Instrumentation Flashcards
Majority of techniques → 4 basic discipline (within field of analytical chemistry)
Photometry, Luminescence, ElectroAnalytic Method, and Chromatography
- study of phenomenon of light absorption by molecules in solution
- absorb light at a particular wavelength
- follows beer lambert’s law
Photometry
transmitted light decreases ex ponentially with increase in concentration of absorbing medium
Beers Law
Other Principle of Beer’s Law
Amount of Absorbance is directly proportional to the Concentration and inversely proportional to the transmitted light
These instruments measures electromagnetic radiation
Spectrophotometry, Atomic Absorbance Spectrometry, Flame Emission Photometry, Mass Spectrometry
Photons of energy traveling in waves
Electromagnetic radiation
involves in electromagnetic radiation, Beer’s law, standard curve, and interaction with lights
Photometry
Very short wavelength
UV
Very long Wavelength
Infrared
Wavelength of Visible Spectrum
400-700 nm
distance traveled by one complete wave cycle (nm)
Wavelength
If shorter the wavelength
The greater energy and light and number of photons
particles of light
Photons
Relationship between wavelength and energy
Planck’s formula = E = hv
-220-380
-Deutrium Lamp
-Quartz
UV
-380-750
-Tungsten
-Borosilicate
Visible Incandescent
-750-200
-Tungsten
-Quartz
Infrared
amount of light absorbed; proportional to the inverse log of transmittance
Absorbance (Optical Density)
Blanking Techniques
Reagent and Sample Blanking
correct absorbance caused by the color of reagents
Reagent Blank
correct absorbance caused by samples
Sample Blank
Not effective blanking process may be due to
Turbidity
Components of Spectrophotometer
Light Source - Monochromator - Cuvette - Photodetector - Reading Device
-provides polychromatic light and must generate sufficient radiant energy or power to measure the analyte of interest.
Light Source
Types of light source
Line and Continuum
emits radiation that changes in intensity; widely used in the laboratory, Examples: TUNGSTEN (VS, IR), deuterium (UV), and xenon lamps.
Continuum
emits limited radiation and wavelength. Examples: Mercury and sodium vapor lamps in spectrophotometers (UV and visible regions), and the hollow cathode lamp (AAS)
Line Source
minimizes unwanted or stray light
Entrance slit
refers to any wavelengths outside the band transmitted by the monochromator
Stray Light
most common cause of loss of linearity at high-analyte concentration.
Stray light
refers to the range of wavelengths passing through the sample. The narrower the bandpass, the greater the photometric resolution.
Bandpass
isolates specific or individual wavelength of light
Monochromator
mast commonly used monochromator; better resolution than prism.
Diffraction Gratings
allows only a narrow fraction of the spectrum to reach the sample cuvette.
Exit slit
It holds the solution whose concentration is to be measured.
Cuvette
most commonly used cuvette (available in 350 mm to 2000 nm)
Alumina-silica
used for measurement of solution requiring visible and ultraviolet spectra
Quartz
It detects and converts transmitted light into photoelectric energy
Photodetector
not as sensitive as PMT but with excellent linearity
Photodiode
simplest type of an absorption spectrophotometer
Single Beam Spectrophotometer
ore beam passes through the sample, and the other through a reference solution or blank
Double Beam Spectrophotometer
with 2 photodetectors, for the sample beam and Reference beam
Double Beam in Space
used to check wavelength accuracy (wavelength quality assuramce calibration).
didymium or holmium oxide filter
Pinciple:-Measures light absorbed by ground state atoms. Analytic technique that measures concentrationof’anayres by detecting absorption of electromagnetic radiation by atoms rather
than molecules,
AAS
Element is not excited by merely dissociated from its chemical bond and place in an unionized, unexcited ground state.
AAS
Light Source for AAS
Hollow Cathode Lamp
More sensitive than FEP
AAS
Excitation of electrons from lower to higher energy state.
FEP
Determines Na, K, and Li
FEP
Light source of FEP
FLame
based on the fragmentation
and ionization of molecules using a suitable source of energy.
Mass Spectrometry
vital in-structral identication and determination of molecular weight
MS
detection and quantification of analytes in human fluids and tissues;
MS
emission of light as by a chemical or physiological process
Luminescence
Pinciple-Chemical reaction that produces light. Usually involves oridation of luminol, acridinium esters, or dioxetanes.
Chemiluminiscence
widely used nowadags due to is high sensitivity while even more sensitive than fluorometry.
Chemiluminiscence
atoms absorb light of specific wavelength, demiight of longer wavelength (lower energy).
Flourometry
-has two monochromators
-1000x sensitive than Spectrophotometer
Flourometry
Flourometry disadvatage
Quenching - ph and temp change
Measures reduction in light transmission by particles in suspension and lightis measured at angle from light source.
Nephelometry
measurement of
LIGHT SCATTERED by a particulate solution.
Nephelometry
unknown sample is made to react with a known solation in the presence of an indicator.
Volumetric
-The measurement of current or voltage generated by the activity of a specific ion.
-It measures blood pH and gases, electrolytes in blood and sweat samples, toxins, glucose, and
urea.
Electroanalytic methods
Migration of charged particles in an electric field.
Electrophoresis
Buffer in Electrophoresis
Barbital (ph 8.6)
In electrophoresis, this is separated from basis of their elictric charge densisties
Proteins
separates by molecular size.
Cellulose Acetate
separates by electrical charge
Agarose Gel
separates on the basis of charge and molecular size;
separates proteins into 20 fractions used to study isoenzymes.
Polyacrylamide gel
for CSF Protein Electrophoresis
Coomassie Blue
very sensitive even to nanogram quantities of proteins
Gold/silver stain
-measures the absorbance of stain - concentration of the dye and protein fraction.
* It scans and quantitates electrophoretic pattern.
Densitometry
The measurement of current or voltage generated by the activity of a specific ion.
Electrochemistry
-measurement of electrical potential due to the activity of free ions - change in voltage indicates activity of each analyte.
* It measures the differences in voltage (potential) at a constant current.
Potentiometry
known concentration of KCI.
SATURATED CALOMEL ELECTRODE
immersed in potassium chloride solution that have been saturated with silver chloride; ideal for temperatures higher than 60° C and react with more sample.
SILVER-SILVER CHLORIDE
employs an adaptation of the pH measurement.
Severinghaus electrode
*Used to assay heavy metals such as lead in blood.
- Lead and iron testing
ANODIC STRIPPING VOLTAMMETRY
separation of soluble components in a solution by specific differences in physical-chemical characteristics
Chromatography
-various ISEs may be covered by immobilized enzymes that can catalyze a specific chemical reaction.
-Selection of the ISE is determined by the reaction product of the immobilized enzyme.
Enzyme electrodes
- electrochemical transducer capable of responding to one given ion.
- Its ionic selectivity depends on the membrane/barrier composition used.
-It is very sensitive and measures the activity of one ion (selective)
ISE
the patient’s serum sample is brought into direct contact with the electrode surface and the activity of the relevant ion is measured in the water portion of the serum.
Direct ISE
POCT electrolyte test and blood gas analvsis
Direct ISE
Use: Routine electrolyte test
Indirect ISE
Valinomycin (Antibiotics)
K+ Analysis
- measurement of the amount of electricity (in coulombs) at a fixed potential.
- electrochemical titratiom
Coloumetry
Interferences of Coloumetry
Bromide cyanide and cysteine
Amount of increase in current proportional to the concentration of analyte.
Polarography
measurement of current after which a potential is applied to an electrochemical cell.
VOLTAMMETRY
used for fractionation of sugar and amino acid. Sorbent (stationary phase): Whatman paper
Planar chromatography
Separation occurs based on differences in absorption at the solid phase surfaces.
GSC
Separation occurs by differences in solute partitioning between the gaseous mobile phase and the liquid stationary phase.
GLC
technique where solutes in a sample are separated for identification based on physical differences that allow their differential distribution between a mobile phase and a stationary phase
Chromatography
-liquid-liquid chromatography.
-Separation of solute is based on relative solubility in an organic (nonpolar) solvent and an aqueous (polar) solvent. In its simplest form,
Partition chromatography
based on the competition between the sample and the mobile phase for adsorptive sites on the solid stationary phase.
Adsorption chromatography
solute mixtures are separated by virtue of the magnitude and charge of ionic species.
Ion exchange chromatography
relative distance of migration from the point application
Retention factor
-It is used for separation of steroids, barbiturates, blood, alcohol, and lipids.
* It is useful for compounds that are naturally volatile or can be easily converted into a volatile form. If the molecule of interest is not volatile enough for direct injection, it is necessary to derivatize or volatilize the sample. Flame ionization is used as a detector for gas liquid chromatography (GLC). Elution order of volatiles is based on their boiling point.
GC
Advantage: Short turnaround time, sensitivity, accuracy, and high resolution
GC
widely used liquid chromatography.
HPlC
utilized for detecting nonvolatile substances in body fluids; used as a
screening and confirmatory method.
LC-MS
- It uses pressure for fast separations, controlled temperature, in-line detectors, and gradient elution technique.
HPLC
