System International d'Unites (SI)
The standard measurement system used in scientific literature & clinical laboratories.
The solubility of a gas in a liquid is directly proportional to the pressure of the gas about the liquid at equilibrium.
Correlation between pressure, temperature, & solubility.
As the pressure of a gas is doubled, its solubility is also doubled. The solubility decreases with an increase in temperature.
Chemicals that have been especially purified to meet specific requirements. Used for chromatography, atomic absorption, & molecular diagnostics.
Chemically pure or Pure Grade
The impurity limitations are not stated. It is not recommended for reagent preparation.
Technical or commercial grade reagents
This method is used primarily in manufacturing & should never be used in the clinical laboratory
Primary reference materials
99.98% pure with a known concentration that has a certificate of analysis for each lot. The substance must be weighed dry preferably at 104-110 degrees Celsius.
Secondary reference materials
Concentrations cannot be prepared by weighing the solute and dissolving a known amount into a volume of solution.
What does OSHA require from a manufacturer.
Lot numbers, physical or biologic health hazard, precautions for the safe use and storage, & a MSDS.
Type 1 water
Used in test methods requiring minimal interference & maximal precision accuracy. Examples include high performance liquid chromotography, and preparation of all calibrators &solutions of reference materials.
Type 2 water
Used for general laboratory testing not requiring type 1 water such as reagent quality control, and standards.
Type 3 water
Used for glassware washing though final rinsing should be done with a higher grade water.
Filtration cartridges are composed of glass; cotton. Activated charcoal, which removes organic materials & chlorine with pores that filter bacteria.
Removes almost all organic materials by boiling the water & vaporizing it. Distillation can be done repeatedly which removes additional impurities.
Passing feed water through columns containing insoluble resin polymers which exchanges H+ & OH- ions for the impurities present in ionized form in the water.
How do you prepare reagent grade water?
To make reagent grade water you must put the water through several purification processes including; prefiltration, reverse osmosis, deionization, & distillation.
Is the temperature at which the vapor pressures of the solid & liquid phases are the same
How is osmotic pressure of a dilute solution?
It's proportional to the concentration of the molecules in a solution
What does "to contain" mean
The container is not meant to measure the volume but to simply contain the substance for a later use
What is a Ostwald-Folin pipette used for?
Used for the accurate measurement of a viscous fluid such as blood or serum
Used to transfer solutions or biologic fluids without consideration of a specific volume. Pasteur pipets have no calibration marks.
What's the difference between a Mohr pipette & a Serological pipette?
A Mohr pipette has no graduation to the tip; while a serological pipette has graduation marks all the way to the tip & is often a blowout pipet.
What is the main material used for filtration?
Filter material is made of cellulose and filter paper differs in pore size
Is a method for separating macromolecules from solvent or smaller sized substances by putting a solution in a bag which contains a semipermeable membrane
Concentration of a substance is directly proportional to right amount of light absorbed or inversely proportional to logarithm of transmitted light
What is wavelength?
Is the linear distance traversed by one complete wave cycle and is usually given in nanometers.
The absorption of radiant energy that plots the absorbance as a function of wavelength.
Is the concentration of the compound in solution increases. The more light absorbed by the solution the less light is transmitted.
What are the inverse proportions of Beer's Law?
If the concentration of a solution is constant & the path length through the solution that the light must traverse is doubled the absorbance is doubled & the absorbance is directly proportional to the path length of the radiant energy.
What are the main components of Beer's Law?
b=light path in cm
Unknown concentration formula?
As=Absorbance of standard Cs=Concentration of standard
Au=Absorbance of unknown Cu=Concentration of unknown
What are some deviations from Beer's Law?
Stray light, radiant energy is not monochromatic, very elevated concentrations, & sides of the cell are not parallel.
A single beam spectrophotometer consist of what?
A source of radiant energy, an entrance slit, wavelength selector, an exit slit, a device to hold the cuvette, a radiant energy detector, & a device to read out the electrical signal generated by the detector.
What are some advantages of the double beam instrument?
The capability of making simultaneous corrections for changes in light intensity, grating efficiency, slit-width variation.
What common light source is used for visible wavelengths?
Tungsten filament lamp. With a range from 360 to 950 nm.
Tungsten halide filament
Longer lasting produce more light at shorter wavelengths & emits a higher intensity radiant energy than tungsten filaments
Hydrogen & deuterium
Emits a continuous spectrum & is used for the UV region of the spectrum 220 to 360 nm
How do you calibrate a prism?
To certify wavelength calibration three different wavelengths must be checked
How to you calibrate a grating?
To certify wavelength calibration two different wavelengths must be checked for accuracy
What does the entrance slit do?
Focuses the light on the grating or prism where it can be dispersed with a minimum of stray light
What does the exit slit do?
It determines the band width of light that will be selected from the dispersed spectrum
Barrier layer (photo voltaic) cells
Detectors consisting of a plate of copper or iron on which a semiconducting layer of cuprous oxide or selenium is placed
A diode array
Is a two-dimensional matrix composed of hundreds of thin semiconductors spaced very closely together. The entire spectrum is essentially recorded within milliseconds.
The mercury lamp
Is substituted for the usual light source, & the spectrum is scanned to locate mercury emission peaks to determine the accuracy of the wavelength indicator control.
What is the most common source of stray light?
reflection of light from scratchy on optical surfaces or from duct particles anywhere the light path & higher-order spectra produced by diffraction grating
Blanking refers to reading the absorbance of a solution that includes all light absorption not due to the desired chromophore
Atomic Absorption Spectrophotometer
Measures concentration by detecting absorption of electromagnetic radiation by atoms rather than by molecules; very sensitive & precise & used to find the concentrations of trace metals
Part of chemical energy generated produces excited intermediates that decay to a ground state with emission of photons
Atomic Absorption spectrophotmetry
Is used for determining calcium, magnesium, lithium, lead, copper, zinc & other metals
What is the principle of Atomic Absorption?
Vaporized atoms in the ground state absorb light at very narrowly defined wavelengths. The radiant energy of the element is measured as it passes through a flame containing the vaporized metals.
A technique in Atomic Absorption that converts the sample into a fine spray or aerosol while it is being introduced into the flame
A technique in Atomic Absorption where the flame evaporates the solvent from the aerosol, leaving microscopic particles that disintegrate under the influence of heat to yield atoms
What is an advantage seen in Flame-less Atomic Absorption?
The same light sources & wavelength is used to measure the total adsorptions & the background absorptions
What are some disadvantages in Flame-less Atomic Absorption?
Sometimes compounds are vaporized with the element to be measured & with "matrix modifiers" the sample vaporizes at higher temperatures, allowing interfering compounds to be burned off at lower temperatures.
What is the principle behind Nephelometry & Turbidimetry
When a photon in the light beam of a spectophotometer strikes a solid particle in the cuvette, the photon is scattered, reflected & scattered back
Uses 2 wavelengths to correct for interference when a color-producing reaction is performed on a patient's sample that is hemolyzed, icteric, or lipemic
What is a disadvantage of Fluorometry?
A decreased fluorescence reading due to absorption of emitted light by either solutes in the cuvette or contaminants from insufficient washing called "quenching"
Why does Fluorometry have greater specificity?
because the secondary monochromator selects only the emitted wavelength of light
Distinguishes different cell types by their surface antigens then different fluorochromes are attached to antibodies that are specific to the surface antigens that identify different cell types.
In electrochemistry Ion-Selective Electrodes are sensitive toward individual ions such as indicator electrodes, liquid junctions, & Nernst equations
Migration of charged solutes of particles in a liquid medium under the influence of an electric field
Ampholyte in different pH
In acidic solution it becomes positively charged & binds protons, while in alkaline solution the ampholyte is negatively ionized & give up protons
What factors effect rate of migration
Net electrical charge of the molecule, the size & shape of the molecule, electrical field strength, properties of the supporting medium, & temperature
The rate of migration per unit field strength; which is directly proportional to net charge & inversely proportional to molecular size & viscosity
How do buffers function in electrophoresis?
Carries the applied current, and establishes the pH at which electrophoresis is performed
Effects of increasing buffer concentration
The ionic cloud increases in size and the molecule becomes hindered in its movement
Effects of high ionic strength buffer
Yields sharper band separations but also produce more Joule heat due to increased current levels, which denaturation of heat-labile proteins
How do you treat cellulose acetate?
Because cellulose acetates membranes are dry, opaque, & brittle you must add acetic anhydride to it
What substances are best separated by agarose?
Serum, urine, cerebrospinal fluid, proteins, hemoglobin variant, isoenzymes, & lipoproteins. Agarose gel has large enough pores for all proteins to pass through unimpeded
is most useful for mixtures of smaller DNA fragments & resolves fragments smaller that 1kbp
What are the most common used dyes for protein electrophoresis?
Amido Black B, or Coomassie Brilliant Blue series of dyes
How do you quantify the individual zones?
Either as a percentage of the total or absolute concentration by direct densitometry, if the total protein concentration is known
What are the five zones of electroporesis using agarose gel?
albumin, Alpha 1, Alpha 2, Beta, & gamma-globulins
Describe the type of gel called PAGE?
Samples are separated in individual gels prepared in open-ended glass tubes & forms a bridge between two buffer reservoirs
Isoelectric Focusing Electrophoresis
Separates amphoteric compounds, such as proteins, with increased resolution in a medium possessing a stable pH gradient
First dimensional electrophoresis is
carried out in a large-pore medium, such as agarose gel or large pore polyacrylamide gel; while ampholytes are added to yield a pH gradient
Second dimensional electrophoresis is
is often polyacrylamide in a linear or gradient format which achieves the highest resolving power for the separation of DNA fragments
occurs when the support media in contact with water take on a negative charge due to adsorption of hydroxyl ions
How should buffers be prepared & stored?
Should be refrigerated when not in use & cold buffer improves resolution & reduces evaporation
Distorted protein zones are caused by what?
bent applicators, air bubble during sample application, over application of sample, & excessive drying of the electrophoretic support