Clinical Chemistry Exam 1

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Standard Clinical Lab operations Spectrophotometry & Photometry Electrophoresis

System International d'Unites (SI)

The standard measurement system used in scientific literature & clinical laboratories.

Henry's law

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.

Ultra-pure Reagents

Chemicals that have been especially purified to meet specific requirements. Used for chromatography, atomic absorption, & molecular diagnostics.

United States Pharmacopoeia and National Formulary

Chemical purity used to manufacture drugs

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.

Prefiltration

Filtration cartridges are composed of glass; cotton. Activated charcoal, which removes organic materials & chlorine with pores that filter bacteria.

Distilled water

Removes almost all organic materials by boiling the water & vaporizing it. Distillation can be done repeatedly which removes additional impurities.

Deionization

Passing feed water through columns containing insoluble resin polymers which exchanges H+ & OH- ions for the impurities present in ionized form in the water.

Reverse osmosis

Water is forced through a semipermeable membrane that acts as a molecular filter.

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.

What wavelength do use for ultraviolet oxidation?

254 nanometers

What is a solute?

A substance that is dissolved in a liquid

What is another word for solute?

Analytes

What is a solvent?

A liquid in which the solute is dissolved in

What do you get from a solute mixed with a solvent?

Solution

Vapor pressure

The pressure at which the liquid solvent is in equilibrium with the water vapor

Freezing point

Is the temperature at which the vapor pressures of the solid & liquid phases are the same

Boiling point

The temperature at which the vapor pressure of the solvent reaches one atmosphere

Osmotic pressure

Is the pressure that allows solvent to flow through a semipermeable membrane

How is osmotic pressure of a dilute solution?

It's proportional to the concentration of the molecules in a solution

What can be used to calibrate a thermometer?

Gallium

What does a class A volumetric flask do?

Calibrated to hold one exact volume of liquid

What class is used for laboratory utensils?

Class A

What does "to deliver" mean?

That the utensil will deliver an exact amount of a substance

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 volumetric transfer pipette used for?

Delivering a fixed volume

What is a Ostwald-Folin pipette used for?

Used for the accurate measurement of a viscous fluid such as blood or serum

Pasteur pipets

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 three variables are applied to centrifugation?

Mass, speed, & radius

How is speed express in centrifugation?

Revolutions per minute (RPM)

Centrifugal force generated is expressed in what?

Relative centrifugal force (RCF)

What is the main material used for filtration?

Filter material is made of cellulose and filter paper differs in pore size

Dialysis

Is a method for separating macromolecules from solvent or smaller sized substances by putting a solution in a bag which contains a semipermeable membrane

What does a spectrometer do?

Isolates a narrow range of incident wavelength

Beer's Law

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.

What is frequency?

The number of cycles occurring per second & is obtained by relationship.

What is velocity?

Varies with the medium through which the radiant energy is passing.

What is the velocity constant in a vacuum?

C=3*10^10 cm/sec

What is the formula for calculating energy?

E=hv

What is plank's constant?

6.62*10^-27

What's the formula for calculation energy involving wavelength

E=hc/£

What wavelength does ultraviolet fall between?

180-390 nanometers

What wavelength does visible light fall between?

390-780 nanometers

Absorption spectrum

The absorption of radiant energy that plots the absorbance as a function of wavelength.

Emission process is used in what absorption methods?

Flame photometry & fluorometric methods

Transmittance

Is the concentration of the compound in solution increases. The more light absorbed by the solution the less light is transmitted.

What are the transmittance equations?

A=log 100%
-log %T
A=2-log%T

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?

A=absorbance
a=absorptivity
b=light path in cm
c=concentration

What type of graph is used to find the concentration of an unknown using Beer's Law?

A linear graph

Unknown concentration formula?

As/Au=Cs/Cu
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.

What does monochromicity mean?

One light source

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.

What light source is used for UV range?

Deuterium lamp or hydrogen discharge

What light source is used for calibration & accuracy?

Holmium oxide didymium or deuterium lamp

Tungsten iodide lamps

Ranges from visible & near UV radiant energy

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

Deuterium lamp

Is more intense than the hydrogen lamp used for the UV region

Mercury vapor lamp

Used for wavelength calibration

What type of filter is used for a monochromator?

A grating or a prism

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

At what wavelengths does a prism begin to be less linear?

over 550 nm

Band pass

Is where the light transmitted is one-half of the peak tansmittance

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

What cuvette must be used to measure wavelengths below 320 nm?

Quartz (silica) cells

What effects occur with a scratched cuvet?

It scatters light

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

Photo-multiplier tube

Is an electron tube that is capable of significantly amplifying a current

Photodiodes

Is a semiconductor that change their charged voltage upon being struck by light

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.

Stray light

any wavelengths outside the band transmitted by the monochromator

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

Blank

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

Chemiluminescence

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.

What is the source emitting the radiant energy in Atomic Absorption?

Hollow cathode lamp

Nebulization

A technique in Atomic Absorption that converts the sample into a fine spray or aerosol while it is being introduced into the flame

Atomization

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

Flame-less Atomic Absorption

Converts the sample into an atomic vapor by chemical reactions

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

Mia Theory

Most of the light appears to be scattered forward

Rayleigh Theory

More light appears scattered in a forward direction than in a backward direction

What degree is light scattered on a Nephelometry?

90 degrees

Bichromatic analysis

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

Flame photometry is mostly used for what metals?

Na, K, or Li

Fluorometry

Energy emission that occurs when certain compounds absorb electromagnetic radiation

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

Flow cytometry

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.

pH Electrodes

In electrochemistry Ion-Selective Electrodes are sensitive toward individual ions such as indicator electrodes, liquid junctions, & Nernst equations

Electrophoresis

Migration of charged solutes of particles in a liquid medium under the influence of an electric field

Cation

is a positively charged ion that moves toward the cathode

Anion

is a negatively charged ion the moves toward the anode

Ampholyte

A molecule that is either positively or negatively charged

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

Electrophoretic mobility

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 are the support media of choice in electroporesis?

Agarose & polyacrylamide

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

Molecular sieving

Proteins are separated when both charge to mass ratio & molecular size

Polyacrylamide

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

Southern Blotting

Requires an electrophoretic separation of DNA or DNA fragments by AGE

Northern blotting

Used to detect ribonucleic acids RNA

Western blotting

Used to separate proteins

Endosmosis

occurs when the support media in contact with water take on a negative charge due to adsorption of hydroxyl ions

What causes discontinuities?

dirty applicators

What causes unequal migration?

dirty electrodes

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

What causes irregularities?

excessively wet agarose gels

What does unusual band forms mean?

patients plasma was used instead of serum

What happens when you overload a sample?

Albumin bands are large & distorted

How do you increase your band migration?

dilute buffer

What does a hemolyzed sample do?

increases beta globulin

What happens if the ionic strength is too high?

denatures proteins

What causes low migration in the bands?

gel is too thick or your pH percentage is wrong

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