UV and Visible Spectroscopy

Ultraviolet and Visible Spectroscopy

• Ultraviolet and visible spectroscopy involves measuring and interpreting the electromagnetic radiation absorbed or emitted when molecules, atoms, or ions transition between energy states.

Spectrophotometry

• A chemical information source goes to the traducer, the signal conditioner, and then to the display system.

Electromagnetic Radiation

• Electromagnetic radiation is a type of energy transmitted through space at enormous velocities.
• It varies in strength from low to high energy.
• It includes radio waves, microwaves, infrared light, visible light, ultraviolet light, x-rays, and gamma rays
• It has a wave nature and is associated with electric and magnetic fields.
• These fields oscillate in mutually perpendicular directions.

Energy of Electromagnetic Radiation (EMR)

• The energy of EMR can be determined by the equation E = hμ, where E is energy, h is Planck's constant, and μ is frequency of radiation.
• Frequency is defined as c/λ, where c is the speed of light in a vacuum and λ is wavelength.
• Energy is hence defined as: E=hc/λ

Electromagnetic Spectrum

• The electromagnetic spectrum arranges various types of electromagnetic waves or radiations by increasing wavelength or decreasing frequency.
• Radiation is the energy that travels and spreads out, such as visible light from a lamp or radio waves from a radio station.

Regions of the Electromagnetic Spectrum

• The electromagnetic spectrum ranges from gamma rays with short wavelengths (10^-11 m) and high frequencies (10^20 s^-1).
• The spectrum progresses through x-rays, ultraviolet, visible light, infrared, microwaves, to radio frequencies with long wavelengths (10^3 m) and low frequencies (10^4 s^-1).
• The visible region spans wavelengths from 400 to 750 nm.
• The visible spectrum includes ultraviolet light from 190-400 nm.
• Violet's range is 400 - 420 nm and Indigo's range is 420 - 440 nm.
• Blue light ranges from 440 - 490 nm, Green light ranges from 490 - 570 nm
• Yellow ranges from 570 - 585 nm and Orange ranges from 585 - 620 nm.
• Red light ranges from 620 - 780 nm.

Types of Spectroscopy

• Atomic spectroscopy involves energy changes at the atomic level, like atomic absorption spectroscopy and flame photometry.
• Molecular spectroscopy involves energy changes at the molecular level, such as UV spectroscopy, colorimetry, and infrared spectroscopy.

Absorption Spectrophotometry

• Absorption spectrophotometry measures the absorption of radiant energy by various substances.
• It involves measuring the absorptive capacity for radiant energy in the visible, UV, and IR regions of the spectrum.

Colorimetry

• Colorimetry is a scientific technique used to determine the concentration of colored compounds in solution.
• The principle is that the colored substance absorbs light of different wavelengths (λ) in different manners, resulting in an absorption curve, with λ ranging from approximately 400-800 nm.
• Wavelength max refers to the specific wavelength at which maximum absorption occurs, characterizing a colored substance

UV Spectroscopy

• UV spectroscopy studies the absorption of UV radiation within the 200-400 nm range.
• Valence electrons absorb energy, causing molecules to transition from a ground state to an excited state.

Types of Electrons

• σ electrons are associated with saturated compounds.
• π electrons are associated with unsaturated compounds.
• n electrons are non-bonded electrons.

Electronic Transitions

• Electronic transitions occur when an electron is excited from one energy level to another.

Types of Electronic Transitions

• σ to σ* transitions involve exciting a σ electron to its corresponding anti-bonding orbital (σ*); this requires substantial energy; and is observed with saturated compounds. Organic compounds with valence shell electrons involved in σ bond formation does not shoow absorption in normal UV region, 200-400 nm.
• π to π* transitions involve exciting a π electron from a bonding orbital to its corresponding anti-bonding orbital (π*); this is less energy when compared to n - σ*, are found in compounds with multiple bonds like alkenes, alkynes, carbonyls, nitriles, and aromatic compounds. Absorption bands occur in unconjugated alkenes (170-190 nm) and carbonyls (180 nm); and are introduced via alkyl groups in Olefinic linkage that produces a bathochromic shift.
• n to σ* transitions involves transition requiring less energy than σ to σ*. Saturated compounds can undergo this with one heteroatom with an unshared pair of electrons (n) like O, N, S, and Halogens. Saturated alkyl halides see the energy required for transition decrease as the size of the halogen atom increases, and its electronegativity decreases. Methyl chloride has a λmax of 173 nm and methyl Iodide has a λmax of 258 nm. This is very sensitive to hydrogen bonding and found in alcohols and amines. Hydrogen bonding shifts UV absorptions to shorter wavelengths.
• n to π* transitions have an electron from a non-bonding orbital promoted to anti-bonding π* orbital and typically has double bonds involving hetero atoms (C=0, N=0) and require minimum transition energy and show absorption at longer wavelengths around 300 nm. Saturated Aldehydes show this type of transition showing around 290 and 180 nm.

Chromophore

• A chromophore defines the nucleus or any isolated covalently bonded group responsible for light radiation absorption.
• Any group exhibits absorption of electromagnetic radiations in the visible or ultraviolet region.
• Examples include C=C, C=O, and NO2.
• Some important chromophores are carbonyls, acids, esters, nitrile, and ethylenic groups.

Auxochrome

• Auxochromes are color enhancing groups that don't absorb radiation themselves, but enhance a chromophore's absorbing properties when present alongside.
• They have one or more non-bonding pairs of electrons, like -NH2, -OH, -OR, -COOH, and extend the conjugation of a chromophore by sharing the non-bonding electrons.
• For example, Benzene has λ_max at 255 nm, Phenol with λ_max at 270 nm and Aniline with λ_max at 280 nm

Absorption and Intensity Shifts

• Bathochromic shift (Red Shift): shifts to a longer wavelength
• Hypsochromic shift (Blue Shift): shifts to a shorter wavelength
• Hyperchromic effect: increases absorption
• Hypochromic effect: decreases absorption

Bathochromic Shift (Red Shift)

• In spectroscopy, the position of a peak or signal shifts to longer wavelengths (lower energy).
• This occurs when the absorption maxima (λmax) of a compound shifts to longer wavelengths.
• It is caused by the presence of an auxochrome or by a change of solvent.
• For instance, the n-π* transition experiences bathochromic shift in carbonyl compounds when decreasing the polarity of the solvent.

Hypsochromic Shift (Blue Shift)

• The absorption maxima (λmax) of a compound shifts to a shorter wavelength.
• The effect is due to the presence of a group that causes removal of conjugation or a change of solvent.
• In acidic medium the Aniline shifts from 280 nm to 203 nm with Anilinium ion.

Hyperchromic Effect

• The absorption intensity (ε) of a compound increases.
• The introduction of an auxochrome usually increases absorption intensity.
• For example, Pyradine has λmax = 257 nm and ε = 2750 comparing to the 2-methyl pyridene which has has λmax = 260 nm ε = 3560

Hypochromic Effect

• The absorption intensity (ε) of a compound decreases.
• The introduction of a group that distorts the geometry of the molecule produces this shift.
• Naphthalene has ε = 19000, while 2-methyl napthalene has ε = 10250

Absorption Bands

• Absorption Bands are ranges of wavelengths, frequencies, or energies in the electromagnetic spectrum exhibiting characteristic transitions.
• Types include K-Bands, R-Bands, B-Bands, and E-Bands.

K-Band

• These are the types of originate due to π-π* transition and contain a conjugated system.
• They generally arise in compounds containing double bonds like Dienes, Polyenes, and Enones.
• For instance, Acetophenone had λmax(nm) with 240, and εmax with 13000, and 1,3-butadiene has λmax(nm) with 217, and εmax with 21000.

R-Band

• R-bands have transitions originate due to n-π* transition of a single chromophoric group with at least one lone pair of electrons on the hetero atom.
• Max value is generally less than 100.
• In Acetone an ŋ-π* Transition has a λ max of 270 and ε max of 15.
• In Acetaldehyde an ŋ-π* Transition has a λ max of 293 and ε max of 12.

B-Band

• B-bands derive from π-π* transitions in aromatic or hetero-aromatic molecules.
• Benzene compounds will show absorption peaks between 230-270 nm when a chromophoric group is attached to the benzene ring.
• These bands are seen with longer wavelengths as a result.
• For instance, Benzene's π-π* Transition has aλ max of 255 and ε max of 215.
• Phenol's, on the otherhand, is π-π* Transition has aλ max of 270 and ε max of 1450

E-Band

• E-bands come through electronic transitions in benzenoid systems with three ethylenic bonds in closed cyclic conjugation.
• E₁ band appears at shorter wavelengths and is more intense that then E2 band.
• Benzene's E1 band is λmax(nm) 184 and εmax of 50000, with E2 Band being λmax(nm) of 204 and εmax of 79000.
• Napthalene's E1 band is λmax(nm) 221 and εmax of 133000, with E2 Band being λmax(nm) of 286 and εmax of 9300.

Beer-Lambert Law

• Beer's Law states when monochromatic radiation passes through a absorbing species solution, the beams of monochromatic intensity light decrease exponentially with increasing the concentration of the absorbing species.
• Lambert's Law states the rate of decreasing light intensity with thickness of the medium is directly proportional to the initial intensity.

Beer-Lambert Law Formula

• A = εbc
• A is absorbance
• ε is the molar absorptivity, measured in L/(mol cm)
• b is the path length, measured in cm
• c is the concentration, measured in mol/L

Modified Beer-Lambert Law for Pharmaceuticals

• A=A(1%,1cm) bc. Concentrations in pharmaceutical products are usually as grams or milligrams rather than in moles. So the Beer-Lambert equation is rewritten for the analysis of these products, where A (absorbance) is measured; A (1%, 1 cm) is the absorbance of a 1% w/V (1 g/100 ml) solution in a 1 cm cell; b is the path length in cm (typically 1 cm); and c is the sample's concentration in g/100 ml

Factors Governing Absorption in UV/Visible Regions

• Radiation in the 200-700 nm range passing through a compound's solution leads to excitation of electrons, occupying higher quantum states and absorbing energy.
• More loosely held elections result in long wavelength absorbtion given a lower energy.

Application in Pharmaceutial Analysis

• A robust method workhorse method in quantifications where interferences isn't a problem.
• Determination of pKa values of some drugs
• Determination of partition coefficients and solubilities of drugs Used to determine the release of drugs from formulations with time, e.g. in dissolution testing
• Monitor reaction kinetics of the drugs.
• The UV spectrum of a drug is often used as one of a number of pharmacopeial identity

Strengths of UV Spectrometry

• Easy to you, cheap and robust
• Good precision for making quantitative measurements of drugs in formulations Routine method for determining some of the physicochemical properties of drugs which need to be know for the purpose of formulation

Limitations of UV Spectrometry

• Moderately selective
• Not readliy applicable to the analysis of mixtures

The Beer-Lambert Law

• It can be expressed as Log(I₀/It) = A = εbc, where I₀ is intensity of incident radiation, and It is the intensity of transmitted radiation.
• A is the a measurement of light absorbtion that passes through a solution of molecules. A is therefore, a measure of the absorbance.
• 'b' is the path length, whilst 'c' is the analyte in moles per 1 liter.

Instrument Calibration

Pharmacopeial monographs usually rely on standard A (1%, 1 cm) values.

Calibration Measures

• Standard values require proper instrument calibration regarding wavelength and absorption scales.
• Checks for stray light and spectral resolution which are often built into the software of UV instruments.

Practical Aspects of UV/Visible Spectrophotometry

• Avoid touching optical surfaces of sample cells to prevent fingerprints from causing significant absorbance.
• Precision of the pathlength of the cell as tolerances should be around 0.01 mm for pathlength
• Use of a cell is recommended that it is of good quality and that the light source is always facing the same direction.
• Distilled water will do for a solvent though some organic solvents can also be used. Methanol and ethanol are good solvents, though can't be used <210 nm.
• factors like solvation, concentration, pH, and temperature influence the measurement and absorption band intensity and should be controlled. Sample cells should have tops, particularly if an organic solvent is being used
• Absences should be measured between 0.4-1.0. Absorbances must be measured within a correct pathlegth to be accurate.
• There must be no scattering or pollution from the solutions used.

Calibration of Absorbance Scale

• The British Pharmacopoeia utilizes potassium dichromate solution to calibrate the absorbance scale of UV spectrophotometers.
• The A (1%, 1 cm) values at specified wavelengths must lie within the ranges specified by the BP.
• Spectrum includes a concentration of 0.006% w/ and a solution of potassium dichromate in 0.005 M H2SO4 is shown in Figure 1.5. The spectrum range is generally between 220-350 nm.
• Wavelengths include 235 nm (122.9-126.2), 257 nm (142.4-145.7), 313 nm (47.0-50.3), 350 nm (104.9-108.2)

Calibration of Wavelength Scale

• To identify the scale, determine the specified wavelength of maxima with a solution made from 5 w/v% to holmium percholate.
• Also, calibrate the according to the spectra lines. They maybe found under cerium or mercury discharge lamps. Such tests should be built with them. This can vary but on a graph you normally get 241.15 ( +- 1 nm), 287.15 ( +-1 nm), and 361.5 ( +-1 nm( values measured.

UV Spectra of Representative Drug Molecules

• Most medications is based of the chromophores of benzene ring chromophore modified. Exceptions can oftern include steroidal androgens, but more frequently are found in corticosteroids.
• Enone is a standard for measuring steroid absorption.

Characteristics of Some Steroids

• Hydrocortisone has a MW of 362.5, with λ max being 240, and A (1%, 1 cm) value being 435.
• Betamethasone has a MW of 392.5, with λ max being 240, and A (1%, 1 cm) value being 390.
• Clobetasone butyrate has a MW of 479.0, with λ max being 236, and A (1%, 1 cm) value being 330.
• Betamethasone has a MW of 516.4, with λ max being 241, and A (1%, 1 cm) value being 296.

Other Drugs

• Similar to benzene, ephedrine's most intense absorption maximum is below 200 nm. Has one of the simplest modifications, and is around 15 nm. Has a weak symmetric, value of around 12
• There are no polar groups attached to or involved in the chromophore, so its vibrational fine structure is preserved because the chromophore does not interact strongly with the solvent
• Drugs having a chromophore like that of ephedrine include Diphenhydramine, Amphetamine, Ibuprofen and Dextropropoxyphene
• ketoprofen: A simple bendzoid, with a bathochormic shift.
• Pralcaine: The amino group of proalcaine has be extended due its benzyne composition.

Analysis of the Aromatic Benzene

• Phenyplephrine: has a group under alkine conditions. The is has, under acidic condions, two lone pairs. Under acid it is extended.
• It is possible, for phenylene to use pH balance through spectrophommetry analysis. Formulae exists when the data exist.

Applications in Quantitative Analysis

• Pharmacopeial methods heavily rely on simple UV/visible spectrophotometry analysis to determine active ingredients in formulations.
• These methods are usually based on the use of a standard A (1%, 1 cm) which relys on an acurate spectromenter. Excipients like colorants ect are often mixed in formulations but this generally is not an issue.
• However, suspended like can be in the light.
• Standard content from the capsules, must be properly labeled.
• Readings were taken at 299 mm of the sample solutions with and without standard addition against reference solutions 2 and 1, respectively.

Derivative Spectra

• Used to clarify absorption bands in UV spectra.
• Applied in the determination of the purity of chromatographic peaks monitored by diode array detection. Main effect is to remove underlying broad absorption bands.
• Often used in spectrophotometry.
• More complex spectraphotormatary may also be used. One such method is UV visible spectraphotometry. UV/Visible is useful for determing UV/Vis is most usefully determing the types of properties.
• A partition coefficience may also be used to determin between water, organice, and organic layering and uv structophotometry.

Solubility

• Solubilty may be used by determing by the concentration of turbidity by light scattering.
• The UV visible is standard method for pre properties of drug measurements.
• Release of a drug is routinely monorted in vitro or in vivo.