CC lec 4 Flashcards
4 basic discipline
- Spectrometry
- Luminescence
- Electromagnetic/Electroanalytic methods
- Chromatography
is transmitted via electromagnetic waves that are characterized by their
frequency and wavelength
energy
distance between two successive peaks
wavelength
visible spectrum
400-700nm
ultraviolet region
<400nm
infrared region
> 700nm
Planck’s formula:
E = hv
h = constant
(6.626x10-34 erg sec)
the number of vibrations of wave motion per second
frequency
the ____ the wave frequency the ___ the wavelength
lower
longer
represents the wavelength in nm at peak transmittance
Nominal wavelength
is used to check wavelength accuracy (wavelength calibration)
Didymium or holmium oxide filter
verify absorbance accuracy on linearity
Neutral density filters and dichromate solution
The primary analytical utility of spectrophotometryor filter photometry is the isolation of discreet portions of the spectrum for purposes of measurement.
Photoelectric Colorimetry
is the measurement of light intensity in a narrower wavelength
Spectrophotometric measurement
is the measurement of light intensity of multiple wavelength
Photometric measurement
6 basic components of single/double beam spectrophotometer:
stable source of radiant energy,
filter that isolates a specific region of electromagnetic spectrum,
sample holder,
radiation detector,
signal processor
and readout device
- It provides polychromatic light and must generate sufficient radiant energy or power to measure the analyte of interest
- An intense beam of light is directed through themonochromator and the sample
Light/Radiant source
Highest or maximum wavelength of tungsten
1200nm
most common light source in visible and near infrared region
tungsten
routinely used to provide UV radiation
Deuterium
covers both the UV and the visible range
Xenon
emits limited radiation and wavelength
line source
2 types of light/ radiant source:
Continuum source
Line source
emits radiation that changes in intensity; most commonly used
continuum source
It minimizes any stray light and prevents the entrance of scattered light into the monochromator system
entrance slit
refers to any wavelengths outside the band transmitted by the monochromator and not by the light source
Stray light
causes absorbance error most common cause of loss of linearity at high analyte concentration
Stray light
Isolates specific or individual wavelength of light
monochromator
Are wedge-shaped pieces of glass, quartz or sodium chloride
prisms
It can be rotated, allowing only the desired wavelength to pass through an exit slit
prisms
made by placing a semi-transparent silver films on both sides of dielectric such as magnesium fluoride
Filters
Are the most commonly used; better resolution than prisms
* Made by cutting grooves into aluminized surface of flat piece of crown glass
* Wavelength are bent as they pass a sharp corner
Diffraction gratings
Also called absorption cell/analytical cell/sample cell
* It holds the solution whose concentration is to be measured
Cuvet
- controls the width of light beam (bandpass)
Exit slit
- It detects and converts transmitted light into photoelectric energy
- It detects the amount of light that passes through the sample in the cuvet
Photodetector
- It displays output of the detection system
- Measures the magnitude of the current generated by the detector
- Converts electrical energy into readable numbers
- Examples: galvanometer, ammeter, light-emitting diode (LED) display
Read-out/Meter device
It states that the concentration of the unknown substance is directly proportional to the absorbed light (absorbance or optical density) or inversely proportional to the logarithm of transmitted light (% transmittance)
Beer’s Law
the ratio of the radiant energy transmitted divided by radiant energy incident on the sample
Percent’s transmittance
➢ is the amount of light absorbed
➢ proportional to the inverse log of transmittance
Absorbance
Didymium glass __nm __ wavelength
✓ Holmium oxide __nm __ wavelength
(600nm) higher
(360 nm) lower
Means that the blank contains serum but without the reagent to complete the assay
Blanking technique
measures absorbance of the sample and reagent in the absence of the end product, and corrects the measurement for optical interference (like hemoglobin) absorbing the wavelength of measurements
Sample blank
corrects absorbance caused by the color of reagents
Reagent blank
measures the light transmitted by a solution to determine the concentration of the light-absorbing substance in the solution.
Spectrophotometry
2 kinds of Spectrophotometry
Single beam spectrophotometer
Double beam spectrophotometer
designed to make one measurement at a time at one specified wavelength
Single beam spectrophotometer
- splits the monochromatic light into two components
- additional beam corrects for variation in light source intensity
Double beam spectrophotometer
2 types of Double-beam spectrophotometer
Double-beam in space
Double-beam in time
uses 2 photodetectors, for the sample beam and reference
beam
Double-beam in space
uses one photodetector and alternately passes the monochromatic light through the sample cuvet and then the reference cuvet using a chopper or rotating sector mirror
Double-beam in time
for light source variation
chopper
lithium =
potassium =
sodium =
calcium =
magnesium =
rubidium
red
violet
yellow
red orange
blue
red
alternative if Lithium will be measured
cesium
