Automation (Clinical Chemistry) Flashcards
References: CC ni Hyacinth Yambao CC ni Rovie Vila
4 analytical techniques
• Spectrometry
• Luminescence
• Electroanalytic methods
• Chromatography
has provided scientists with a means to use both qualitative and quantitative methods of measuring analytes in body fluids. (McPherson & Pincus, 2017)
Absorption spectroscopy
2 types of Spectroscopy
- Photometric measurement
- Spectrophotometric measurement
measurement of light intensity without consideration of wavelength
Photometric measurement
• measurement of light intensity in a narrower wavelength.
• Spectrum of light
Spectrophotometric measurement
It is transmitted by via electromagnetic waves that are characterized by their frequency and waves
Energy
It is characterized by waves and frequency
Electromagnetic waves
is described as photons of energy traveling in waves—Electromagnetic waves.
Electromagnetic Radiation
It states that the relationship between wavelength and energy.
Planck’s law
It is the number of vibrations of wave motion per second.
Frequency
distance between two peaks.
- Lower frequency = Longer wavelength
(Ex. Red)
- Higher frequency = Shorter wavelength (Ex. Violet)
Wavelength
Visible: 400 – 700 nm =
visible spectrum
<400 nm =
ultraviolet region (UV)
> 700 nm =
infrared region (IR)
Light source for spectrophotometry
Light Amplification by Stimulated Emission of Radiation (LASER)
Who invented the Beer’s law?
Lambert and Beer
Describes the relationship between absorption of light by a solution and the concentration of that solution.
Beer’s Law
Beer’s law state that the concentration of a substance is _____ proportional to the amount of light absorbed
directly
Beer’s law state that the concentration of a substance is _______ proportional to the logarithm of the transmitted light.
inversely
It is the light absorbed by the solution
Absorbance
Ratio of the incident light and light transmitted.
Transmittance
- Characteristic of a substance to absorb a specific fraction of a specific wavelength
- Varies from one analyte to another
- Constant
Molar absorptivity
- Length that the light needs to travel through the solution
- Dependent on the cuvet
- Constan
Path length
used to measure the light transmitted by a solution to determine the concentration of the light-absorbing substance in the solution.
Spectrophotometer
measured through the different components of the spectrophotometer
Radiant energy
- provides the energy that the sample will modify or attenuate by absorption.
- Provides incident light for the system.
Light source
2 types of light source in spectrophotometer
- Continuum source
- Line source
emits radiation that changes in intensity very slowly as a function of wavelength.
Continuum source
Provides a vast array of wavelength – visible to near IR region
Incandescent tungsten or tungsten iodide lamp
Alternative for UV spectrum
▪ Mercury arc (+ visible)
▪ Deuterium lamp
▪ Hydrogen lamp
▪ Xenon lamp
Spectrum usually used for enzymatic reactions
UV spectrum
Alternatives for IR spectrum
▪ Tungsten lamp (+visible)
▪ Nernst/ Merst glower
▪ Globar lamp (Si Carbide)
emit a limited number of discrete lines or bands of radiation, each of which spans a limited range of wavelengths
Line sources
• minimizes stray light
• prevents the entrance of scattered light
• where light passes through
Entrance slit
- refers to any wavelengths outside the band
- from the monochromator system
- absorbance error
Stray light
Entrance slit that prevents stray light
Nickel sulfate
It is an anti-stray light
Cutoff filter
It is 1⁄2 peak transmittance
Bandpass
Use to isolate an individual wavelength of light
Monochromator
Degree of isolation of the wavelength is affected by the:
1) monochromator
2) the width of the entrance
3) exit slits
Produces a monochromatic light based on the principle of constructive interference of waves
Colored Glass Filters
when 2 electromagnetic waves meet, synergism will occur
Constructive interference
transmit multiples of the desired wavelengths, they require accessory filters to eliminate these harmonic wavelengths.
Interference Filter
- wedge-shaped glass, quartz or sodium chloride.
- separates white (visible) light into a
continuous spectrum
Prisms
- most commonly used: better resolution than prism
- Parallel grooves or slits into an aluminized surface of a plat piece of a crown glass
- Wavelengths are bent as they pass a
sharp corner
Diffraction Gratings
the separation of light into component wavelengths, is based on the principle that wavelengths bend as they pass a sharp corner
Diffraction
- Controls the width of the light beam (band pass)
- Allows only a fraction of the spectrum to reach the sample cuvette.
Exit slit
- Also known as analytical cell
- Holds the solution of which the absorption is to be measured
Sample cell/cuvet
2 shapes of Sample cell
Round and rectangular cuvets
Why rectangular cuvets are the most common shape used in spectrophotometry?
it’s easier to maintain the length of light
or the measurement of the cuvet.
Kinds of sample cell used for visible range
Glass cuvette
Kind of cuvette used for UV range
Quartz or fused silica
kind of cuvette that is used for 350-2000 nm wavelength
Borosilicate
Converts transmitted radiant energy into an equivalent amount of electrical energy
Photodetector
- Barrier layer cell/Photovoltaic cell
- Simplest and least expensive
- Low sensitivity and fatigue are two
distinct disadvantages of these cells.
Photocell
- anode and cathode enclosed in a glass tube
- It gives off electron when light energy
strikes it.
Phototube
- more sensitive than vacuum phototubes
but less sensitive than the PMTs - multitude of wavelength
Phototransistors/photodiode
- most common type (visible and UV)
- commonly used when radiant power is very low, which is characteristic of very low-analyte concentrations.
- highly sensitive to ultraviolet and visible
radiation - amplifies radiant energy (200x sensitive)
Photomultiplier Tube (PMT)
Multiplies the radiant energy
Dynode
It displays the output of the detection system.
Read-out device
implies that a photometer is measuring at the wavelength that it is set to.
Wavelength accuracy
can be assessed easily using special glass-type optical filters.
Photometric accuracy
performed using glass filters or solutions that have known absorbance values for a specific wavelength.
Absorbance check
defined as the ability of a photometric system to yield a linear relationship between the radiant power incident upon its detector and the concentration
Linearity
Used to measure concentration by detecting the absorption of electromagnetic radiation by atoms rather than by molecules
Atomic absorption spectrophotometry (AAS)
Used to measure concentration by detecting the absorption of electromagnetic radiation by atoms rather than by molecules
Atomic absorption spectrophotometry (AAS)
Things that are usually measure using AAS
- Al
- Ca
- Cu
- Pb
- Mg
- Li
- Zn
2 types of light source in AAS
- Electrophoresis discharge lamp
- Hollow-cathode lamp