QC 2 M1L3 Lec

Spectroscopy

Study of spectra

Spectroscopy

It is often used in physical and analytical chemistry for identification of substances, through the spectrum emitted or absorbed.

UV-Vis Spectroscopy

A branch of spectrometry which embraces the measurement of the absorption by chemical species of radiant energy of definite and narrow wavelength, approximating “monochromatic radiation”

UV-Vis Spectroscopy

It refers to the analysis of colored solutions that absorb light at specific wavelength

Spectrophotometer or Spectrometer

Are instruments which have a radiant energy dispersing device, such as prism or grating, and the associated electronics that permit the measurement of wavelength and radiant power

Colorimeter & Photometer

Are instruments that permit measurement of radiant power but which use a filter instead of a prism or grating for the purpose of increasing the sensitivity of the measurement

Colorimeter

Device used for measuring colors (colorimetry), it measures the absorbance of different wavelengths of light in a solution.

Colorimeter

It can be used to measure the concentration of a known solute.

Photometer

Device that measures the intensity of light (brightness) produced by an unknown solution in terms of a standard source

Power Supply

A simple transformer that provides current at the proper voltage for several components such as the detector, radiant devices and lamps

Lamps

The “light source” generates a broad band of electromagnetic radiation.

Lamps

TYPES:

• Tungsten lamp (vis)

• Deuterium lamp (uv)

• Xenon lamp (alternative source)

Monochromator

Used to isolate the desired wavelength from the broadband radiation.

Monochromator

It consists of the following:

• Entrance slit

• Dispersion device

• Exit slit

Detector or Microprocessor

• It produces a current in response to the light impinging upon it and converts a light signal into electrical signal

Detector or Microprocessor

• It contains a light sensitive surface that releases electrons in numbers proportional to the intensity of light impinging upon it.

Spectrophotometer or Spectrometer, Colorimeter & Photometer

Instruments that are used in UV-Vis Spectroscopy

Power Supply, Lamps, Monochromator, Detector or Microprocessor

Parts of Spectrophotometer

Photocell

• Also known as “barrier layer cell” or “selenide cell”

Photocell

• The simplest of all the detectors.

Phototube

It consists of a curved cathode of metal coated with a photosensitive material.

Phototube

More sensitive than the photocells.

It combines signal conversion, with several stages of amplification in the body of the tube

Photomultiplier Tube

It is required when low levels of light or quick bursts of light must be measured

Photomultiplier Tube

It is sensitive and produces a fast response.

Photodiodes

Small, durable and capable of high amplification.

Photodiodes

• Newest of the light detectors.

Photocell, Phototube, Photomultiplier Tube, Photodiodes

Detector Types

Optics or Optical System

Relay or focus light through the instrument

Optics or Optical System

• Uses either:

  • Achromatic lenses

  • Concave mirrors

Readout Devices

Measures the magnitude of the current generated by a detector

Readout Devices

• Examples:

  • Galvanometer

  • Ammeter with a meter needle

  • Recorder

  • Digital readout

Cuvette

• Also known as “cells”

Cuvette

It contains the solution to be analyzed

Glass Cuvettes, Quartz or fused silica cuvettes, Plastic cuvettes

Types of Cuvette

Glass cuvettes

• Round bottom

• Stoperred, prism-shaped glass cuvettes

Open-Topped

Rectangular Cell - 10 nm is the most popular pathlength used.

Apertured Cell

• For a limited volume of samples.

Micro Cell

• For extremely limited samples, it can reduce the aperture of the sample to a very small cross-section area.

Flow Through Cell

For automated applications

Double Beam Spectrophotometer

• Operates like a single-beam spectrophotometer but they are designed to compensate for possible variations in intensity of the light-source.

Double Beam Spectrophotometer

• Accomplished by splitting the light beam from the lamp and directing one portion to a reference cuvette and the other to the sample cuvette.

Infrared (IR) Spectroscopy

Measures different types of inter-atomic bond vibrations at different frequencies.

Infrared (IR) Spectroscopy

Useful in organic chemistry analysis of IR absorption spectra, for the identification of the types of chemical bonds present in the sample.

NIR

Interest in the NIR has grown in recent years particularly in the food and feedstuff industry where it is routinely used for quantitative analysis

MIR

Most widely used region with molecular vibration typically involved in organic molecules, it provides a wealth of structural information as well as quantitative data

FIR

Principally concerned with rotational spectral and crystal lattice vibrations

Skeletal Vibrations

8 to 15 um (Vibrations)

Skeletal Vibrations

• A region that gives a spectrum and identification of the molecule as a whole

Characteristic Group Vibrations

3 to 8 um (Vibrations)

Characteristic Group Vibrations

A region that gives a spectrum and identification of the functional groups found in organic compounds

Water is NOT USED as solvent.

• It strongly absorbs most of the IR radiation

• It would destroy the sodium chloride cells which hold the sample

DISPERSING THE FINELY GROUND SOLID IN LIQUID PETROLATUM

Also known as “Mull technique”

Nujol, Fluorolube

Mulling Agents

Nujol

a white hydrocarbon mineral oil

Fluorolube

a perfluorinated hydrocarbon oil

semi-transparent to visible light

The mull should be _____.

KBr, KCl, NaCl, CsI

Matrix Materials for Pellet-Making

KBr Pellet

• Die Assembly

Flame Spectroscopy

• It is used in the assay of metallic elements in the blood (Na+, K+, Li+, Ca++)

Flame Spectroscopy

It is useful in clinical chemistry.

Flame Spectroscopy

• TYPES:

  • Atomic emission spectrophotometry

  • Atomic absorption spectrophotometry

• Also known as “Flame photometry”

Atomic Emission Spectrophotometry

It deals with the measurement of emitted light, whose intensity is proportional to the number of atoms.

Atomic Emission Spectrophotometry

It is used to identify a certain element.

• Aspirator

• Premix burner

• Flame

• Monochromator

• Exit slit

• Detector (a phototube)

• Readout system

Components for Atomic Emission Spectrophotometry

Cesium or lithium

• used as an internal standard to compensate for the variations in sample feed, gas pressure or fuel.

Detergent

reduces the viscosity of the solution and improves aspiration of the sample.

Procedure for Atomic Emission Spectrophotometery

It is diluted with a nonionic detergent (wetting agent) containing a specified concentration of a cesium or lithium salt.

Atomic Absorption Spectrophotometry

It is the measurement of the absorption of light by free metallic atoms

Atomic Absorption Spectrophotometry

• Commonly used for the analysis of certain trace elements in aqueous samples and various liquid samples

Atomic Absorption Spectrophotometry

• Its advantages include good sensitivity and selectivity

• Light Source (hollow cathode lamp)

• Burner

• Monochromator

• Detector

• Readout Device

Components of Atomic Absorption Spectrophotometry

Fluorometry

• Fluorometry or spectrofluorometry

Fluorometry

• A type of electromagnetic spectroscopy that analyzes fluorescence from a sample

Fluorometry

It involves the use of a beam of light that excites the electron/s in a certain compound that causes them to emit light of lower energy.

Lamp, Detector, Readout Device, Monochromators,

Components of Fluorometry

Lamp

• Light source

Lamp

• Types:

  • Mercury arc discharge lamp

  • Xenon arc tube

Detector

• Uses a phototube or photomultiplier tube.

Detector

• It is placed at right angles to the beam of light from the lamp to the sample.

Monochromators

Excitation Monochromator

• Allows the passage of light of the proper wavelength for the absorption by the molecule.

Emission Monochromator

• Transmits light of specific wavelength emitted by the sample

Excitation Monochromator

Allows the passage of light of the proper wavelength for the absorption by the molecule

Emission Monochromator

• Transmits light of specific wavelength emitted by the sample.

Filter Type, Single Wavelength, Spectro Type

Sampe Holders in Fluorometers

Turbidimetry

A branch of spectrometry which deals with the measurement of transmitted light by a turbid solution or suspension

Turbidimetry

Based on an optical detection system that measures “turbidity”

Turbidimetry

• A light beam that passes through a solution is scattered, depending on the degree of turbidity.

Turbidimetry

• A photodetector measures the reduction in the intensity of the light beam.

Turbidimetry

• The transmitted light represents a decreased signal. (The more turbid the solution, the more light will be absorbed, the less light will be transmitted).

Turbidimetry

• Absorbance is in increasing quantity, in relation to the concentration of the sample.

Turbidimetry

Official method of assay for the majority of antibiotics.

Turbidimetry

• Official method of assay for calcium pantothenate and cyanocobalamin (vit B12).

Turbidimetry

Standardization of bacterial concentrations of the inoculum used in microbial assays.

Turbidimetry

• Applied to certain official chemicals to ensure the absence of excessive chlorides and sulfates.

Nephelometry

A branch of spectrometry which deals with the measurement of the brightness of light reflected by a turbid solution.

Nephelometry

Used in the clinical laboratory to quantitate the rate of insoluble antigen-antibody complex formation during the assay of specific serum proteins.

Mass Spectrometry

Analytical technique for the determination of the elemental composition of a sample or molecule.

Mass Spectrometry

• Used for the elucidation of the chemical structures of molecules (peptides or other chemical compounds)

Mass Spectrometry

It relies on the production of ions from a parent compound and the subsequent characterization of the patterns that are produced.