Instrumental Chemical Analysis

Transcription

1 L6 page 1 Instrumental Chemical Analysis Ultraviolet and visible spectroscopy Dr. Ahmad Najjar Philadelphia University Faculty of Pharmacy Department of Pharmaceutical Sciences 2 nd semester, 2016/2017

2 L6 page 2 Spectrophotometry Spectroscopy is a general term referring to the interactions (absorption, emission) of various types of electromagnetic radiation with matter. Electromagnetic spectrum refers to the full range of all frequencies of electromagnetic radiation, which is refers to the waves of the electromagnetic field, propagating through space carrying electromagnetic energy. Spectrophotometry is a method to measure how much a chemical substance absorbs or emits light by measuring the intensity of light (electromagnetic radiation).

3 L6 page 3 Spectrophotometry Electromagnetic radiation (EMR) has been described in terms of a stream of photons that travel in a wave-like pattern. Each photon contains a certain amount of energy, and all electromagnetic radiation consists of these photons. All electromagnetic radiations travels in a straight line at the speed of light (3 x 10 8 m/s). The only difference between the various types of electromagnetic radiations is the amount of energy found in the photons. Crest Trough Crest λ ν ν wavelength (units: m, cm, frequency (units of wavenumber (number of waves per cm; unit : cm velocity of light in vacuum μm, nm) cycles/sec, sec c ν λ 1, Hertz m.s 1 1 ) ν 1/λ c ν λ ν /ν c Energy (E) h ν h h c ν λ h is planck's constant 6.62x10-34 J.s

4 L6 page 4 Spectrophotometry Electromagnetic radiation in the domain ranging between 180 and 780 nm, has been studied extensively. This portion of the electromagnetic spectrum, designated as the UV/Visible. Generally provide little structural information but is very useful for quantitative measurements.

5 L6 page 5 Problem 1: Calculate the wavenumber of a beam of IR radiation with a wavelength of 3μm. Problem 2: The frequency of a radiation is 3x10 12 s -1. Calculate the wavelength of the radiation. Problem 3: Calculate the energy of 530-nm photon of visible radiation Legend: γ = Gamma rays HX = Hard X-rays SX = Soft X-Rays EUV = Extreme-ultraviolet NUV = Near-ultraviolet Visible light (colored bands) NIR = Near-infrared MIR = Mid-infrared FIR = Far-infrared EHF = Extremely high frequency (microwaves) SHF = Super-high frequency (microwaves) UHF = Ultrahigh frequency (radio waves) VHF = Very high frequency (radio) HF = High frequency (radio) MF = Medium frequency (radio) LF = Low frequency (radio) VLF = Very low frequency (radio) VF = Voice frequency ULF = Ultra-low frequency (radio) SLF = Super-low frequency (radio) ELF = Extremely low frequency(radio) Answer: wavenumber = 1/ λ = 3,333 cm -1 Answer: λ = c/ = 10-4 m Answer: E = h = h c/λ = 3.75 x J

6 L6 page 6 Spectrophotometric methods A group of techniques that relies on the interaction of EMR and matter. There are many types of methods based on either molecular or atomic interactions: Absorption (excitation) Emission (luminescence, relaxation or deactivation): Non-radiative Relaxation (vibrational or internal conversion) Radiative photoluminescence (luminescence after absorption) : Fluorescence: Resonance fluorescence Non-resonance fluorescence Phosphorescence

7 L6 page 7 Spectrophotometric methods Molecular orbital energies: Electrons in atoms exist in atomic orbitals (consist of electronic levels only) while electrons in molecules exist in molecular orbitals (consist of electronic, vibrational and rotational levels). Each molecular orbital has energy level represent electronic state S. Between each electronic states there lies several vibrational levels V, themselves also sub-divided into a collection of rotational levels R.

8 L6 page 8 Molecular orbital energies:

9 L6 page 9 Molecular orbital energies:

10 L7 page 1 Spectrophotometric methods

11 L7 page 2 Spectrophotometric methods A molecule absorbs a photon by undergoing an energy transition exactly equal to the energy of the photon. The energy captured during the photon absorption can be expressed as E tot = E rot + E vib + E elec Promotion of an electron from one occupied orbital (HO) to an unoccupied a- radiative process b- internal conversion c- inter system crossing orbital (LU) with the apparition of a singlet state giving rise rapidly to a more stable triplet state. This process corresponds to a return of an excited species to the ground state.

12 L7 page 3 Spectrophotometric methods

13 L7 page 4 The UV/Vis spectrum UV/Vis spectrometers collect the data (transmittance or absorbance) over the required range of wavelengths and generate the spectrum of the compound under analysis as a graph. The spectrum exhibit peaks over the investigated wavelengths range. The wavelength at which the top of the peak occurs is called l max (lambda max). Some compounds show more than one l max. Spectrum profile is affected by several conditions like : sample state, ph, solvent nature, presented metal ions, temperature and concentration.

14 L7 page 5 The UV/Vis spectrum The recorded spectra of compounds in the condensed phase, whether pure or in solution, generally present absorption bands that are both few and broad, while those spectra obtained from samples in the gas state yield spectra of detailed fine structure. Examples:

15 L7 page 6 Electronic transitions of organic compounds Organic compounds represent the majority of the studies made in the UV/Vis. The observed transitions involve electrons engaged in or or non-bonding n electron orbitals of light atoms such as H, C, N, O. The character of each absorption band will be indicated in relation to the molecular orbitals (MO) concerned and the molar absorption coefficient.

16 L7 page 7 Electronic transitions of organic compounds * Appears in saturated hydrocarbons. Hexane (gas state): l max =135nm. All solvents have this transition. It is strong transition and needs high energy. n * mainly if n electron from an atom of O, N, S, Cl present in saturated hydrocarbons system. Examples: methanol: l max = 183nm, ether: l max = 190nm, ethylamine: l max =210nm. Weak transition. n * this transition is usually observed in molecules containing a hetero atom carrying lone electron pairs as part of an unsaturated system. Example: ethanal: l max =293nm. Weak transition. * for unsaturated systems. Example: ethylene: l max =165nm. Strong transition. d d inorganic salts containing electrons engaged in d orbitals are responsible for transitions of weak absorption located in the visible region. These transitions are generally responsible for their colours. That is why the solutions of copper salt [Cu(H 2 O) 6 ] 2+ is blue, while potassium permanganate yields violet solutions.

17 L7 page 8 Electronic transitions of organic compounds

18 L7 page 9 Chromophore groups Chromophore: unsaturated groups or any functional group that absorbs at near UV or Vis region when it is attached to non absorbing saturated residue with no unshared pair of e.

19 L7 page 10 Chromophore groups More chromophores in the same molecule cause bathochromic effect (Red shift: shift to longer wavelength) and hyperchromic effect (increase in intensity). In contrast the shift to shorter wavelengths (Blue shift) is called Hypsochromic effect and the decrease in intensity is called Hypochromic effect. In the conjugated chromophores electrons are delocalized over larger number of atoms causing a decrease in the energy of to * transitions and an increase in due to an increase in probability for transition. They are groups that do not confer color but increase the coloring power of a chromophore, they called Auxochromes. They are functional groups that have nonbonded valence electrons and show no absorption at l> 220 nm; they absorb in the far UV. (e.g. -OH and -NH 2 groups cause a red shift)

20 L7 page 11 Chromophore groups

21 L7 page 12 Chromophore groups

22 L7 page 13 Chromophore groups

23 L8 page 1 Fieser Woodward rules Empirical rules to set up a correlation between structures and positions of the absorption maxima. Many system were studied and rules were established for these systems such as: Heteroannular Diene (Transoid and Cisoid), Polyene, and unsaturated carbonyl (enone). In such systems, the chemical structure was fragmented to basic structure and substituents. λ max = Base value + Σ Substituent Contributions + Σ Other Contributions For enones and dienones we could start with the following basic structures:

24 L8 page 2 Fieser Woodward rules For enones and dienones we could start with the following basic structures:

25 L8 page 3 Fieser Woodward rules Component Base- cyclohexenone Substituents at α-position: 0 Substituents at β-position: 1 alkyl group Substituents at γ-position: 0 Substituents at δ-position: 0 Substituents at ε-position: 0 Substituents at ζ-position: 1 alkyl group Contribution nm + 12 nm + 18 nm Other Effects: 2 Double bonds extending conjugation 2 x 30 = + 60 nm Homoannular Diene system in ring B + 35 nm 1 Exocyclic double bond + 5 nm Calculated λ max 345 nm

26 L8 page 4 Solvent effects: solvatochromism Solvents decrease the sharpness and fine details in the spectrum peaks due to the large interaction between molecules, the strong intermolecular forces cause the electronic peaks to blend, giving only a single smooth absorption band. Polar solvents stabilize both non-bonding electrons in the ground state and * electrons in the excited state. This will lowering the energy state for both n and * electrons, but n state will be affected strongly.

27 L8 page 5 Solvent effects: solvatochromism

28 L8 page 6 Solvent effects: solvatochromism

29 L8 page 7 Solvent effects: solvatochromism The choice of solvents should take into account their cutoff point s!!

30 L8 page 8 Effect of ph ph of the solution could affect the chemical structure of the molecule. Rings may opened or closed, saturation and conjugation could be affected, also charges may appeared and this with affect the polarity and electrons delocalization. Actually this is what happens for acid/base indicator molecules, like phenolphthalein. In basic solution, the central carbon becomes part of a double bond becoming sp2 hybridized instead of sp3 hybridization and leaving a p orbital to overlap with the -bonding in the rings. This makes the three rings conjugate together to form an extended chromophore absorbing longer wavelength visible light to show a fuchsia color.

31 L8 page 9 Effect of ph An animation of the ph dependent reaction mechanism: H 3 In + H 2 In In 2 In(OH) 3 Methyl orange is a different example. What's happened here?? See:

32 L8 page 10 Effect of ph

33 L8 page 11 Effect of ph

34 L8 page 12

35 L9 page 1 Instrumentation in the UV/Visible UV/Vis spectrometers main components are : Source, Wavelength selector (Dispersive system or Discriminator or Monochromator), Sample container and Radiation transducer (Detector) Two optical schemes are well-known in UV/Vis spectrometers design. In the first design on which the majority of instruments are based, the spectrum is obtained in a sequential manner as a function of time (one wavelength after another). In the second, the detector sees all of the wavelengths simultaneously.

36 L9 page 2 Light sources: Instrumentation in the UV/Visible for the visible region of the spectrum, an incandescent lamp fitted with a tungsten filament; for the UV region (<350nm) a deuterium arc lamp under a slight pressure; alternatively, for the entire region 200 to 1100 nm, a xenon arc lamp can be used. Dispersive systems and monochromators Sequential instruments: the light emitted by the source is dispersed through either a planar or concave grating which forms part of a monochromator assembly. This device permits the extraction of a narrow interval of the emission spectrum. The wavelength or more precisely the width of the spectral band, which is a function of the slit width, can be varied gradually by rotating the grating. Simultaneous instruments: this category of instrument functions according to the spectrograph principle. The light beam is diffracted after travelling through the measuring cell.

37 L9 page 3 Instrumentation in the UV/Visible

38 L9 page 4 Instrumentation in the UV/Visible Detectors: The detector converts the intensity of the light reaching it to an electrical signal. Photoelectric effect: light incident on the surface of a metal causes electrons to be ejected. Two types of detector are used, either a photomultiplier tube or a semiconductor (charge transfer devices or silicon photodiodes). Photomultiplier tubes (PMTs) amplifies the number of photoelectrons through the use of a dynode chain. When a dynode struck by a single energetic electron, it will emit several electrons. If 6-8 dynodes are chained together, then a single photoelectron incident on the first can generate electrons at the anode.

39 L9 page 5 Instrumentation in the UV/Visible Optical Materials: Lenses, mirrors, wavelength-selecting elements and sample containers, which are usually called cells or cuvettes, must transmit radiation in the wavelength region being investigated. In UV/Visible spectrophotometers, cells were made of quarts, glass or plastic for visible radiations, while it should be only quartz when using UV radiations.

40 L9 page 6 Instrumentation in the UV/Visible Block Diagrams: A- Sequential Spectrometer Single-Beam Instruments Double-Beam Instruments

41 L9 page 7 Instrumentation in the UV/Visible Block Diagrams: A- Sequential Spectrometer

42 L9 page 8 Instrumentation in the UV/Visible Block Diagrams: B- Simultaneous Spectrometer (also called multichannel)

43 L9 page 9 Quantitative analysis: laws of molecular absorption Lambert Beer law Example Calculate the absorbance of a solution having a %T of 89 at 400 nm. A = log (100/%T) = log(100/89) = Example A solution of Co(H 2 O) 2+ has an absorbance of 0.20 at 530 nm in a 1.00 cm cell. Is known to be 10 L mol -1 cm -1. What is its concentration? A = bc C = A/( b) = 0.20/(1.00x10) = M

44 L9 page 10 Quantitative analysis: laws of molecular absorption Lambert Beer law Example The Absorbance of an unknown MnO - 4 solution is at 525 nm. When measures under identical conditions, a 1.0x10-4 M MnO - 4 is found to have an absorbance of Determine the concentration of the unknown. Aunknown. b. Cunknown Cunknown A. b. C C known known known Cunknown -4 C 4 unknown In general, when the absorbance is to be measured at a single wavelength, the absorption maximum is chosen. This is the point of maximum response so better sensitivity and lower detection limits. We will also have reduced error in our measurement (Why!!) M

45 L9 page 11 Quantitative analysis: laws of molecular absorption Lambert Beer law Conditions for applying Beer-Lambert law The light used must be monochromatic The concentrations must be low The solution must be neither fluorescent or heterogeneous The solute must not undergo to photochemical transformations The solute must not undertake variable associations with the solvent Deviations from linearity are divided into three categories: Fundamental Chemical Instrumental Ideally, according to Beer's law, a calibration curve of absorbance versus the concentration of analyte in a series of standard solutions should be a straight line with an intercept of zero and a slope of ab or εb.

46 L9 page 12 Quantitative analysis: laws of molecular absorption Lambert Beer law At high concentrations the individual particles of analyte no longer behave independently of one another. The resulting interaction between particles of analyte may change the value of a or ε. The absorptivity, a, and molar absorptivity, ε, depend on the sample's refractive index. Since the refractive index varies with the analyte's concentration, the values of a and ε will change. For sufficiently low concentrations of analyte, the refractive index remains essentially constant, and the calibration curve is linear.

47 L10 page 1 Quantitative analysis: laws of molecular absorption Additivity of absorbances Example We need to measure a metal-reagent complex (MR) which absorbs at 522 nm ( = 1.18x10 4 ). The solution also contains 1.00x10-4 M excess reagent (R) with an of 5.12x10 2 at 522 nm. If the total absorbance is at 522 nm in a 1.00 cm cell, what is the concentration of MR?. A A A bc bc Total C MR MR ( R MR 4 ) (1.00) C M MR R MR R ( ) (1.00) ( )

48 L10 page 2 Quantitative analysis: laws of molecular absorption Additivity of absorbances At two different wavelength max Example Two metal complexes (X & Y) demonstrate at least some absorption over the entire visible range. A mixture was measured at two using a 1 cm cell and the following data was obtained. A 1 = A 2 = Determine the concentration of each species. At λ At λ 1 C X (3.55x x x (5.64x10 by substituting C X 3 2 )C 3 C )C X Y X (2.96x10 (1.45x )C )C Y Y 1 2 X 3.55x x10 2 Y 2.96x x x x x10-5 C 3.60x10 M Y (2.96x10 )(3.60x10 And CX x10-4 C 1.20x10 M X 3 C Y -5 ) 1.45x10 4 C Y

49 L10 page 3 Quantitative analysis: laws of molecular absorption Isobestic point An isosbestic point is the wavelength in which the absorbance of two or more species are the same. Assume compound A, which is transformed by a reaction of first order to compound B. The separately recorded spectra of A and B are cross over at a point I when one is superimposed upon the other. For the wavelength of point I, the absorbances of the two solutions are the same and by corollary the coefficients A and B are equal. A will always be of the same value at the isobestic point. isosbestic point is observed when studying coloured indicators as a function of ph, or kinetic studies of particular reactions. The isobestic point is useful to measure the total concentration of two species in equilibrium, i.e. an isomerization reaction.

50 L10 page 4 Quantitative analysis: laws of molecular absorption Spectrophotometric Titrations useful for locating the equivalence points of titrations. This application of absorption measurements requires that one or more of the reactants or products absorb radiation or that an absorbing indicator be added to the analyte solution. A photometric titration curve is a plot of absorbance (corrected for volume change) as a function of titrant volume. Typical photometric titration curves. Molar absorptivities of the substance titrated, the product, and the titrant are A, P, and T, respectively.

51 L10 page 5 Derivative spectrometry The principle of derivative spectrometry consists of calculating, by a mathematical procedure, derivative graphs of the spectra to improve the precision of certain measurements. This procedure is applied when the analyte spectrum does not appear clearly within the spectrum representing the whole mixture in which it is present. This can result when compounds with very similar spectra are mixed together. The traces of the successive derived spectral curves are much more uneven than the one of the original spectrum (called zeroth order spectrum). These derivative plots amplify the weak slope variations of the absorbance curve. The procedure of obtaining the first derivative graph, da/d =(d /d )bc, can be extended to successive derivatives (nth derivatives).

52

53

54

55

56

57