron Multi-charged ions are produced. o Degree of fragmentation of molecule depends on energy of bombarding electrons. At low energies (1-2x 10 -18 J) only one electron is removed from molecule. 5  Mass Spectrometry o Method is sensitive and uses: As little as 10-6 to 10-9 g of material. o A mass spectrum is: A plot of the abundance of the fragments and molecular ions against mass. o Fragmentation pattern in a mass spectrum is characteristic of compound Structure of molecules can be deduced from it. 6  Mass Spectrometry A mass spectrum of paracetamol 7 Instrumentation 8  Instrumentation o All mass spectrometers are made of 3 major parts 1. An ionization chamber or source 2. A mass analyzer 3. A detector 9  Instrumentation Ionization Chamber (Source) Ions may be produced either by: 1. Removing an electron from molecule to produce a cation or 2. Adding an electron to form an anion Only one kind of ion may (+/-) be accelerated out of the source region at any time, despite formation of both ion types in ionization process. o Cations (+) will be accelerated in either An increasing negative gradient field (attracted towards a negative electrode) or A decreasing positive gradient field (repelled from a positive electrode). o Exactly the converse is true for anions (-). 10  Instrumentation Ionization Chamber (Source) o Use of electrons in ionization process, produce different amounts of cations and anions because: Removal of an electron to form a cation is more efficient Electron capture to form an anion is less efficient o However, many more cations are produced than anions Explains why positive ion electron impact (EI) mass spectrometry is more common 11  Instrumentation Ionization Chamber (Source) o Removal/addition of protons (H+) also produces ions Mass of resultant ion will differ by ± 1 from the mass of original entity. o Ions are produced by adduct formation with NH4+ or CH5+ in a process known as chemical ionizations Additional masses of new entities are 18 or 17, respectively. 12  Instrumentation Ionization Chamber (Source) There are several forms of ionization: o Electron Impact(EI) Ionization o Chemical Ionization (CI) o Electrospray Ionization (ESI) o Fast-atom bombardment (FAB) o Field ionization o Laser ionization o Matrix-assisted laser desorption ionization (MALDI) o Atmospheric pressure chemical ionization (APCI) 13  Instrumentation Electron Impact o EI is simply done by volatilizing a sample into its gas phase. These gas molecules are then bombarded with a beam of electrons which causes them to eject an ion and form a radical ion. o Heated filament (2000 kw) lose electrons by diffusion from their surface. o If subjected to an appropriate potential gradient, the electrons removed rapidly from surface Gain an energy directly related to potential applied. a 70 V potential produces beams containing 70 eV electrons. 14  Instrumentation Electron Impact o Electrons stream across evacuated chamber into which: Molecules of substance of interests are allowed to diffuse in a vapor state. o 70 eV electrons interact with molecules of substance to be analyzed resulting in either Loss of an electron (to produce a cation) or Electron capture (to produce an anion). o Ionization potential of most organic molecules = +20 eV Excess energy in beam of bombarding electrons = 50 eV o Possible events which may occur are as follows: 15  Instrumentation Electron Impact o Possible events which may occur are as follows: 1. M + e- (bombarding electrons) 2. M·+ e- + e- = M·+ + 2 e- (electron removal) OR 3. M + e- = M·- (electron capture) o Chemical bonds in organic molecules are formed by pairing of electrons. Formation of a cation (+) requires loss of an electron from one of these bonds leaving a bond with single unpaired electron This is a radical as well as a cation hence the representation M·+ 16 7/15/2024 17  Instrumentation Chemical Ionization o Similar to EI but source filled, prior to analysis, with suitable reagent gas such as methane (CH4 ) or NH3. o Normal generation of ions of these gases by EI gives rise to species such as CH4+. or NH3+. CH4 + e- → CH4 +. + 2e- NH3 + e- → NH3 +. + 2e- o Secondary reagents ions may also be formed NH3 + NH3 +. → NH4 + + NH2 18  Instrumentation Chemical Ionization o CH5+ and NH4+ are formed because of removal of protons by original radical ions from neutral molecules. These are powerful proton donors in the vapor state o Addition of material for analysis into the source (electron beam switched off), causes ionization of material by protonation Gives rise to a thermodynamically relatively stable parent plus one pseudomolecule ion. M + CH5 + → CH4 + [M + H]+ o This type of ionization is used in the study of drugs and secondary metabolites. 19  Instrumentation Acceleration out of Source o Ions formed will have to be accelerated out of the source. By establishing an electric potential across the source o Ions emerge through slits with a given terminal velocity Directly related to accelerating force applied. o Terminal velocities differ because not all ions are subjected to same force. Ion A arising at the 0 V plate will express full accelerating force. Ion B half way between plates will experience a force of -4 kV Ion C, about 10% of distance from -8 kV potential plate will only experience about 10% of the force (-0.8 kV). o Ions emerge with varying terminal velocities Have varying momentum and kinetic energies. 20 Instrumentation 21  Instrumentation Electric sector analyzer o To overcome this variation in terminal velocities, the emergent ion beam is energy analyzed. Achieved by electric sector analyzer - consists of 2 stainless steel plates o Ions follow a circular trajectory between these plates, whose radius (Re) is given by: Re = 2 v/E Where v = accelerating voltage E = electrostatic field 22  Instrumentation Electric sector analyzer o Electric sector analyzer is usually referred to as electro-static analyzer (ESA) Packets of ions emerging from ESA have different masses - but same velocity (different kinetic energies) Magnetic sector analyzer o A given packet with appropriate velocity then enters the magnetic sector analyzer (MSA) Undergoes mass analysis o Double focusing mass spectrometer (electrostatic + magnetic fields) can be used to: Distinguish between compounds with same nominal mass - but different accurate mass Determine chemical groups lost during fragmentation 23  Instrumentation Detectors o Most detectors are of the impact or ion collection type. o All detectors require a surface on which the ions impinge, and charge is neutralized either by: Collection of electrons or Donation of electrons. o Electron transfer occurs and an electron current flows May be amplified and ultimately converted into - a signal recorded on a chart or processed by a computer o Total ion current (TIC) is sum of all currents Carried by all ions 24  Instrumentation Electron multiplier o The original ions cause a shower of new electrons to be produced. o These electrons impinge on a second dynode and Produce yet more electrons o Continues until a sufficient large current for normal amplification is obtained. 25  Instrumentation https://youtu.be/RuwbeA22rew 26  Instrumentation Link for video on ionization types https://youtu.be/SQucmCTpdgg 27  Mass Spectrometry A mass spectrum of paracetamol 28  Mass Spectrometry Fragmentation pattern of paracetamol 29  Mass Spectrometry A mass spectrum of aspirin 120 138 43 92 30  Mass Spectrometry o Modern instruments have built-in data systems which allows experimental fragmentation data to be compared with stored reference data. Example is GNPS (Global Natural Products Social) molecular networking platform o Tables compiled for fragmentation patterns of a wide range of compounds Help to elucidate structure of an unknown compound 31  Mass Spectra o A mass spectra usually contains a series of peaks/lines corresponding to m/z value of positive/negative ions produced o Height of peaks = relative abundance of ions o A reference ion of similar m/z ratio to parent ion is used to calibrate the mass axis of spectrum. o Parent peak/Molecular ion peak (M+): analyte molecule that has not undergone fragmentation and is usually the peak with greatest mass o Base peak: peak with most abundant fragment 32  Mass Spectra o Ion intensities in a mass spectrum are usually recorded as: % intensity of base peak 33  Mass Spectrometry o Resolving Power = (m1 /m2 - m1) Is the ability of a mass spectrometer to separate 2 ions of similar mass, m1 and m2 o This is usually a function of detector slit width. o Essential requirement in obtaining a mass spectrum is to: Produce ions in a gaseous phase Accelerate them to a specific velocity using electric fields Mass analyze and separate them based on mass Detect each charged entity of a particular mass sequentially in time. 34  Applications 1.Chemical structure o Identifications of compounds Qualitative analysis of small amounts of relatively complex organic molecules. o All elements have a non-whole number atomic weights Accurate determination of the molecular ion (to 4 decimal places) gives a unique molecular formula. o By studying the accompanying fragment ions and their relative abundance Functional groups and Whole molecular structures, can be deduced; e.g. For steroids, ubiquinones and TAGs 35  Applications 2. Amino acid sequence o Used to determine sequence of oligo-peptides derived from: Protein hydrolysates and other sources o Peptides made more volatile by acetylation and permethylation, Makes peptide bonds most susceptible to cleavage on electron bombardment. Fragmentation occurs from c-terminal end of peptide. 36  Infra-Red Spectroscopy Principle o When infra-red radiation interacts with molecules Specific frequencies are absorbed, and the rest are transmitted The absorbed energy is not sufficient to cause excitation as in the UV but however causes vibrational motion of bonds between atoms o This type of motion can be visualized as a simple diatomic molecule in which the Two atoms are small spheres connected by a spring (bond) 37  Infra-Red Spectroscopy o Frequency (and therefore the energy, E = hν) of the vibration, according to Hooke's Law, is related to: Strength of the bond (stiffness of the spring) and Masses of the atoms attached o In simple molecules the common vibrations can be categorized and some examples are illustrated in the next slide o In more complex molecules additional vibrations may occur simultaneously and interfere with one another Makes the picture very complicated 38 Infra-Red Spectroscopy Bending 39  Infra-Red Spectroscopy Wave Numbers o Infra-red absorptions are generally given in two units: wave-numbers and wavelengths; But are however reported in wavenumbers, cm-1 Wave-numbers, cm-1 are proportional to energy and frequency o A listing of major regions of absorption are shown in the next slide 40  Infra-Red Spectroscopy Wave Numbers o An IR spectrum is usually plotted using transmittance; hence absorption band appears as dips rather than maxima. Each dip is called band or peak. o Absorbance (A)= log 10 (1/T) 41  Infra-Red Spectroscopy Table of some major absorptions in IR Frequency Absorption Appearance Group Compound range (cm-1) class 4000-3000 3700-3584 Medium,sharp O-H stretching alcohol 3400-3300 medium N-H Aliphatic strectching primary amine 3100-3000 strong C-H stretching Alkyne 2400-2000 2349 strong O=C=O Carbon dioxide 2000-1650 2000-1650 weak C-H bending Aromatic compound 1760 strong C=O Carboxylic acid 1465 medium C-H bending alkane 42  Infra-Red Spectroscopy o Similar molecules have aspects of their structures which are similar. Can be recognized by patterns in their IR spectra. o The uniqueness of molecular structure of a given compound Makes an IR spectrum like a fingerprint for the substance o Correlation charts give group and absorbance resulting from various structures. Large absorbance at about 3300 cm-1 is typical of OH stretching seen in alcohols like cyclohexanol. 43  Instrumentation An infra-red device is relatively simple and is based on a design similar to most optical absorption spectrometers Source Common infra-red source is an inert solid (metal/ceramic) Heated electrically to temperatures between 1500 and 2000 K. Monochromator Quartz and prisms of alkali metal/halide crystals and gratings are employed for dispersing IR radiation 44  Instrumentation Sample preparation/Cell o A variety of cells are available for this purpose Path lengths range from a few centimetres to several metres. o Spectrum of a low-boiling liquid/gas can be obtained After permitting sample to expand into an evacuated cell. o Pure liquids may be run, but path length must be very small (≤ 0.01 mm) Often achieved by sandwiching a thin film of the pure liquid between two rock salt plates. o Solids which cannot be dissolved in a suitable solvent Can be suspended in a transparent medium called a mull. 45  Instrumentation Sample preparation/Cell o Solid may also be dissolved in a volatile solvent and drops of solution placed onto a plastic film on a card. Solvent evaporates leaving crystals suitable for infra-red analysis. o Solutions provide reproducible spectra But solvents that do not absorb in region of interest are rare o NaCl windows are frequently employed They become cloudy after a time due to absorption of water; - thus frequently stored in desiccators. o Sample is placed in beam of infra-red radiation which is varied in frequency by turning on grating. 46  Instrumentation Detectors o Various types of thermal detectors are used. o As grating moves, detector determines decreases in intensity of infra-red radiation passing through sample. Sends a signal to a chart recorder - plots absorbance (or % transmittance) vs. wavelength/wave number. o Each "dip" in line along top of chart indicates A wavelength of infra-red radiation absorbed by molecule due to some vibrational motion. 47 Instrumentation 48 Instrumentation 49 IR spectrum of Cyclohexanol 50  IR spectrum of a cyclic peptide 1208.74cm- 1 1643.50cm-1, 1542.82cm-1, 3298.91cm-1, 84.48% T 90.28% T 89.23% T 2958.14cm-1, 2927.08cm-1, 88.38% T 88.03% T 51  Applications o Identification of functional groups and structure elucidation of organic compounds o Studying the progress of a reaction o Detection of impurities in a compound o Study the presence of water in a molecule o Qualitative analysis 52