Clinical Chemistry (Instrumentation and Analytical Principles) Flashcards
No.1 - 76
Which of the following lamps provides a continuous spectrum of radiant energy in the visible, near IR, and near UV regions of the spectrum?
A. Tungsten-filament
B. Hydrogen
C. Deuterium
D. Mercury vapor
A.
A tungsten-filament lamp is the most common light source for photometry in the visible region. It provides a continuous spectrum (360-800 nm) from the near infrared (IR) through the visible to the near ultraviolet (UV) region. Most of the radiant energy is in the near IR. Only about 15% is in the visible region—the region usually used. Because of the large emission in the near IR, tungsten lamps generate a significant amount of heat.
Hydrogen and deuterium lamps are used for work in the 200-375 nm range. The mercury vapor lamp does not provide a continuous spectrum, emitting radiation at specific wavelengths.
Which of the following isolates light within a narrow region of the spectrum?
A. Photomultiplier tube
B. Monochromator
C. Photovoltaic cell
D. Detector
B.
Photometric methods are based on the use of Beer’s law, which is applicable only for monochromatic light. A monochromator is a device for selecting a narrow band of wavelengths from a continuous spectrum. The three kinds of monochromators are filters, prisms, and diffraction gratings.
Which of the following is not descriptive of a photomultiplier tube?
A. Emits electrons proportionally to
initial light absorbed
B. Must be shielded from stray light
C. Cannot be used with a chopper
D. Amplifies the initial signal received
C.
A photomultiplier tube (PMT) responds to
the radiant energy (light) it absorbs by emitting electrons in a proportional amount to the initial light absorbed. These electrons then go through a series of stages where amplification occurs. The cascade effect, as the electrons go through 10 to 15 stages, results in a final current that may be one million times the initial current. The PMT exhibits rapid response time and sensitivity. These qualities also dictate that this type of detector be shielded from stray light and room light to prevent burnout. The rapid response time of a PMT makes it able to monitor interrupted light beams produced by a chopper
Which of the following is false about a photomultiplier tube?
A. Converts radiant energy (light)
to electrical energy (current)
B. Amplifies the current significantly
C. Has a very rapid response time
D. Is composed of an iron plate and
a layer of selenium
D. A photomultiplier tube (PMT) has two functions: (1) It is a transducer that converts light to electricity; and (2) it amplifies the signal within the tube. Amplification can be as great as one million times. The emission of electrons by a light-sensitive surface—that is, the conversion of light energy to electrical energy—is virtually instantaneous. Hence, PMTs have a very rapid response time. An iron plate and a layer of selenium are partial descriptions of the composition of a photocell or barrier layer cell
Which type of photodetector employs a linear arrangement that allows it to respond to a specific wavelength resulting in complete UV/visible spectrum analysis?
A. Photomultiplier tube
B. Phototube
C. Barrier layer cell
D. Photodiode array
D. Photodiode array detectors are designed
with 256 to 2048 photodiodes that are arranged in a linear fashion. This arrangement allows each photodiode to respond to a specific wavelength that results in a continuous UV/visible spectrum. Resolution is generally 1-2 nm.
When performing spectrophotometer quality assurance checks, what is the holmium oxide glass filter used to assess?
A. Linearity
B. Stray light
C. Absorbance accuracy
D. Wavelength accuracy
D. Wavelength calibration of a spectrophotometer is performed to verify that the radiant energy emitted from the monochromator through the exit slit is the same as the wavelength selector indicates. The glass filters holmium oxide, used in the UV and visible ranges, and didymium, used in the visible and near IR regions, are employed to check wavelength accuracy. Solutions of stable chromogens such as nickel sulfate may be used. Source lamps may be replaced with mercury-vapor or deuterium lamps. These lamps have strong emission lines and provide the most accurate method of wavelength calibration
In spectrophotometric analysis, what is the
purpose of the reagent blank?
A. Correct for interfering chromogens
B. Correct for lipemia
C. Correct for protein
D. Correct for color contribution of the
reagents
D. The reagent blank contains the same reagents as those used for assaying the specimen. By adjusting the spectrophotometer to 100% T (or 0 absorbance) with the reagent blank, the instrument automatically subtracts the color contributed by the reagents from each succeeding reading of specimens, controls, and standards. This technique is used both in manual procedures and automated instruments. Because the reagent blank does not contain sample, there is no correction for interfering chromogens or lipemia.
In regard to bichromatic analysis, which of the following is false?
A. Absorbance is measured at the
spectral absorbance peak for a blank
and the sample using the same
wavelength.
B. Eliminates background interferences
C. Sample concentration determined
from difference in two measured
absorbances
D. Functions as a reference blank for
each sample
A. Measurement of an assay at two different
wavelengths is termed bichromatic. The wavelengths chosen for absorbance readings will represent the peak and base of the spectral absorbance curve for the particular assay. By determining the difference between the two measured absorbances, the sample’s concentration can be calculated with elimination of background interference from such substances as bilirubin and hemoglobin. Thus, bichromatic analysis functions as a reference blank for each individual sample
The bandpass of a spectrophotometer is
10 nm. If an instrument is set at 540 nm,
the wavelengths that are permitted to impinge on the sample will be within what wavelength range?
A. 530-540 nm
B. 530-550 nm
C. 535-545 nm
D. 540-550 nm
C. The bandpass or bandwidth is the range of wavelengths that are passed by a monochromator. In the example given, the bandpass will permit a 10-nm range of wavelengths to pass through the monochromator and impinge on the sample solution in the cuvet. Thus, 540 ± 5 nm (10-nm bandpass) will be equivalent to a wavelength range of 535-545 nm.
Which of the following formulas is an expression of the Beer-Lambert law that is routinely applied to spectrophotometric analysis?
A. Au x Cs/As = Cu
B. Cu x Cs/As = Au
C. As X Cs/Cu = Au
D. A = 2 - log% T
A. When the absorbance of a sample in solution varies directly with the concentration of the sample, Beer’s law is followed. In turn, when the absorbance increases exponentially with an increase in the light path, the Lambert law is followed. Incorporation of these two laws may be stated as A = abc, where A = absorbance, a= absorptivity of the substance being measured, b = light path in cm, and c = concentration of the measured substance. When the Beer-Lambert law is applied to spectrophotometric analyses of standards and unknown samples that are being measured, the following equation is derived: Au x Cs/As = Cu where Au= absorbance of
unknown, Cu = concentration of unknown, As = absorbance of standard, and Au = absorbance of unknown. This formula is applied to assays that exhibit linear relationships between changes in absorbance with changes in concentration to calculate the concentration of the unknown sample
In spectrophotometry, which of the following is a mathematical expression of the relationship between absorbance and transmittance?
A. A = abc
B, Au/Cu = As/Cs
C. A = 2 - log % T
D. A= log % T
C. In spectrophotometry, molecules in solution will cause incident light to be absorbed while the remaining light energy will be transmitted. Absorbance is the term used to describe the monochromatic light that is absorbed by the sample, and transmittance describes the light that passes through the sample. The mathematical relationship between absorbance and transmittance is expressed by A = 2 — log %T
Which of the following is not a problem inherent in turbidimetry?
A. Variation in particle size of samples
B. Variation in particle size of standards
C. Rate of aggregation or settling of particles
D. Need to maintain a constant and specific temperature
D. Turbidimetry is the measurement of the
amount of light blocked by particulate matter in passing through a turbid solution. The amount of light blocked depends on the number and the size of the particles. Hence the particle size in samples and standards must be comparable. Consistent timing of sample preparation and assay helps to avoid errors resulting from aggregation or settling of particles. The procedure is usually carried out at room temperature. Slight variations in temperature are not critical
Which of the following may be associated with reflectance spectrophotometry as it relates to the dry reagent slide technique?
A. Light projected to the slide at 180-degree angle
B. Dye concentration directly proportional to reflectance
C. Unabsorbed, reflected light detected by photodetector
D. Reflectance values are linearly proportional to transmission values
C. In the dry reagent slide technique, as light from a radiant energy source passes through an interference filter, it is projected to the slide at a 45-degree angle. The light then follows a path through the clear support material and reagent layer and hits a white spreading layer; the unabsorbed light is then reflected back through the reagent and support layers. This reflected light impinges on the photodetector, which is positioned at a 90-degree angle to the slide. Because reflectance values are neither linearly proportional to transmission values nor consequently to dye concentration, the microcomputer utilizes an algorithm as a linearizing transformation of reflectance values to transmission values so that concentration may be calculated.
Fluorometers are designed so that the path of the exciting light is at a right angle to the path of the emitted light. What is the purpose of this design?
A. Prevent loss of emitted light
B. Prevent loss of the excitation light
C. Focus emitted and excitation light upon the detector
D. Prevent excitation light from reaching
the detector
D. In a fluorometer, light from the excitation
lamp travels in a straight line, whereas the fluorescent light is radiated in all directions. If
the detector for the emitted fluorescent light is placed at a right angle to the path of the excitation light, the excitation light will not fall on the detector. In addition, baffles can be placed around the cuvet to avoid reflection of the exciting light from the surface of the cuvet to the detector. The right-angle configuration does not prevent loss of the exciting or the emitted light
Which of the following represents a primary advantage of performing fluorometric over absorption spectroscopic methods of analysis?
A. Increased specificity and increased sensitivity
B. Increased specificity and decreased sensitivity
C. Purity of reagents used not as critical
D. Ease of performing assays
A. Fluorescence occurs when a molecule absorbs light of a particular wavelength and is thereby stimulated to emit light of a longer wavelength. The emitted light has a characteristic spectrum, the emission spectrum, that is unique for each fluorescing molecule. Hence, fluorometric methods are extremely sensitive and highly specific. Because of this extreme sensitivity, reagents used must be of a higher degree of purity than is required for spectroscopy, because even slight traces of impurities may fluoresce
Which of the following may be associated with fluorescence polarization?
A. Plane-polarized light is used for sample excitation.
B. Small molecular complexes show a greater amount of polarization.
C. It is a heterogeneous technique employed in fluorophore-ligand immunoassays.
D. Polarized light detected is directly
proportional to concentration of ligand
in sample.
A. Instrumentation employing fluorescence
polarization is used for such testing as therapeutic drug levels and fetal lung maturity analysis. In these immunologic assays, plane-polarized light excites fluorophors in the sample cuvet. The free fluorophore-labeled ligands rotate freely because of their small size and primarily emit depolarized light. The labeled ligand-antibody complexes rotate more slowly because of their large size and emit polarized fluorescent light. Because of the differences in emitted light, it is not necessary to separate free from bound fluorophore-labeled ligands, allowing for use of the homogeneous assay technique. The emitted fluorescence intensity is measured by a polarization analyzer in the vertical plane, followed by its 90-degree movement for measurement in the horizontal plane. The amount of polarized light detected is inversely proportional to the concentration of ligand in the serum sample
Which of the following may be associated with bioluminescence?
A. Light emission produced due to enzymatic oxidation of a substrate
B. Less sensitive than direct fluorescent assays
C. Electron excitation caused by radiant energy
D. Employs a radioactive label
A. Bioluminescence is a type of chemiluminescence in which the excitation energy is supplied by an enzymatic chemical reaction rather than by radiant energy, as in fluorescence and phosphorescence. Bioluminescence assays may employ such systems as NADH:FMN
oxidoreductase bacterial luciferase or adenosine triphosphatefirefly luciferase. Bioluminescence assays are nonradioactive, having sensitivity levels in the attomole (10^-18) to zeptomole (10^~21) ranges, which makes them more sensitive than direct fluorescence assays. Bioluminescence has been applied in the development of immunoassays
Nephelometry is based on the measurement of light that is
A. Absorbed by particles in suspension
B. Scattered by particles in suspension
C. Produced by fluorescence
D. Produced by excitation of ground-state atoms
B. Nephelometry is the measurement of the amount of light scattered by particles in suspension. The amount of light scattered depends on the size and shape of the particles and on the wavelength of the incident light. Ultraviolet light should not be used because it might produce some fluorescence, which would lead to erroneously high results.
Which of the following instruments is used in the clinical laboratory or in reference laboratories to detect beta and gamma emissions?
A. Fluorometer
B. Nephelometer
C. Scintillation counter
D. Spectrophotometer
C. Radionuclides are quantified by measuring the amount of energy that they emit. This can be in the form of alpha emission 42He²+, beta emission (electrons ejected from the nucleus of a radioisotope during radioactive decay), or gamma emission (electromagnetic radiation emitted during radioactive decay). Beta and gamma emissions can be detected by scintillation counters. The sensing element of a scintillation counter is a fluor, a substance capable of converting radiation energy to light energy. The light energy is converted to electrical energy and amplified by a photomultiplier tube. A fluor commonly employed in solid scintillation counters is a large crystal of sodium iodide containing a small amount of thallium as an activator; it is used for gamma counting. Beta emission is counted by liquid scintillation counters using fluors dissolved in organic solvents. Alpha emission has very low penetrating power
and is not measured in the clinical laboratory.
Although radioimmunoassay (RIA) is no longer
used for routine analyses and has been replaced by nonradioactive immunoassays, it is still used in a limited manner in some clinical reference laboratories and in research settings.
Which of the following best describes chemiluminescence?
A. Electron excitation caused by radiant energy
B. Enzymatic oxidation of a substrate produces light emission
C. Chemical energy excites electrons that emit light upon return to ground state
D. Employs a fluorescent label that produces light
C. Chemiluminescence is a type of luminescence where excitation does not require absorption of radiant energy. Chemiluminescence is the process where the chemical energy of a reaction produces excited atoms, and upon electron
return to ground state photons of light are emitted. Chemiluminescence has been applied in the development of immunoassays and has ultrasensitivity in the attomole (10~18) to zeptomole (10~21)
ranges.
In assaying an analyte with a single-beam atomic absoiption spectrophotometer, what is the instrument actually measuring?
A. Intensity of light emitted by the analyte on its return to the ground state
B. Intensity of light that the analyte absorbs from the hollow-cathode lamp
C. Intensity of light that the analyte absorbs from the flame
D. Intensity of the beam from the hollow cathode lamp after it has passed through the analyte-containing flame
D. Atomic absorption spectrophotometry (AAS) is based on the principle that atoms in a basic ground state are capable of absorbing energy in the form of light at a specific wavelength. In a single-beam AAS, the amount of light that the analyte absorbs from the hollow-cathode lamp is what we wish to know. However, what is actually measured is the intensity of the beam after it
has passed through the flame. This measurement is made with and without sample in the flame. In this way, the instrument calculates the amount of light absorbed because of the presence of the analyte in the flame. Because most samples usually have the analyte in the form of a compound or an ion, the analyte must first be converted to nonionized atoms. This is achieved by heating in a flame. About 99% of the atoms of analyte in the flame are in the ground state and, therefore, are capable of absorbing energy at the appropriate wavelength. Hence, light absorbed is essentially proportional to the concentration of the analyte. The light source in AAS is a hollow cathode lamp in which the cathode contains the element that is to be measured.
What is the function of the flame in atomic absorption spectroscopy?
A. Absorb the energy emitted from the metal analyte in returning to ground state
B. Supply the thermal energy needed to excite the metal analyte
C. Bring the metal analyte to its ground state
D. Supply the light that is absorbed by the metal analyte
C. The basis of AAS is the measurement of
light, at a specific wavelength, that is absorbed by an element whose atoms are in a ground state. The flame in AAS serves two functions—to accept the sample, thus serving as a cuvet, and to supply heat for converting the element, which is usually present in the sample in molecular form, into its atomic form at ground-state energy level. The hollow-cathode lamp supplies the emission line of light required for the analysis. The metal element of interest is coated on the cathode of the lamp. When the inert gas, either argon or neon, becomes ionized, it is drawn toward the cathode. The impact excites the metal element coated on the cathode, resulting in the emission of spectral lines specific for the element. This light emission is then absorbed by the metal element in the sample. A flameless AAS employs a carbon rod (graphite furnace), tantalum, or platinum to hold the sample in a chamber. The temperature is raised to vaporize the sample being analyzed. The atomized sample then absorbs the light energy from the hollow-cathode lamp. This
technique is more sensitive than the flame
method
Most atomic absorption spectrophotometers incorporate a beam chopper and a tuned amplifier. The purpose of these components is to avoid errors that would be caused by
A. Variations in flame temperature
B. Deterioration of the hollow-cathode lamp
C. Stray light from the hollow-cathode lamp
D. Measurement of light emitted by the analyte
D. A beam chopper is a device for interrupting a beam of light so that a pulsed beam is produced. In an atomic absorption spectrophotometer, if the light entering the flame from the hollow cathode lamp is pulsed, then the light leaving the flame will consist of unabsorbed pulsed light and impulsed light from the flame and from a
small amount of emission by excited atoms of
the analyte. The detector has an amplifier that is tuned to recognize and amplify only the pulsed signal. Thus errors caused by light from the flame and light emitted by the analyte are avoided. However, the beam chopper and tuned amplifier do not compensate for errors introduced by variations in flame temperature or deterioration of the hollow-cathode lamp. AAS
may be used to measure such analytes as lead, zinc, copper, aluminum, magnesium, calcium, and lithium
In potentiometry, which of the following is considered the standard electrode?
A. Hydrogen electrode
B. Calcium electrode
C. Potassium electrode
D. Copper electrode
A. A half-cell, also called an electrode, is composed of a single metallic conductor surrounded by a solution of electrolyte. An electrochemical cell consists of two half-cells. If two different kinds of half-cells are connected in such a way as to make a complete circuit, a current will flow
because of the potential difference between the two electrodes. The connection must be between the two metallic conductors and also between the two electrolyte solutions, usually by means of a salt bridge. In the analytical technique of potentiometry, a comparison is made between the voltage of one half-cell connected to another half-cell. It is customary that all half-cell potentials be compared to the potential generated by a standard electrode. The universally accepted standard half-cell with which all other half cells are compared is the standard hydrogen electrode, arbitrarily assigned a potential E° of 0.000 volt
In an electrolytic cell, which of the following is the half-cell where reduction takes place?
A. Anode
B. Cathode
C. Combination electrode
D. Electrode response
B. Oxidation involves the loss of electrons, and reduction the gain of electrons. In an electrolytic cell composed of two different half-cells—for example, zinc in zinc sulfate and copper in copper sulfate—electrons will flow from the anode to the cathode. Thus reduction takes place at the cathode, whereas oxidation occurs at the anode.
“Combination electrode” refers to the combining of indicator and reference electrodes into a single unit. “Electrode response” refers to the ability of an ion-selective electrode to respond to
a change in concentration of the ion being measured by exhibiting a change in potential.
Mercury covered by a layer of mercurous chloride in contact with saturated potassium chloride solution is a description of which of the following types of electrodes?
A. Sodium
B. Calomel
C. Calcium
D. Silver/silver chloride
B. In practical applications of potentiometry, it
is desirable to use one half-cell with a known
and constant potential that is not sensitive to the composition of the material to be analyzed. This is called the reference electrode. One type of reference electrode is the calomel electrode, which consists of mercury covered by a layer of mercurous chloride in contact with a saturated solution of potassium chloride. The other half-cell, called the indicator electrode, is selected on the basis of the change in its potential with change in the concentration of the analyte of interest. The silver-silver chloride electrode is a commonly used type of reference electrode. The sodium and calcium electrodes are types of ion selective electrodes
When a pH-sensitive glass electrode is not actively in use, in what type of solution should it be kept?
A. Tap water
B. Physiologic saline solution
C. The medium recommended by the manufacturer
D. A buffer solution of alkaline pH
C. For optimum performance, pH-sensitive glass electrodes that are not actively in use should be kept immersed in an aqueous medium. Because the exact composition of the pH-sensitive glass varies from one manufacturer to another, the glass electrode should be maintained in the medium recommended by the manufacturer.
Usual media are deionized water, dilute HC1, and buffer with a pH near the pH of the solution to be measured. The functioning of a glass electrode depends on the properties of the pH-sensitive glass. A typical glass electrode is made by sealing a thin piece of pH-sensitive glass at the end of a piece of glass tubing and filling the tube with a solution of hydrochloric acid saturated with silver chloride. A silver wire is immersed in the solution in the tube, with one end extending outside the tube for external connection. This is essentially a silver/silver chloride reference electrode sealed within the tube with the pH-sensitive glass tip. This pH-sensitive glass functions appropriately only when it is saturated with water. Then each surface of the glass develops a hydrated lattice, where exchange of alkaline metal ions in the lattice for hydrogen ions in the test solution can occur
When measuring K+ with an ion-selective electrode by means of a liquid ion-exchange membrane, what antibiotic will be incorporated into the membrane?
A. Monactin
B. Nonactin
C. Streptomycin
D. Valinomycin
D. The ion-exchange electrode is a type of
potentiometric, ion-selective electrode that consists of a liquid ion-exchange membrane that is made of an inert solvent and an ion-selective neutral earner material. A collodion membrane may be used to separate the membrane solution from the sample solution being analyzed. Because of its ability to bind K+, the antibiotic valinomycin is used as the neutral carrier for the K+-selective membrane. The antibiotics non-actin and monactin are used in combination as the neutral carrier for the NH+4 selective membrane. A special formulation is used to make a selective glass membrane for the measurement of sodium
Which of the following is false about ion-selective electrode analysis of sodium?
A. Uses a glass membrane
B. Errors occur from protein buildup on
the membrane.
C. Membrane coated with valinomycin
D. Principle based on potentiometry
C. Ion-selective electrodes for the measurement of sodium are glass membrane electrodes with selective capability. They are constructed from glass that consists of silicon dioxide, sodium oxide, and aluminum oxide. This type of electrode is based on the principle of potentiometry. Measurement errors may occur from protein buildup on the membrane surface. Potassium is measured using an ion-exchange electrode where the liquid ion-exchange membrane consists of valinomycin as the ion-selective carrier.
What are the principles of operation for a chloride analyzer that generates silver ions as part of its reaction mechanism?
A. Potentiometry and amperometry
B. Amperometry and polarography
C. Coulometry and potentiometry
D. Amperometry and coulometry
D. A chloride coulometer employs a coulometric system based on Faraday’s law, which states that in an electrochemical system, the number of equivalent weights of a reactant oxidized or reduced is directly proportional to the quantity of electricity used in the reaction. The quantity of electricity is measured in coulombs.
The coulomb is the unit of electrical quantity;
1 coulomb of electricity flowing per minute constitutes a current of 1 ampere. Thus, if the current is constant, the number of equivalent weights of reactant oxidized or reduced depends only on the duration of the current. In the chloride coulometer, the electrochemical reaction is the generation
of Ag+ ions by the passage of a direct current
across a pair of silver electrodes immersed in a conducting solution containing the sample to be assayed for chloride. As the Ag+ ions are generated, they are immediately removed from solution by combining with chloride to form insoluble silver chloride. When all the chloride is precipitated, further generation of Ag+ ions causes an increase in conductivity of the solution. Thus the instrument provides an electrometric titration, in which the titrant is Ag+ ions and the endpoint of the titration is indicated by the increase in conductivity of the solution. Amperometry is used to measure the increase in conductivity. The amperometric circuit includes a second pair of silver electrodes that are immersed
in the solution. They are provided with a small, steady, and constant voltage. The appearance of free Ag+ ions in the solution generates a sharp increase in conductivity, which, in turn, causes a sudden rise in the current between the electrodes in the amperometric circuit. This increase in current activates a relay that stops the further generation of Ag+ ions and also stops an automatic timer placed in the circuit to measure the total duration of current in the coulometric circuit. Although this system is no longer used for routine analysis of serum, it is still employed for sweat chloride analysis.
