PHA6122 LEC: Ultraviolet-Visible Spectrometry

SPECTROSCOPY

It is the study of the absorption and emission of light and other radiation by matter

SPECTROMETRY

This is the measurement (may be qualitative or quantitative) of the interactions between light and matter, and the reactions and measurements of radiation intensity and wavelength.

SPECTROPHOTOMETRY

• It is the quantitative measurement of the reflection or transmission properties of a material as a function of wavelength. 

ABSORPTION OF RADIATION

Particles are moved from their normal r temperature state, room or ground state, to one or more higher-energy excited states.

Electromagnetic energy

is transferred to the atoms, ions or molecules of the sample when radiation passes through a layer of solid, liquid, or gas.

EMISSION OF RADIATION

Electromagnetic radiation is produced when excited particles (atoms, ions or molecules) relax to lower energy levels by giving up their excess energy as photons.

Bombardment

_ with electrons or elementary particles, which leads to emission of X-radiation.

photon

Outer shell electron moves down to fill the ejected electronʼs space. The energy from this is released as a characteristic energy _.

Exposure

_ to an electric current or intense heat source producing UV, visible or IR radiation.

Irradiation

_ with a beam of electromagnetic radiation.

chemiluminescence

An exothermic chemical reaction that produces _.

SCATTERING OF RADIATION

Electromagnetic radiation is transmitted at all angles from the original path; usually observed when atomic or molecular particles are small relative to the wavelength of the radiation.

DIFFRACTION OF RADIATION

A parallel beam of radiation is bent as it passes by a sharp barrier or through a narrow opening.

Electromagnetic radiation

is the type of energy that is commonly known as light.

SINUSOIDAL WAVE MODEL

Includes wavelength, frequency, velocity, and amplitude.

PARTICLE MODEL

Viewed as a stream of discrete particles, or wave packets of energy called photons.

AMPLITUDE

This is the height of a vector in an electrical plane through the highest point of the wave.

PERIOD

It is the time in second wherein two successive crests or troughs are propagated. (sec)

FREQUENCY

This refers to the number of oscillations per second. 1/sec or 1 cps or 1 Hz)

Oscillation

pertains to one wave completion.

WAVELENGTH

It is the distance between two crests or two troughs.

WAVENUMBER

This is the reciprocal of wavelength.

RADIO, IR, VISIBLE, UV, X-RAYS

Used in pharmaceutical analysis. Gamma rays are only for radioactivity and nuclear medicine.

IONIZING RADIATION

Gamma rays, X-rays, and portions of UV rays (blocked by sunblock)

NON-IONIZING RADIATION

Radio, Micro, IR, Visible Light, and some UV

QUANTUM THEORY

The energy of a photon absorbed or emitted during a transition from one molecular energy level to another is given by the equation.

Ultraviolet-visible UV-Vis spectroscopy

is an electronic transition spectroscopic technique in which the interaction between incident radiation and electrons results in the promotion of one or more of the outer or the bonding electrons from a ground state into a higher-energy state.

quantum effect

results in a specific absorption of radiation, the frequency and wavelength of which are governed by the equation:

200-700 nm

Radiation in the wavelength range _ is passed through a solution of a compound.

higher quantum state

The electrons in the bonds within the molecule become excited so that they occupy a _ and in the process absorb some of the energy passing through the solution. 

longer

The more loosely held the electrons are within the bonds of the molecule, the _ the wavelength (lower the energy) of the radiation absorbed.

Fluorescent materials

have the capacity to have wavelength. When it releases a photon, that would be the glow in the dark

Chromophores

It is an extended system of double bonds that causes absorption of light at UV-visible regions.

Pi bonds

have loosely held electrons since the bonds are not that strong. Those with double bonds and loosely held electrons are the ones that can be analyzed by UV-Vis.

Collimator

technical term for the lens. It leads the light to the monochromator.

slit

is the small opening where the wavelength will get in. Some particular wavelengths will not get in. Light behaves like a wave, and sometimes it will not enter.

Incident light

is the light before passing the sample.

CONTINUUM SOURCE

Continuous or pulsed

EXAMPLES OF CONTINUOUS CONTINUUM

• Tungsten Halogen for Visible Light  

• Deuterium Arc for UV 

• Xenon Arc for Both UV and Visible Light

EXAMPLES OF PULSED CONTINUUM

Xenon flash lamp

MONOCHROMATOR

Is rotated so that a range of wavelengths is passed through the sample as the instrument scans across the spectrum.

MONOCHROMATOR

Used to disperse the light into its constituent wavelengths, which are further selected by the slit.

POLYCHROMATOR

Have multiple exit slits, each of which allows a different wavelength to pass through it.

Photoelectric detectors

_ generate an electric current that is directly proportional to the intensity of the radiant energy incident upon them.

DIODE-ARRAY INSTRUMENTS

The optical configuration is reversed from that in a conventional spectrophotometer, and the light beam passes through the sample before being dispersed by the polychromator. 

DIODE-ARRAY INSTRUMENTS

Gives the benefit of fast, full spectral data with no moving parts that can wear out.

HPLC DETECTORS

High-stability, low signal-to noise ratio output at high transmittance levels through a small-aperture flow cell.

ADVANTAGES of FIBER-OPTIC BASED MODULAR SYSTEMS

• Flexible and easy to use  

• Allow measurements to be performed on microplates  

• Customized systems

DISADVANTAGES of FIBER-OPTIC BASED MODULAR SYSTEMS

• There might be room light interference 

• Custom-built systems do not have additional shuttering, stray light filtering

• Light levels transmitted directly down fibers from high-intensity sources such as Xenon flash lamps may cause photodegradation

• Cant be used in samples that have the tendency to degrade when exposed to light.

SPECTRAL BANDWIDTH

• The width of the band of light at one-half the peak maximum (or full width at half maximum [FWHM]).

•  2 nm is adequate.

STRAY LIGHT

Radiant energy at wavelengths other than those indicated by the monochromator setting and all radiant energy that reaches the detector without having passed through the sample or reference solutions.

CAUSES of STRAY LIGHT

• Any scattered radiation from imperfections in the dispersing medium 

• Light leaks in the system

• Incorrect wavelength calibration

• Incorrect optical alignment

• Reduced source output

• Reduced detector response

OPTIMUM WORKING PHOTOMETRIC RANGE

The center plateau is the photometric range in this spectrum of molar absorptivities at different concentrations.

FLUORESCENCE AND LIGHT SCATTERING

The measured signal usually will contain a contribution from fluorescence.

FLUORESCENCE AND LIGHT SCATTERING

Suspended particles scatter light by the Tyndall effect, causing a decrease in the measured intensity that increases as the wavelength decreases.

CELLS

• Improved sensitivity generally can be obtained by increasing the cell path length.

• Errors are caused by dirty windows that can absorb a significant proportion of the incident light beam.

• Incorrect choice of cell material for the wavelength required (UV - quartz; Vis - glass or plastic) 

• Nonrepeatability of cell positioning

• Differences in cell window thickness

• Nonparallel optical windows

Impurities in cell window materials

BEER-LAMBERT’S LAW

The quantity of light absorbed by a substance dissolved in a fully transmitting solvent is directly proportional to the concentration of the substance and the path length of the light through the solution.

BEER’S LAW

The concentration of a chemical solution is directly proportional to its absorption of light.

Epsilon (ε)

• also known as molar absorptivity

• L/mol • cm (unit)

BEER’S LAW

A=εbc

LAMBERT’S LAW

Each layer of equal thickness of the medium absorbs an equal fraction of the energy traversing it.

LAMBERT’S LAW

A=log I0/It

APPLICATIONS IN PHARMACEUTICAL ANALYSIS

Quantification of drugs where there is no interference from excipients

APPLICATIONS IN PHARMACEUTICAL ANALYSIS

Determination of pKa values

APPLICATIONS IN PHARMACEUTICAL ANALYSIS

Determination of partition coefficients and solubilities 

APPLICATIONS IN PHARMACEUTICAL ANALYSIS

To determine drug release from formulations over time

APPLICATIONS IN PHARMACEUTICAL ANALYSIS

To monitor reaction kinetics of drug degradation

APPLICATIONS IN PHARMACEUTICAL ANALYSIS

Part of pharmacopeial identity checks

STRENGTHS of UV-Vis

Easy-to-use, cheap and robust method with good precision for quantitative measurements of drugs in formulations.

LIMITATIONS

Only moderately selective; Not readily applicable to the analysis of mixtures.