[1S] UNIT 4 Analytical Techniques & Automation Flashcards
Four Major Disciplines of Analytic Techniques
- Spectrometry
- Luminescence
- Electroanalytic Methods
- Chromatography
Is the distance between 2 successive peaks and it is expressed in terms of nanometer (nm)
Wavelength
<400 nm =
400-700 nm =
>700 nm =
UV
Visible
Infrared
The longer the wavelength, the ___________ the energy
lower
The higher the frequency, the _______ the energy
higher
Planck’s formula
E = hv
BEER’S LAW
Concentration of the unknown substance is ________ ________ to the absorbed light (absorbance or optical density) and ________ ________ to the amount of transmitted light.
directly proportional
inversely proportional
T/F: A solution transmits light corresponding in wavelength to its color, and usually absorbs light of wavelengths complementary to its color
T
Which wavelength would be absorbed strongly by a yellow-colored solution?
a. 450 nm c. 600 nm
b. 585 nm d. 650 nm
A
A blue-colored solution would show highest transmittance at:
a. 475 nm c. 585 nm
b. 525 nm d. 620 nm
A
Parts of the Spectrophotometer
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Parts of the Spectrophotometer
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LIGHT SOURCE: CONTINUUM SOURCE
Tungsten
Infrared & Visible Light
LIGHT SOURCE: CONTINUUM SOURCE
Deuterium & Xenon
UV
LIGHT SOURCE: LINE SOURCE
Mercury-vapor lamps
Visible & UV
Which of the following light source provides wavelengths of light that fall under the infrared region?
a. Mercury
b. Xenon
c. Deuterium
d. Silicon Carbide
D
Which of the following light source provides wavelengths of light that fall under the infrared region?
a. Mercury
b. Xenon
c. Deuterium
d. Silicon Carbide
D
PARTS OF SPECTROPHOTOMETER
Minimizes unwanted stray of light
Prevents entrance of scattered light on the monochromator system
Entrance Slit
ENTRANCE SLIT
- Any wavelength outside the band transmitted by the monochromator
- Causes absorbance error
Stray Light
ENTRANCE SLIT
- Limits the maximum absorbance that a spectrophotometer can achieve
- Most common cause of lost linearity at high analyte concentration
Stray Light
PARTS OF SPECTROPHOTOMETER
It isolates specific or individual wavelength of light
Monochromator
PARTS OF SPECTROPHOTOMETER
Kinds of Monochromator
- Prisms
- Diffraction Gratings
- Filters
- Holographic Gratings
PARTS OF SPECTROPHOTOMETER
Controls the width of the light beam (bandpass)
Exit Slit
PARTS OF SPECTROPHOTOMETER
Furthermore, a bandpass can be described as the ________________.
Exit Slit; total range of wavelengths transmitted
PARTS OF SPECTROPHOTOMETER
Allows only a narrow fraction of the spectrum to reach the sample cuvette
Exit Slit
PARTS OF SPECTROPHOTOMETER
holds the solution whose concentration is to be measured
Cuvette
PARTS OF SPECTROPHOTOMETER
Kinds of Cuvette
- Alumina Silica Glass
- Quartz Plastic
- Borosilicate glass
- Soft glass
PARTS OF SPECTROPHOTOMETER: KINDS OF CUVETTE
Capable of withstanding light with wavelength of 350-2000 nm
Alumina Silica Glass
PARTS OF SPECTROPHOTOMETER: KINDS OF CUVETTE
Visible-UV
Quartz Plastic
PARTS OF SPECTROPHOTOMETER
detects and converts transmitted light into photoelectric energy
Photodetector
PARTS OF SPECTROPHOTOMETER
Kinds of Photodetectors
- Photocell
- Phototube
- Photomultiplier tube
- Photodiode
PARTS OF SPECTROPHOTOMETER: PHOTODETECTORS
Most sensitive; used in fluorometric assay
Photomultiplier Tube
Compare single beam and double beam
Single Beam = 1 cuvet
Double Beam = 2 cuvets (Sample & Reference)
DOUBLE BEAM
2 photodetectors & cuvettes
In Space
DOUBLE BEAM
Only 1 photodetector
In Time
INSTRUMENTATION
Measures light emitted by one atom burned in a flame
Flame Emission Photometry
INSTRUMENTATION PRINCIPLE
Measures the excitation of electrons from lower to higher state
Flame Emission Photometry
INSTRUMENTATION PHOTODETECTOR & LIGHT SOURCE
Photo cell
Flame
Flame Emission Photometry
INSTRUMENTATION PHOTODETECTOR & LIGHT SOURCE
Photo cell
Flame
Flame Emission Photometry
INSTRUMENTATION
Measures light absorbed by one atom in a dissociated by heat
Atomic Absorption Spectrophotometry
INSTRUMENTATION
Measures light absorbed by one atom in a dissociated by heat
Atomic Absorption Spectrophotometry
INSTRUMENTATION
Most sensitive & specific for electrolytes and trace elements
Atomic Absorption Spectrophotometry
INSTRUMENTATION PRINCIPLE
Dissociation of subatomic bonds in electrolytes
Atomic Absorption Spectrophotometry
INSTRUMENTATION PHOTODETECTOR & LIGHT SOURCE
Photomultiplier Tube
Hollow Cathode Lamp
Atomic Absorption Spectrophotometry
When measuring calcium by atomic absorption spectrophotometry, which is required?
a. An organic extraction reagent to deconjugate calcium from protein
b. An internal standard
c. A magnesium chelator
d. Lanthanum oxide to chelate phosphates
D
INSTRUMENTATION PRINCIPLE
The unknown sample is made to react with a known solution in the presence of an indicator
Volumetric (Titremetric)
INSTRUMENTATION: VOLUMETRIC
For determination of fluoride concentration in the bloodstream
Schales and Schales Method
INSTRUMENTATION: VOLUMETRIC
For determination of fluorife concentration in the bloodstream
Schales and Schales Method
INSTRUMENTATION: VOLUMETRIC
Chelates calcium
EDTA Titration Method
INSTRUMENTATION PRINCIPLE
- It determines the amount of scattered light by a particulate matter suspended in a turbid solution
- Measures protein in a sample
Nephelometry
INSTRUMENTATION: NEPHELOMETRY
Light Scattering depends on the following factors?
a. Particle Size
b. Wavelength
INSTRUMENTATION
➢ Measures the amount of Antigen-Antibody Complexes.
➢ Measures the angle at 15-90 degrees.
Nephelometry
Used to descrive a molecule that invoke an immune response
Antigens
Most anti antigens to least (d k alam bt gnyn pero yn nsa notes k bhala kau)
- Proteins
- Polysaccharides
- Nucleic Acids & Lipids
- Direct neutralization
- Opsonization
- Complement activation
- Somatization
- Control of inflammatory response
NEPHELOMETRY: Antibody Molecule
INSTRUMENTATION PRINCIPLE
Measures reduction (not specific protein) in light transmission by one particle formation
Turbidimetry