5.1 Chemistry - Instrumentation Part 1 (1-40)

Which wavelength would be absorbed strongly by a red-colored solution? A. 450 nm B. 585 nm C. 600 nm D. 650 nm

A. 450 nm A A solution transmits light corresponding in wavelength to its color, and usually absorbs light of wavelengths complementary to its color. A red solution transmits light of 600-650 nm and strongly absorbs 400-500 nm light.

A flameless atomic absorption spectrophotometer dehydrates and atomizes a sample using: A. A graphite capillary furnace B. An electron gun C. A thermoelectric semiconductor D. A thermospray platform

A. A graphite capillary furnace A Flameless atomic absorption uses a hollow tube of graphite with quartz ends. The tube is heated in stages by an electric current to successively dry, ash, and atomize the sample. During the ash and atomization steps, argon is injected into the tube to distribute the atoms. The furnace is more sensitive than a flame atomizer and more efficient in atomizing thermostable salts. However, it is prone to greater matrix interference and is slower than the flame atomizer because it must cool down before introduction of the next sample.

Which of the electrodes below is a currentproducing (amperometric) rather than a voltage-producing (potentiometric) electrode? A. Clark electrode B. Severinghaus electrode C. pH electrode D. Ionized calcium electrode

A. Clark electrode A The Clark electrode is composed of two half cells that generate current, not voltage. It is used to measure partial pressure of oxygen (PO2), and is based upon an amperometric method called polarography. When -0.8 V is applied to the cathode, O2 is reduced, causing current to flow. Current is proportional to the PO2 of the sample.

The reference potential of a silver-silver chloride electrode is determined by the: A. Concentration of the potassium chloride filling solution B. Surface area of the electrode C. Activity of total anion in the paste covering the electrode D. The concentration of silver in the paste covering the electrode

A. Concentration of the potassium chloride filling solution A The activity of any solid or ion in a saturated solution is unity. For a silver electrode covered with silver chloride paste, the Nernst equation is E = E° - RT/nF x 2.3 log10 [Ag° × Cl-]/[AgCl]. Because silver and silver chloride have an activity of 1.0, and all components except chloride are constants, the potential of the reference electrode is determined by the chloride concentration of the filling solution. E = Eo - RT/nF × 2.3 log10[Cl-] = E° - 59.2 mV × log[Cl-] (at room temperature)

Light scattering when the wavelength is greater than 10 times the particle diameter is described by: A. Rayleigh's law B. Te Beer-Lambert law C. Mie's law D. Te Rayleigh-Debye law

A. Rayleigh's law A Rayleigh's law states that when the incident wavelength is much longer than the particle diameter, there is maximum backscatter and minimum right-angle scatter. The Rayleigh-Debye law predicts maximum right-angle scatter when wavelength and particle diameter approach equality. In nephelometry, the relationship between wavelength and diameter determines the angle at which the detector is located

Which of the following components is not needed in a chemiluminescent immunoassay analyzer? A. Source lamp B. Monochromator C. Photodetector D. Wash station

A. Source lamp A Chemiluminescence is the production of light following a chemical reaction. Immunoassays based upon chemiluminescence generate light when the chemiluminescent molecule becomes excited; therefore, a light source is not used. In immunoassay platforms, chemiluminescent molecules such as acridinium can be used to label antigens or antibodies. Alternatively, chemiluminescent substrates such as luminol or dioxetane phosphate may be used. Light will be emitted when the enzyme-labeled molecule reacts with the substrate. In such assays, free and bound antigen separation is required and is usually accomplished using paramagnetic particles bound to either antibody or reagent antigen

Bichromatic measurement of absorbance can correct for interfering substances if: A. The contribution of the interferent to absorbance is the same at both wavelengths B. Both wavelengths pass through the sample simultaneously C. The side band is a harmonic of the primary wavelength D. The chromogen has the same absorbance at both wavelengths

A. The contribution of the interferent to absorbance is the same at both wavelengths A In bichromatic photometry, the absorbance of sample is measured at two different wavelengths. The primary wavelength is at or near the absorbance maximum. An interfering substance having the same absorbance at both primary and secondary (side band) wavelengths does not affect the absorbance difference (Ad).

The half-band width of a monochromator is defined by: A. The range of wavelengths passed at 50% maximum transmittance B. One-half the lowest wavelength of optical purity C. The wavelength of peak transmittance D. One-half the wavelength of peak absorbance

A. The range of wavelengths passed at 50% maximum transmittance A Half-band width is a measure of bandpass made using a solution or filter having a narrow natural bandpass (transmittance peak). The wavelength giving maximum transmittance is set to 100%T (or 0 A). Then, the wavelength dial is adjusted downward, until a readout of 50%T (0.301 A) is obtained. Next, the wavelength is adjusted upward until 50%T is obtained. The wavelength difference is the half-band width. The narrower the half-band width, the better the photometric resolution of the instrument.

A solution that has a transmittance of 1.0 %T would have an absorbance of: A. 1.0 B. 2.0 C. 1% D. 99%

B. 2.0 B A = 2.0 - log %T A = 2.0 - log 1.0 The log of 1.0 = 0 A = 2.0

Interference in atomic absorption spectrophotometry caused by differences in viscosity is called: A. Absorption interference B. Matrix effect C. Ionization interference D. Quenching

B. Matrix effect B Significant differences in aspiration and atomization result when the matrix of sample and unknowns differ. Differences in viscosity and protein content are major causes of matrix error. Matrix effects can be reduced by using protein-based calibrators and diluting both standards and samples prior to assay.

The term RT/nF in the Nernst equation defines the: A. Potential at the ion-selective membrane B. Slope of the electrode C. Decomposition potential D. Isopotential point of the electrode

B. Slope of the electrode B In the term RT/nF, R = the molar gas constant, T = temperature in degrees Kelvin, F = Faraday's constant, and n = the number of electrons donated per atom of reductant. The slope depends upon the temperature of the solution and the valence of the reductant. At room temperature, the slope is 59.2 mV for a univalent ion and 29.6 mV for a divalent ion.

Which instrument requires a highly regulated DC power supply? A. A spectrophotometer with a barrier layer cell B. A colorimeter with multilayer interference filters C. A spectrophotometer with a photomultiplier tube D. A densitometer with a photodiode detector

C. A spectrophotometer with a photomultiplier tube C When AC voltage regulators are used to isolate source lamp power, light output fluctuates as the voltage changes. Because this occurs at 60 Hz, it is not detected by eyesight or slow-responding detectors. Photomultiplier tubes are sensitive enough to respond to the AC frequency and require a DC-regulated power supply.

Which component is required in a spectrophotometer in order to produce a spectral absorbance curve? A. Multiple monochromators B. A reference optical beam C. Photodiode array D. Laser light source

C. Photodiode array C There are two ways to perform spectral scanning for compound identification. One is to use a stepping motor that continuously turns the monochromator so that the wavelength aligned with the exit slit changes at a constant rate. A more practical method is to use a diode array detector. This consists of a chip embedded with as many as several hundred photodiodes. Each photodiode is aligned with a narrow part of the spectrum produced by a diffraction grating, and produces current proportional to the intensity of the band of light striking it (usually 1-2 nm in range). The diode signals are processed by a computer to create a spectral absorbance or transmittance curve.

When measuring lead in whole blood using atomic absorption spectrophotometry, what reagent is required to obtain the needed sensitivity and precision? A. Lanthanum B. Lithium C. Triton X-100 D. Chloride

C. Triton X-100 C A graphite furnace is preferred over a flame for measuring lead because it is sufficiently sensitive to detect levels below 5 μg/dL, the cutoff needed for lead screening of children. The matrix modifier consists of Triton X -100, ammonium phosphate and nitric acid. This allows for release of Pb from the RBCs, and solubilization of cell stroma. The matrix modifier also prevents loss of Pb caused by formation of lead halides and promotes interaction between Pb and the tube wall, preventing its loss during the ashing cycle.

Which monochromator specification is required in order to measure the true absorbance of a compound having a natural absorption bandwidth of 30 nm? A. 50-nm bandpass B. 25-nm bandpass C. 15-nm bandpass D. 5-nm bandpass

D. 5-nm bandpass D Bandpass refers to the range of wavelengths passing through the sample. The narrower the bandpass, the greater the photometric resolution. Bandpass can be made smaller by reducing the width of the exit slit. Accurate absorbance measurements require a bandpass less than one-fifth the natural bandpass of the chromophore

In absorption spectrophotometry: A. Absorbance is directly proportional to transmittance B. Percent transmittance is directly proportional to concentration C. Percent transmittance is directly proportional to the light path length D. Absorbance is directly proportional to concentration

D. Absorbance is directly proportional to concentration D Beer's law states that A = a × b × c, where a is the absorptivity coefficient (a constant), b is the path length, and c is concentration. Absorbance is directly proportional to both b and c. Doubling the path length results in incident light contacting twice the number of molecules in solution. This causes absorbance to double, the same effect as doubling the concentration of molecules.

Which formula correctly describes the relationship between absorbance and %T ? A. A = 2 - log %T B. A = log 1/T C. A = -log T D. All of these options

D. All of these options D Absorbance is proportional to the inverse log of transmittance. A = -log T = log 1/T Multiplying the numerator and denominator by 100 gives: A = log (100/100 X T) 100 X T = %T, substituting %T for 100 X T gives: A = log 100/%T A = log 100 - log %T A = 2.0 - log %T For example, if %T = 10.0, then: A = 2.0 - log 10.0 log 10.0 = 1.0 A = 2.0-1.0 = 1.0

A linearity study is performed on a visible spectrophotometer at 650 nm and the following absorbance readings are obtained: Concentration of Standard 10.0 mg/dL 20.0 mg/dL 30.0 mg/dL 40.0 mg/dL 50.0 mg/dL Absorbance 0.20 0.41 0.62 0.79 0.92 The study was repeated using freshly prepared standards and reagents, but results were identical to those shown. What is the most likely cause of these results? A. Wrong wavelength used B. Insufficient chromophore concentration C. Matrix interference D. Stray light

D. Stray light D Stray light is the most common cause of loss of linearity at high-analyte concentrations. Light transmitted through the cuvette is lowest when absorption is highest. Therefore, stray light is a greater percentage of the detector response when sample concentration is high. Stray light is usually most significant when measurements are made at the extremes of the visible spectrum because lamp output and detector response are low.

Which statement regarding nephelometry is true? A. Nephelometry is less sensitive than absorption spectrophotometry B. Nephelometry follows Beer's law C. The optical design is identical to a turbidimeter except that a HeNe laser light source is used D. The detector response is directly proportional to concentration

D. The detector response is directly proportional to concentration D In nephelometry, the detector output is proportional to concentration (as opposed to turbidimetry where the detector is behind the cuvette). The detector(s) is (are) usually placed at an angle between 25° and 90° to the incident light, depending upon the application. Nephelometers, like fluorometers, are calibrated to read zero with the light path blocked, and sensitivity can be increased up to 1,000 times by amplification of the detector output or increasing the photomultiplier tube dynode voltage

The purpose of the nebulizer in an atomic absorption spectrophotometer that uses a flame is to: A. Convert ions to atoms B. Cause ejection of an outer shell electron C. Reduce evaporation of the sample D. Burn off organic impurities

A. Convert ions to atoms A The atomizer of the atomic absorption spectrophotometer consists of either a nebulizer and flame or a graphite furnace. The nebulizer disperses the sample into a fine aerosol, distributing it evenly into the flame. Heat from the flame is used to evaporate water and break the ionic bonds of salts, forming ground state atoms. The flame also excites a small percentage of the atoms, which release a characteristic emission line.

SITUATION: A technologist is performing an enzyme assay at 340 nm using a visible-range spectrophotometer. After setting the wavelength and adjusting the readout to zero %T with the light path blocked, a cuvette with deionized water is inserted. With the light path fully open and the 100%T control at maximum, the instrument readout will not rise above 90%T. What is the most appropriate first course of action? A. Replace the source lamp B. Insert a wider cuvette into the light path C. Measure the voltage across the lamp terminals D. Replace the instrument fuse

A. Replace the source lamp A Visible spectrophotometers are usually supplied with a tungsten or quartz halogen source lamp. Tungsten lamps produce a continuous range of wavelengths from about 320-2,000 nm. Output increases as wavelength becomes longer peaking at around 1,000 nm, and is poor below 400 nm. As the lamp envelope darkens with age, the amount of light reaching the photodetector at 340 nm becomes insufficient to set the blank reading to 100%T. Quartz halogen lamps produce light from 300 nm through the infrared region. Deuterium or hydrogen lamps produce ultraviolet-rich spectra optimal for ultraviolet (UV) work. Mercury vapor lamps produce a discontinuous spectrum that includes a high output at around 365 nm that is useful for fluorescent applications. Xenon lamps generate a continuous spectrum of fairly uniform intensity from 300-2,000 nm, making them useful for both visible and UV applications.

The reagent blank corrects for absorbance caused by: A. The color of reagents B. Sample turbidity C. Bilirubin and hemolysis D. All of these options

A. The color of reagents A When a spectrophotometer is set to 100%T with the reagent blank instead of water, the absorbance of reagents is automatically subtracted from each unknown reading. The reagent blank does not correct for absorbance caused by interfering chromogens in the sample such as bilirubin, hemolysis, or turbidity.

A green-colored solution would show highest transmittance at: A. 475 nm B. 525 nm C. 585 nm D. 620 nm

B. 525 nm B Green light consists of wavelengths from 500-550 nm. A green-colored solution with a transmittance maximum of 525 nm and a 50-nm bandpass transmits light of 525 nm and absorbs light below 475 nm and above 575 nm. A solution that is green would be quantitated using a wavelength that it absorbs strongly, such as 450 nm.

The response of a sodium electrode to a 10-fold increase in sodium concentration should be: A. A 10-fold drop in potential B. An increase in potential of approximately 60 mV C. An increase in potential of approximately 10 mV D. A decrease in potential of approximately 10 mV

B. An increase in potential of approximately 60 mV B The Nernst equation predicts an increase of approximately 60 mV per 10-fold increase in sodium activity. For sodium: E = E° + RT/nF × 2.3 log10[Na+] RT/nF × 2.3 = 60 mV at 37°C. Therefore: E = E° + 60 mV × log10[Na+]. If sodium concentration is 10 mmol/L, then: E = E° + 60 mV × log10[10] = E° + 60 mV. If sodium concentration increases from 10 mmol/L to 100 mmol/L, then: E = E° + 60 mV × log10[100] = E° + 60 mV × 2 = E° + 120 mV.

Ion selective analyzers using undiluted samples have what advantage over analyzers that use a diluted sample? A. Can measure over a wider range of concentration B. Are not subject to pseudohyponatremia caused by high lipids C. Do not require temperature equilibration D. Require less maintenance

B. Are not subject to pseudohyponatremia caused by high lipids B Ion-selective analyzers measure the electrolyte dissolved in the fluid phase of the sample in millimoles per liter of plasma water. When undiluted blood is assayed, the measurement is independent of colloids such as protein and lipid. Hyperlipemic samples cause falsely low sodium measurements when assayed by flame photometry and ion-selective analyzers requiring dilution because lipids displace plasma water containing the electrolytes. One drawback to undiluted or direct measuring systems is that the electrodes require more frequent deproteinization and usually have a shorter duty cycle.

Why do many optical systems in chemistry analyzers utilize a reference light path? A. To increase the sensitivity of the measurement B. To minimize error caused by source lamp fluctuation C. To obviate the need for wavelength adjustment D. To reduce stray light effects

B. To minimize error caused by source lamp fluctuation B A reference beam is used to produce an electrical signal at the detector to which the measurement of light absorption by the sample is compared. This safeguards against measurement errors caused power fluctuations that change the source lamp intensity. Although reference beams increase the accuracy of measurements, they do so at the expense of optical sensitivity since some of the incident light must be used to produce the reference beam

Which statement regarding reflectometry is true? A. The relation between reflectance density and concentration is linear B. Single-point calibration can be used to determine concentration C. 100% reflectance is set with an opaque film called a white reference D. The diode array is the photodetector of choice

C. 100% reflectance is set with an opaque film called a white reference C Reflectometry does not follow Beer's law, but the relationship between concentration and reflectance can be described by a logistic formula or algorithm that can be solved for concentration. For example, K/S = (1 - R) 2/2R, where K = Kubelka-Munk absorptivity constant, S = scattering coefficient, R = reflectance density. K/S is proportional to concentration. The white reference is analogous to the 100%T setting in spectrophotometry and serves as a reference signal. Dr = log R0/R1, where Dr is the reflectance density, R0 is the white reference signal, and R1 is the photodetector signal for the test sample.

Which condition is a common cause of stray light? A. Unstable source lamp voltage B. Improper wavelength calibration C. Dispersion from second-order spectra D. Misaligned source lamp

C. Dispersion from second-order spectra C Stray light is caused by the presence of any light other than the wavelength of measurement reaching the detector. It is most often caused by second-order spectra, deteriorated optics, light dispersed by a darkened lamp envelope, and extraneous room light.

Which instrument requires a primary and secondary monochromator? A. Spectrophotometer B. Atomic absorption spectrophotometer C. Fluorometer D. Nephelometer

C. Fluorometer C A fluorometer uses a primary monochromator to isolate the wavelength for excitation, and a secondary monochromator to isolate the wavelength emitted by the fluorochrome.

Select the equation describing the potential that develops at the surface of an ion-selective electrode. A. van Deemter equation B. van Slyke equation C. Nernst equation D. Henderson-Hasselbalch equation

C. Nernst equation C The van Deemter equation describes the relation between the velocity of mobile phase to column efficiency in gas chromatography. The Henderson-Hasselbalch equation is used to determine the pH of a solution containing a weak acid and its salt. van Slyke developed an apparatus to measure CO2 and O2 content using a manometer.

Which type of filter is best for measuring stray light? A. Wratten B. Didymium C. Sharp cutoff D. Neutral density

C. Sharp cutoff C Sharp cutoff filters transmit almost all incident light until the cutoff wavelength is reached. At that point, they cease to transmit light. Because they give an "all or none effect," only stray light reaches the detector when the selected wavelength is beyond the cutoff.

A plasma sample is hemolyzed and turbid. What is required to perform a sample blank in order to correct the measurement for the intrinsic absorbance of the sample when performing a spectrophotometric assay? A. Substitute deionized water for the sample B. Dilute the sample 1:2 with a standard of known concentration C. Substitute saline for the reagent D. Use a larger volume of the sample

C. Substitute saline for the reagent C A sample blank is used to subtract the intrinsic absorbance of the sample usually caused by hemolysis, icterus, turbidity, or drug interference. On automated analyzers, this is accomplished by measuring the absorbance after the addition of sample and a first reagent, usually a diluent. For tests using a single reagent, sample blanking can be done prior to the incubation phase before any color develops. Substituting deionized water for sample is done to subtract the absorbance of the reagent (reagent blanking). Diluting the sample with a standard (standard addition) may be done when the absorbance is below the minimum detection limit for the assay. Using a larger volume of sample will make the interference worse.

Which of the following statements about fluorometry is accurate? A. Fluorometry is less sensitive than spectrophotometry B. Fluorometry is less specific than spectrophotometry C. Unsaturated cyclic molecules are often fluorescent D. Fluorescence is directly proportional to temperature

C. Unsaturated cyclic molecules are often fluorescent C Increasing temperature results in more random collision between molecules by increasing their motion. This causes energy to be dissipated as heat instead of fluorescence. Temperature is inversely proportional to fluorescence. Fluorescence is more sensitive than spectrophotometry because the detector signal can be amplified when dilute solutions are measured. It is also more specific than spectrophotometry because both the excitation and emission wavelengths are characteristics of the compound being measured.

The ion-selective membrane used to measure potassium is made of: A. High-borosilicate glass membrane B. Polyvinyl chloride dioctylphenyl phosphonate ion exchanger C. Valinomycin gel D. Calomel

C. Valinomycin gel C Valinomycin is an antibiotic with a highly selective reversible-binding affinity for potassium ions. Sodium electrodes are usually composed of a glass membrane with a high content of aluminum silicate. Calcium and lithium ion-selective electrodes are made from organic liquid ion exchangers called neutral carrier ionophores Calomel is made of mercury covered with a paste of mercurous chloride (Hg°/Hg2Cl2) and is used as a reference electrode for pH.

Which type of monochromator produces the purest monochromatic light in the UV range? A. A diffraction grating and a fixed exit slit B. A sharp cutoff filter and a variable exit slit C. Interference filters and a variable exit slit D. A prism and a variable exit slit

D. A prism and a variable exit slit D Diffraction gratings and prisms both produce a continuous range of wavelengths. A diffraction grating produces a uniform separation of wavelengths. A prism produces much better separation of high-frequency light because refraction is greater for higher-energy wavelengths. Instruments using a prism and a variable exit slit can produce UV light of a very narrow bandpass. The adjustable slit is required in order to allow sufficient light to reach the detector to set 100%T.

All of the following are required when measuring magnesium by atomic absorption spectrophotometry except: A. A hollow cathode lamp with a magnesium cathode B. A chopper to prevent optical interference from magnesium emission C. A monochromator to isolate the magnesium emission line at 285 nm D. A 285-nm reference beam to correct for background absorption

D. A 285-nm reference beam to correct for background absorption D Atomic absorption requires a lamp with a cathode made from the metal to be assayed. The lamp emits the line spectrum of the metal, providing the wavelength that the atoms can absorb. The chopper pulses the source light, allowing it to be discriminated from light emitted by excited atoms. A monochromator eliminates light emitted by the ideal gas in the lamp. Deuterium (wide bandpass light) or Zeeman correction (splitting the incident light into side bands by a magnetic field) may be used to correct for background absorption.

Which of the following materials is best suited for verifying the wavelength calibration of a spectrophotometer? A. Neutral density filters B. Potassium dichromate solutions traceable to the National Bureau of Standards reference C. Wratten filters D. Holmium oxide glass

D. Holmium oxide glass D Wavelength accuracy is verified by determining the wavelength reading that gives the highest absorbance (or transmittance) when a substance with a narrow natural bandpass (sharp absorbance or transmittance peak) is scanned. For example, didymium glass has a sharp absorbance peak at 585 nm. Therefore, an instrument should give its highest absorbance reading when the wavelength dial is set at 585 nm. Holmium oxide produces a very narrow absorbance peak at 361 nm; likewise, the hydrogen lamp of a UV spectrophotometer produces a 656-nm emission line that can be used to verify wavelength. Neutral density filters and dichromate solutions are used to verify absorbance accuracy or linearity. A Wratten filter is a widebandpass filter made by placing a thin layer of colored gelatin between two glass plates and is unsuitable for spectrophotometric calibration

When measuring calcium by atomic absorption spectrophotometry, which is required? A. An organic extraction reagent to deconjugate calcium from protein B. An internal standard C. A magnesium chelator D. Lanthanum oxide to chelate phosphates

D. Lanthanum oxide to chelate phosphates D An acidic diluent such as hydrochloric acid (HCl) will displace calcium bound to albumin. However, calcium forms a thermostable bond with phosphate that causes chemical interference in atomic absorption. Lanthanum displaces calcium, forming lanthanum phosphate, and eliminates interference from phosphates. Unlike in some colorimetric methods for calcium (e.g., o-cresolphthalein complexone), magnesium does not interfere because it does not absorb the 422.7 nm emission line from the calcium-hollow cathode lamp.

Which photodetector is most sensitive to low levels of light? A. Barrier layer cell B. Photodiode C. Diode array D. Photomultiplier tube

D. Photomultiplier tube D The photomultiplier tube uses dynodes of increasing voltage to amplify the current produced by the photosensitive cathode. It is 10,000 times as sensitive as a barrier layer cell, which has no amplification. A photomultiplier tube requires a DC-regulated lamp because it responds to light fluctuations caused by the AC cycle.

Which substance is used to generate the light signal in electrochemiluminescence? A. Acridinium B. Luminol C. Dioxetane phosphate D. Ruthenium

D. Ruthenium D All of these substances are chemiluminescent. Dioxetane phosphate is excited by alkaline phosphatase. Acridinium and luminol are excited by hydrogen peroxide. In electrochemiluminesence, ruthenium is used to label antibody or antigen. Antigen-antibody complexes containing the ruthenium label are bound to paramagnetic particles via a strepavidin-biotin reaction. The paramagnetic particles are attracted to an electrode surface. The flowcell is washed with a solution containing tripropylamine (TPA) to remove unbound ruthenium label. At the electrode surface, the TPA is oxidized and the electrons excite the ruthenium, causing production of 620-nm light