FLAME SPECTROSCOPY

1. AAS
- Also known as “flame photometry”
- It deals with the measurement of emitted light
COMPONENTS OF AES SPECTROPHOTOMETER:

a. Aspirator
b. Premix burner
c. Flame
d. Monochromator
e. Exit slit
f. Detector (a phototube)
g. Readout system

Flame Spectroscopy | Fluorometry | Nephelometry | Turbidimetry | Mass spectroscopy

Principle:

 The aspiration into the flame of a salt solution initiated the process.
 A small percentage of the atoms is transformed into a temporary excited state; the atoms immediately return
to the ground state and in the process release light.
 The wavelength of the emitted light is specific for each excited state of the element and can be quantified
under carefully controlled conditions.
Sample preparation:

 It is diluted with a nonionic detergent (wetting agent) containing a specified concentration of a cesium or
lithium salt.
 Cesium or lithium is used as an internal standard to compensate for the variations in sample feed, gas
pressure or fuel.

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2. AAS

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

Components of AES Spectrophotometer:

1. Source Hollow cathode lamp [that is unique for AAS]; lamp is filled with inert gas at low pressure
(Argon, Neon)
2. Burner-it is where the sample is introduced [unique also for AAS]
Parts:
a. Nebulizer- It is where the sample is actually introduced or aspirated and nebulized by a stream of oxidant
flowing across a sample capillary tube
b. Premix Chamber- This is where actual mixing of oxidant and fuel happens; Large droplets are trapped
and drained off
c. Burner Head
(see figure 9.7)
3. Monochromator - blocks the light of wavelength different from the desired resonance lines [emission lines
unique for each element] from reaching the detector
4. Detector

Flame Spectroscopy | Fluorometry | Nephelometry | Turbidimetry | Mass spectroscopy

5. Readout Device

Principle:
 A monochromatic light for a particular element is produced by a hollow-cathode lamp using that element
as the cathode.
 The monochromatic light is beamed through a long flame which is aspirated by the solution to be
analyzed.
 The heat energy dissociates the molecules and converts the components to atoms.
 The ground state atom of an element absorbs light energy which is measured in the detector.

FLUOROMETRY

-Fluorescence Spectroscopy or Fluoroscopy.

-type of electromagnetic spectroscopy that analyzes FLUORESCENCE from a sample.
-involves the use of a beam of light (UV) that excites the electrons in the molecules of certain compounds.
This causes to emit light of lower energy, typically (but not the VIS light).

General Procedures of Fluorometry:

1. Illumination of the sample with monochromatic radiant UV energy.
2. Photo-electrical measurements at right angles to the incident radiation (fluorescent light) produced by the
sample.
3. An official reference standard is also illuminated in the same manner, and the fluorescent light produced is
measured.
4. Transmittance values are directly proportional to the concentration values taken.

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FLUOROMETER INSTRUMENTATION:

1. Light Source – lasers, photodiodes or lamps (Xenon arcs or mercury vapor lamps)
2. Filters / Monochromators - for the selection of specific wavelengths; diffraction gratings are used
3. Detectors – single channeled or multi channeled
4. Sample holder
5. Detector

APPLICATIONS OF FLUOROMETRY:
1. In biochemistry and chemistry fields, for the analysis of organic compounds.
2. In medicine, for differentiating malignant, bashful skin tumors from benign.
4. In pharmacy, analysis of vitamins, particulary in the assay of thiamine and riboflavin (as its official method)

TURBIDIMETRY / NEPHELOMETRY

Flame Spectroscopy | Fluorometry | Nephelometry | Turbidimetry | Mass spectroscopy

A light beam that passes through a solution is scaterred, depending on the degree of turbidity.
A photodetector measures the reduction in the intensity of the light beam.
See the illustration of the instrumentation below:

ADVANTAGES OF NEPHELOMETRY AND TURBIDIMETRY:

1. simple and fast

2. with high sensitivity

APPLICATIONS OF TURBIDIMETRY:
1. Official method of assay for majority of antibiotics. 3
The greater the turbidity, the lesser the effectivity of antibiotics. Because turbidity indicates microbial growth.
2. Official method of assay for Calcium pantothenate and Cyanocobalamine (Vit B12).
3. Standardization of bacterial concentrations of the inoculum used in microbial assays.
4. Applied to certain official chemicals to ensure the absence of excessive chlorides and
sulfates in inorganic ions, organic compounds, compounds of immunological importance and biomass.
5. Water analysis
6. Used to locate potential precipitants in soft drinks and alcoholic beverages
7. Measurement of suspended particles in gasses, smog and fog.

Both instruments must comprise of the following parts:

1. Light source –emits in the VIS region
2. Cell compartment
3. Detector – is placed at a 90-degree angle relative to the path of incident radiation
4. Wavelength selector – usually present in between the source and cell compartment
5. Read-out device

MASS SPECTROMETRY /MASS SPECTROSCOPY

Analytical technique for the determination of the elemental composition of a sample or molecule. Used for the
elucidation of the chemical structures of molecules (peptides or other chemical compounds)
It relies on the production of IONS from a parent compound and the subsequent characterization of the patterns
that are produced.
In order for an MS to function, it must be conducted under VACUUM conditions of -10-4 to -10-5 torr.

PRINCIPLE OF MASS SPECTROMETRY:

a. Ionization of chemical compounds to generate charged molecules or molecule fragments.
b. Measurement of their mass-to-charge (m/z) ratios.

TYPICAL PROCEDURES OF MASS SPECTROMETERS:

1. Sample is loaded onto the MS instrument.
2. The components of the sample are ionized by one of the variety of methods, resulting to the formation of
charged particles / molecules.
3. Directing the ions into a magnetic and/or electrical field.

Flame Spectroscopy | Fluorometry | Nephelometry | Turbidimetry | Mass spectroscopy

4. Computation of mass-to-charge ratios of the particles, based on the details of the motion of ions, as they transit
through the electromagnetic field.
5. Detection of ions.

MASS SPECTROMETER: INSTRUMENTATION

1. ION SOURCE - a small sample of compound is ionized, usually to cations, by loss of an electron.
2. MASS ANALYZER- the ions are sorted and separated according to their mass and charge.
3. DETECTOR - the separated ions are then detected and tallied and the results are displayed on chart.

APPLICATIONS OF MASS SPECTROMETRY:

1. Identification of unknown compounds

2. Determination of the isotopic composition of elements in a molecule.
3. Determination of the structure of a compound by observing its fragmentation.
4. Quantitative assay of a compound in a sample.
5. Study of the fundamentals of the “Gas Phase Ion Chemistry”
6. Other physical, chemical and Biologic Properties
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MASS SPECTROMETER: Principle of Operation

1. Ionization:
The atom /molecule is ionized by knocking one or more electrons off to produce the cation (positive ion)
2. Acceleration:
The ions are accelerated so that they all possess the same kinetic energy.
3. Deflection:
The ions are then deflected by the magnetic field according to their masses. The lighter the ions, the
more ions are charged, the more they are deflected.
4. Detection:
The beam of ions passing through the machine is detected electrically.

Flame Spectroscopy | Fluorometry | Nephelometry | Turbidimetry | Mass spectroscopy

Supplemental videos:

Mass Spectroscopy: https://www.youtube.com/watch?v=J-wao0O0_qM

https://www.youtube.com/watch?v=EzvQzImBuq8
AAS: https://www.youtube.com/watch?v=HBegTB_WDxQ