Ultraviolet and visible spectroscopy
Dr. Ahmad Najjar Philadelphia University Faculty of Pharmacy Department of Pharmaceutical Sciences 1st semester, 2020/2021 Spectrophotometry Spectroscopy is a general term referring to the interactions (mainly absorption and emission) of various types of electromagnetic radiation with matter.
Spectrophotometry is a method to measure how much a chemical substance absorbs or emits light by measuring the intensity of light (electromagnetic radiation). It refers to the use of light (electromagnetic radiation) to measure chemical concentrations.
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. Electromagnetic radiation
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 radiations consists of these photons.
All electromagnetic radiations travels in a straight line at the Crest Crest speed of light (3 x 108 m/s). The only difference between the various types of electromagnetic radiations is the amount of energy found in the photons.
Electromagnetic radiations are described by several terms Trough such as wavelength, frequency, wave number and energy.
λ wavelength (units : m,cm, μm, nm) ν 1/λ ν frequency (units of cycles/sec, sec1, Hertz c ν λ ν /ν c ν wavenumber (number of waves per cm; unit : cm1) Energy (E) h ν h h c ν λ 8 1 velocityof light in vacuum c ν λ 3.00 10 m.s h is planck's constant 6.62x10-34 J.s Electromagnetic radiation Crest Crest Electromagnetic wave is also characterized by several fundamental properties, including its velocity, amplitude, phase angle, polarization, and direction of propagation. Trough
Wave amplitude measures the magnitude of oscillation of a particular wave. Larger amplitude means higher energy and lower amplitude means lower energy. Amplitude is important because it tells you the intensity or brightness of a wave in comparison with other waves.
Power, P, and Intensity, I, of light give the flux of energy from a source of EMR. • P is the flux of energy per unit time • I is the flux of energy per unit time per area Electromagnetic radiation spectrum 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. 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)
Problem 1: Calculate the wavenumber of a beam of IR radiation with a wavelength of 3μm. Problem 2: The frequency of a radiation is 3x1012 s-1. Calculate the wavelength of the radiation.
Problem 3: Calculate the energy of 530-nm photon of visible radiation
J 10 x 3.75 = / h = h = E Answer: 흀 풄
19 19 -
m 10 = / = Answer: 풄 흀
4 4 -
= 3,333 cm 3,333 = 1/ = wavenumber Answer: 흀
1 - Spectrophotometric methods
A group of techniques that relies on the quantitative interaction of EMR and matter, these are mainly: . Absorption (excitation process) . Emission (deactivation (or de-excitation) process): Incandescence (luminescence after thermal heating) Chemiluminescence (luminescence after chemical reaction) Photoluminescence (luminescence after light absorption): Fluorescence: Resonance fluorescence Non-resonance fluorescence Phosphorescence
Note: deactivation of absorbed energy could also be done through non-radiative (radiationless) process, such as relaxation, internal conversion (quenching) and intersystem crossing. Spectrophotometric methods: Absorption
When a photon is absorbed by an analyte, it is "destroyed," and its energy is acquired by the analyte. This energy promoting the analyte electron from a lower-energy state (ground state) to a higher-energy, (or excited) state.
Na atomic HCOH molecular energy levels energy levels Spectrophotometric methods: Absorption
. The energy levels have well-defined values (i.e., they are quantized). . Absorption only occurs when the photon's energy matches the difference in energy, E, between two energy levels. . A plot of absorbance as a function of the photon's energy (expressed as wavelength, ) is called an absorbance spectrum.
Absorption spectrum of Hg gas
Absorption spectrum of chlorophyll a
Why atomic spectrum has sharper peaks!!! Molecular orbital
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. Molecular orbital Spectrophotometric methods: Absorption
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
Etot = Erot + Evib + Eelec
In atoms (no sub-levels present), the energy captured during the photon absorption can be expressed as
Etot = Eelec
The reason why electronic absorption bands are usually very broad is that many different vibrational and rotational levels are available at slightly different energies. Therefore, a molecule could absorb photons with a fairly wide range of energies and still be promoted from the ground electronic state to one particular excited electronic state. Spectrophotometric methods: Absorption Absorbing a photon of visible light causes a valence electron in the analyte to move to a higher-energy level.
When an analyte absorbs infrared radiation one of its chemical bonds experiences a change in vibrational energy level. Spectrophotometric methods: Emission
Emission of a photon occurs when an analyte in a higher-energy state returns to a lower-energy state.
The higher-energy state can be achieved in several ways including thermal energy, radiant energy from a photon, or by a chemical reaction.
Emission following the absorption of a photon is also called photoluminescence, and that following a chemical reaction is called chemiluminescence.
Spectrophotometric methods: Emission
In many cases molecules in the excited states loss some energy before emission process occurs. This will give emitted radiations with lower energies than those generated the absorption in first place.
The loss of energy could be happened through several radiative or non- radiative deactivation processes such as: vibrational relaxation, internal or external conversions and through intersystem crossing process.
In general Eemission = Eabsorption - Erelaxation (and/or -Einternal conversion and/or -Eintersystem crossing)
Note that, as the E decrease, the will increase. Spectrophotometric methods: Jablonski diagram Spectrophotometric methods: Jablonski diagram
. Absorption Radiative transition from ground state to higher states with the same spin quantum number
(S0 S0+n). This may include changing vibrational levels also. . Relaxation (R)
Radiationless transition between vibrational levels in the same electronic state ( Vn Vn-1). The definition also cover the transition to Vn-2, Vn-3….until V0. . Internal Conversion (IC)
Radiationless transition between states with same spin quantum numbers ( Sn Sn-1). The transition includes changing the vibrational levels. . Intersystem Crossing (ISC)
Radiationless transition between states with different spin quantum numbers ( S1 T1). Changing vibrational levels is expected also. . Fluorescence
Radiation transition between states with the same spin quantum number (S1 S0). The transition may include changing in the vibrational levels. . Phosphorescence
Radiation transition between states with different spin quantum number (T1 S0) including changing vibrational levels. 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 max (lambda max). Some compounds show more
than one max.
. Spectrum profile is affected by several conditions like : sample state, pH, solvent nature, presented metal ions, temperature and concentration. The UV/Vis spectrum . Other examples:
. 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’. 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 . Electronic transitions of organic compounds Electronic transitions of organic compounds
* Appears in saturated hydrocarbons. Hexane (gas state): 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: max= 183nm, ether: max= 190nm, ethylamine: 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: max =293nm. Weak transition.
* for unsaturated systems. Example: ethylene: max=165nm. Strong transition.
dd 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 colors. That is why the solutions of copper salt 2+ [Cu(H2O)6] is blue, while potassium permanganate yields violet solutions. Electronic transitions of organic compounds 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 electrons. 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. . Auxochromes are groups that do not confer color but increase the coloring power of a chromophore especially if they are in direct conjugation with the -system of the chromophore. They are functional groups that have non-bonded valence electrons and show no absorption at
> 220 nm; they absorb in the far UV. (e.g. -OH and -NH2 groups cause a red shift) Chromophore groups Chromophore groups Chromophore groups 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.
λmax = Base value + Σ Substituent Contributions + Σ Other Contributions Fieser–Woodward rules • For enones and dienones we could start with the following basic structures: Fieser–Woodward rules Component Contribution Base- cyclohexenone + 215 nm Substituents at α-position: 0 Substituents at β-position: 1 alkyl group + 12 nm Substituents at γ-position: 0 Substituents at δ-position: 0 Substituents at ε-position: 0 Substituents at ζ-position: 1 alkyl group + 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 λmax 345 nm 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. Solvent effects: solvatochromism Solvent effects: solvatochromism Solvent effects: solvatochromism
The choice of solvents should take into account their cut-off points!! 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. Effect of pH Effect of pH Instrumentation in the UV/Visible
UV/Vis spectrometers main components are : ❶Source, ❷Wavelength selector (Dispersive system or Discriminator or Monochromator), ❸Sample container and ❹Radiation 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. Instrumentation in the UV/Visible
Examples: Single beam simultaneous spectrometer Double beam sequential spectrometer
Single beam sequential spectrometer Instrumentation in the UV/Visible Light sources: . Sources of radiation should be stable and of high intensity. . Sources are of two types: continuum sources, which emit radiation that changes in intensity only slowly as a function of wavelength, and line sources, which emit a limited number of spectral lines, each of which spans a very narrow wavelength range. . Sources can also be classified as continuous sources, which refer to the fact that they emit radiation continuously with time, or pulsed sources, which emit radiation in bursts. . The most common radiation sources used in UV/Visible spectrophotometers are the Tungsten lamps, Deuterium lamps and Xenon lamps. Instrumentation in the UV/Visible Light sources: . For the visible region of the spectrum, an incandescent lamp fitted with a tungsten filament operated at 3500K; it is also called tungsten/halogen lamp or quarts/halogen lamp. Its lifetime is very long.
. For the UV region (160-380nm) a deuterium arc lamp under a slight pressure; The lamp emits continuum radiation when deuterium (or hydrogen) is
stimulated by electrical energy to produce excited molecule of D2 * (or H2* ). The excited state species then dissociates to give two deuterium or hydrogen atoms plus an ultraviolet photon. Typically it has lifetime of about 2000 working hors.
. For the entire region 200 to 1100 nm, a xenon arc lamp can be used. The lamp makes an arc through ionized xenon gas in a very high pressure bulb. The high pressure give the lamp high efficiency. The light is highly intense and close in frequency to that of sunlight. Its lifetime is about 600 working hors. Instrumentation in the UV/Visible
Dispersive systems and monochromators . Dispersion of different wavelengths is accomplished with the separating capability of refraction (prism) or diffraction (diffraction grating). Typical applications are isolation of a narrow band of radiation from a continuum light source for absorption measurements, or analysis of the emission from excited atoms or molecules. . Three different types of wavelength selectors are: diffraction gratings, prisms or colored filters. . In 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. . In Simultaneous instruments: this category of instrument functions according to the spectrograph principle. The light beam is diffracted after travelling through the measuring cell and the whole spectrum is recorded at the same moment. Instrumentation in the UV/Visible 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. 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 106-108 electrons at the anode. Instrumentation in the UV/Visible Block Diagrams: A- Sequential Spectrometer . Single-Beam Instruments
. Double-Beam Instruments Instrumentation in the UV/Visible Block Diagrams: A- Sequential Spectrometer
http://pharmacydocs.blogspot.com/2017/01/ultraviolet-visible-spectroscopy.html Instrumentation in the UV/Visible Block Diagrams: B- Simultaneous Spectrometer (also called multichannel) https://www.wikihow.com/Do-Spectrophotometric-Analysis 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) = 0.051 Example 2+ A solution of Co(H2O) 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) = 0.020 M Quantitative analysis: laws of molecular absorption Lambert–Beer law
Example - The Absorbance of an unknown MnO4 solution is 0.500 at 525 nm. When measures under -4 - identical conditions, a 1.0x10 M MnO4 is found to have an absorbance of 0.200. Determine the concentration of the unknown. A .b.C C unknown unknown unknown Aknown .b.Cknown Cknown 0.500 C unknown C 2.5104 M 0.200 1.0104 unknown
Concentration of analyte could be determined by using value and point-to-point determination as indicated in pervious examples. However, in point-to-point determinations, the chosen standard point should be whiten the linear calibration range of the analyte and near the expected unknown concentration.
Otherwise we should use the calibration curve method. Quantitative analysis: laws of molecular absorption Lambert–Beer law
Example 30.5 mg sample of impure Drug X was dissolved in 250.0 mL water and 5.0 mL of this solution was further diluted to 50.0 ml by water. The absorbance of the final solution was measured at 380nm using 1-cm cuvettes. The absorbance was 0.230. Use the attached calibration curve to calculate the purity of Drug X in the sample.
-The concentration of the final solution is = (0.230-0.0012)/0.0796 = 2.874 ppm - The concentration of the sample solution = (2.874x50)/5 =28.74 ppm (mg/L g/mL) -The mass of Drug X in the sample solution = 28.74 g/mL x 250mL = 7185g (or 7.185 mg) -Purity of Drug X in the sample = (7.185/30.5)x100% = 23.55%
. 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 (depending on the concentration unit used). Quantitative analysis: laws of molecular absorption Additivity of absorbances
Example We need to measure a metal-reagent complex (MR) which absorbs at 522 nm ( = 1.18x104). The solution also contains 1.00x10-4M excess reagent (R) with an of 5.12x102 at 522 nm. If the total absorbance is 0.727 at 522 nm in a 1.00 cm cell, what is the concentration of MR?.
ATotal AMR AR MRbCMR RbCR 4 2 4 0.727 (1.1810 )(1.00)CMR (5.1210 )(1.00)(1.0010 ) 5 CMR 5.7210 M 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.
A1 = 0.533 A2 = 0.590 1 2 X 3.55x103 5.64x102 Determine the concentration of each species. Y 2.96x103 1.45x104 2 3 5.64x10 0.533 2.96x10 CY 4 3 3 0.590 3 1.45x10 CY At λ1 0.533 (3.55x10 )CX (2.96x10 )CY 3.55x10 0.533 2.96x103 C 5 Y CY 3.60x10 M CX 3 3.55x10 0.533 (2.96x103 )(3.60x105 ) 2 4 At λ 0.590 (5.64x10 )C (1.45x10 )C And CX 3 2 X Y 3.55x10 4 by substituting CX CX 1.20x10 M Quantitative analysis: laws of molecular absorption
Lambert–Beer law If we have a small variation of during our measurement, . In general, when the absorbance is to be measured at a there can be a large single wavelength, the absorption maximum, i.e. at λ difference in response if we are
max, is chosen. This is the point of maximum response note at the max. so better sensitivity and lower detection limits. We will also have reduced error in our measurement. Identical variations in wavelength
. 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 Quantitative analysis: laws of molecular absorption Deviations from linearity (law limitations) are divided into three categories: . Fundamental (real) 1. 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. 2. The absorptivity, a, and molar absorptivity, ε, depend on the sample's refractive index (η). 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. Quantitative analysis: laws of molecular absorption Deviations from linearity (law limitations) are divided into three categories: . Chemical Chemical deviations occur due to chemical phenomenon involving the analyte molecules due to association, dissociation and interaction with the solvent to produce a product with different absorption characteristics. . Instrumental 1. Due to polychromatic radiation (also the reason why absorbance measurements are
taken at the wavelength of maximum absorbance λmax) 2. Due to presence of stray radiation. Usually, this radiation is due to reflection and scattering by the surfaces of lenses, mirrors, gratings, filters and windows. 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. 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. 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).