Analytical methods Chapter 5 Flashcards
What is Spectrophotometric measurement?
It is a measurement of light intensity in a narrower wavelength
What is Photometric measurement?
It is a measurement of light intensity without consideration of its wavelength
Is described as photons of energy traveling in waves
Electromagnetic radiation
What is the formula of the Beer’s law?
Concentration of a substance is directly proportional to the amount of light absorbed or inversely proportional to the logarithm of the transmitted light
What is the use of Light source?
Incident light for the system
What are the common source of light used near infrared regions?
Incandescent tungsten and Tungsten light bulb
What are the alternatives used for UV spectrum?
Deuterium discharge lamp and mercury arc lamp
What is the use of Entrace slit?
It minimizes the unwanted straylight
What is Straylight?
It is a wavelength outside the band which can affect the measurement and cause absorbance error
What is the use of monochromator?
It isolates specific or individual wavelength of light
Kinds of monochromator
Prisms, Diffraction gratings and Interference Filters
The principle of prism
A narrow beam of light focused on a prism is refracted as it enters the more dense glass
Most commonly used monochromator that consists of many parallel grooves
Diffraction gratings
Principle of constructive interference
Light waves enter one side of the filter and are refracted at the 2nd surface
What is the use of Exit Slit?
It controls the width of the light beam or band pass
The narrower the light beam
the greater the resolution
sample cell/ absorption cell/ analytical cell
cuvet
kinds of cuvet and some definition
Alumina Silica glass- simple/ older
Quartz/ Plastic- used for UV spectrum
Borosilicate glass- can stand increase temperature
Soft glass- Common glass
scatter light and should be discarded
scratches
Produces etching and dissolves glass
Alkaline solutions
What is a Photodetector?
It converts Light energy to Photoelectric Energy
Kinds of Photodetectors:
Photocell, Phototube and Photomultiplier tube
Requires external voltage and it gives off electron when light energy strikes it
Phototube
Simplest detector and no external voltage
Photocell
On a plate of iron covered with transparent layer of silver
Selenium
Most common type which can detect low level of light and amplifies radiant energy 200x
Photomultiplier tube
What is Sensitivity and Specificity?
Sensitivity is a term that can measure even the smallest movement of light while Specificity is a term that can only detect a specific light
It detects less amount of light and not as sensitive as photomultiplier tube
Phototransistors and Photodiode
moving needle on a dial or a digital display that indicates the amount of light passing
Galvanometer/ Ammeter
Splits the monochromator light into two components
Double-beam Spectrophotometer
Beam passes through:
1st-sample
2nd- reference solution or blank
Used to check wavelength accuracy
Didychium or holmium oxide filter
Reagent blank
Blanking technique
light emitted by a single atom burned in flame and measurement of excited atoms (Na, K, Li)
Flame-emission spectrophotometry
Principle of Flame-emission photometry
Excitation of electrons from lower to higher energy state
Serves as a light source and cuvette for FEP?
Flame
Serves as a monochromator in FEP?
Filter
Components of FEP
Nebulizer, Burner and Monochromator system
Deliver a fine spray of sample
Nebulizer
Strikes the photomultiplier tube
Monochromator system
What is the light source used in Atomic absorption spectrophotometry?
Hollow cathode lamp
Light is not excited, unionized and at a ground state?
Atomic absorption spectrophotometry
Unexcited trace metals
Ca++ and Mg++
Converts Ions to atoms
Atomizer (Nebulizer grapite furnace)
Used to modulate light source
Chopper
Principle of Fluorometry
amount of light emitted by a molecule after excitation by electromagnetic radiation and uses 2 monochromator
light source used in fluorometry
mercury arc and xenon lamp
light detector used in fluorometry
Photomultiplier tube
difference between primary filter and secondary filter
The primary filter is the selection of wavelength that is best absorbed by the solution while the secondary filter prevent incident light from striking the photodetector
Advantage of Fluorometry
1000x more sensitive than most spectophotometry
Disadvantage of Fluorometry
Quenching - sudden change in temperature and reduces intensity of fluorescence
Principle of Chemiluminescence
Exciting molecules by chemicals and requires no excitation radiation and monochromator (MOLECULES IN UNEXCITED STATE)
Advantage of Chemiluminescence
Speed (10s) - Picomdar detection
Simple instrumentation
Principle of Turbidimetry
Amount of Light blocked by suspension particles and is dependent on particle size and concentration
Abundant in blood sample
Albumin
Principle of Nephelometry
Light scattered by the small particles is measured at an angle forward to the incident light
the higher the scattered light equals the no. of particle
the increase in concentration
Process of separating by electric current
Electrophoresis
2 types of Electrophoresis
Iontophoresis and Zone Electrophoresis
Migration of small ions
Iontophoresis
Migration of charged macromolecules in porous support
Zone electrophoresis
Protein are negatively charged and they moved toward the _______
anode
The 2 Buffers and their corresponding pH
Barbital (veronal) pH 8.6
Tris-boric EDTA pH 8.7
Acidic and basic, determine the net change on a protein (electrophoretic mobility)
Electrophoresis
3 supporting media
Cellulose acetate, Agarose gel, Polyacrylamide gel
separates serum proteins into 5 bands (no. of fractions)
Cellulose acetate
Agarose gel has how many bands?
10-15 bands
Polyacrylamide gel has how many bands?
720 bands
Migration is controlled by
net change of the partilly, Particle size and shape, Electric fields, supporting media and Temperature
movement of buffer and solvent relative to their fixed support
Electroendosmosis
separating molecules migrate(PH gradient) and proteins of identical size but with different net changes
Isoelectric Focusing
Separation is performed in narrow-bore fuse silica capillaries
capillary electrophoresis
Separation sample of mixtures between mobile and stationary phase
Chromatography
Modes of separation
Adsorption (Liquid-Solid Chromatography), Partition ( Liquid-Liquid Chromatography), Steric Exclusion, Ion exchange
Based on the competition of adsorptive sites (stationary phase)
Adsorption (Liquid-solid Chromatography)
Separation of solute based on the relative solubility of the compound
Partition
Separation by magnitude and charge of ionic species
Ion exchange
Gel filtration, Gel permeation or molecular chromatography and separates solutes
Steric Exclusion
2 stationary phase
cation- exchange resin
Anion- exchange resin
Side chains of cation
H+ ions
Side chains of Anion exchange
OH-
2 Types of Chromatography
Planar and Columnar
2 types of Planar Chromatography
:Paper and Thin layer Chromatography (drug screening)
Intube or coated column separation
Columnar Chromatography
distribution of solute between a liquid mobile phase and a stationary phase
Liquid Chromatography
used for fraction of drugs, hormones, lipids, carbohydrates and proteins
HPLC (High performance liquid chromatography)
pressure for fast separation of thermos substance
HPLC
used to separate mixture of compounds that are volatile
Gas chromatography
