Medical Laboratory Science Review Harr 5.1 Chemistry - Instrumentation Part 2 (41-80)

In real-time PCR, what value is needed in order to determine the threshold? A. Background signal B. Melt temperature C. Maximum fluorescence D. Treshold cycle

A. Background signal A In real-time PCR, the fluorescence of the reporter probe is proportional to the concentration of PCR products. For quantitation of PCR products, a well factor and background fluorescence must be determined. Well-factor values are analogous to cuvette blanks. They are used to correct the measurements from each well so that the same concentration of fluorescent dye gives the same signal intensity regardless of the well. The threshold is the lowest signal that indicates the presence of product. It can be calculated manually from a real-time amplification curve by finding the average standard deviation of the fluorescent signal (RFU) from cycles 2-10. This is multiplied by 10 to give the threshold value in RFUs.

In gas chromatography, the elution order of volatiles is usually based upon the: A. Boiling point B. Molecular size C. Carbon content D. Polarity

A. Boiling point A The order of elution is dependent upon the velocity of the analyte. Usually, the lower the boiling point of the compound, the greater its velocity or solubility in carrier gas.

Which of the following compounds can interfere with the coulometric chloride assay? A. Bromide B. Ascorbate C. Acetoacetate D. Nitrate

A. Bromide A Chloride assays based upon either coulometric or chemical titration are subject to positive interference from other anions and electronegative radicals that may be titrated instead of chloride ions. These include other halogens such as bromide, cyanide, and cysteine.

What component is used in a GC-MS but not used in an LC-MS? A. Electron source B. Mass filter C. Detector D. Vacuum

A. Electron source A The mass spectrometer requires a sample that is suspended in a gas phase, and therefore, the sample from a GC can be directly injected into the mass spectrometer. While chemical ionization of the sample is possible, most GC-MS instruments utilize electron ionization. Electrons are produced by applying 70 electron volts to a filament of tungsten or rhenium under vacuum. The electrons collide with the neutral molecules coming from the GC, splitting them into fragments. The array of fragments is a unique identifier of each molecule

What process is most often used in LC-MS to introduce the sample into the mass filter? A. Electrospray ionization B. Chemical ionization C. Electron impact ionization D. Fast atom bombardment

A. Electrospray ionization A HPLC instruments use solvent rather than gas to separate molecules. The sample is converted into a gaseous state by electrospray ionization before it enters the mass filter. Electrospray ionization uses a small-bore tube that forms a 1-4 μ nozzle at the mass filter inlet and which is charged by several kilovolts. The sample enters the tube along with inert drying gas. The tube is heated to help evaporate solvent, but unlike electron impact used in GC-MS, the ionizer is not under vacuum. When a droplet of the sample reaches the nozzle, it becomes highly charged. The size of the droplet is decreased owing to evaporation. This causes the charge density to become excessive, and the droplets break apart. The tiny charged droplets repel each other and break apart again, forming a plume. These particles are drawn into the mass filter by "ion optics" (a system of repeller plates, counter electrode, and magnets). ESI does not result in extensive fragmentation, producing mostly the parent or "molecular" ion, a process called soft ionization.

In addition to velocity, what variable is also needed to calculate the relative centrifugal force (g force) of a centrifuge? A. Head radius B. Angular velocity coefficient C. Diameter of the centrifuge tube D. Ambient temperature in degrees Centigrade

A. Head radius A The relative centrifugal force (number times the force of gravity) is proportional to the square of the rotor speed in revolutions per minute and the radius in centimeters of the head (distance from the shaft to the end of the tube). RCF = s2 x r x 1.118 x 10-5 where s is the speed in RPM, r is the radius in cM, and 1.118 x 10-5 is a conversion constant.

All of the following compounds contribute to the osmolality of plasma except: A. Lipids B. Creatinine C. Drug metabolites D. Glucose

A. Lipids A Osmolality is the concentration (in moles) of dissolved solute per kilogram solvent. Proteins and lipids are not in solution, and do not contribute to osmolality. The nonionized solutes such as glucose and urea contribute 1 osmole per mole per kilogram water, whereas dissociated salts contribute 1 osmole per mole of each dissociated ion or radical.

The term reverse phase is used in HPLC to indicate that the mobile phase is: A. More polar than the stationary phase B. Liquid and the stationary phase is solid C. Organic and the stationary phase is aqueous D. A stronger solvent than the stationary phase

A. More polar than the stationary phase A In reverse-phase HPLC, the separation takes place using a nonpolar sorbent (stationary phase) such as octadecylsilane (C18). Solutes that are nonpolar are retained longer than polar solutes. Most clinical separations of drugs, hormones, and metabolites use reverse phase because aqueous mobile phases are far less toxic and flammable.

Which component is needed for a thermal cycler to amplify DNA? A. Programmable heating and cooling unit B. Vacuum chamber with zero head space C. Sealed airtight constant-temperature chamber D. Temperature-controlled ionization chamber

A. Programmable heating and cooling unit A The polymerase chain reaction for DNA amplification consists of three phases. Denaturation requires a temperature of 90°C 94°C and separates the double-stranded DNA. Annealing requires a temperature between 40°C-65°C and allows the primers to bind to the target base sequence. Extension requires a temperature of 72°C and allows the heat-stable polymerase to add complementary bases to the primer in the 5' to 3' direction. A cycle consists of each temperature stage for a specific number of minutes and most procedures require 30 or more cycles to generate a detectable quantity of target DNA. Rapid heating and cooling is usually achieved using a thermoelectric block that is cooled by forced air flow.

Which measurement principle is employed in a vapor pressure osmometer? A. Seebeck B. Peltier C. Hayden D. Darlington

A. Seebeck A The Seebeck effect refers to the increase in voltage across the two junctions of a thermocouple caused by a difference in the temperature at the junctions. Increasing osmolality lowers the dew point of a sample. When sample is cooled to its dew point, the voltage change across the thermocouple is directly proportional to osmolality.

What component of a freezing point osmometer measures the sample temperature? A. Thermistor B. Thermocouple C. Capacitor D. Electrode

A. Thermistor A A thermistor is a temperature sensitive resistor. The resistance to current flow increases as temperature falls. The temperature at which a solution freezes can be determined by measuring the resistance of the thermistor. Resistance is directly proportional to the osmolality of the sample.

What is the purpose of an internal standard in HPLC and GC methods? A. To compensate for variation in extraction and injection B. To correct for background absorbance C. To compensate for changes in flow rate D. To correct for coelution of solutes

A. To compensate for variation in extraction and injection A Internal standards should have the same affinity as the analyte for the extraction reagents. Dividing peak height (or area) of all samples (standards and unknowns) by the peak height (or area) of the internal standard reduces error caused by variation in extraction recovery and injection volume.

Which reagent is used in thin-layer chromatography (TLC) to extract cocaine metabolites from urine? A. Acid and sodium chloride B. Alkali and organic solvent C. Chloroform and sodium acetate D. Neutral solution of ethyl acetate

B. Alkali and organic solvent B Alkaline drugs such as cocaine, amphetamine, and morphine are extracted at alkaline pH. Ideally, the pH for extracting alkaline drugs into an organic solvent should be 2 pH units greater than the negative log of dissociation constant (pKa) of the drug. More than 90% of the drug will be nonionized and will extract in ethyl acetate or another organic solvent.

Persistent noise from an ion selective electrode is most often caused by: A. Contamination of sample B. Blocked junction at the salt bridge C. Overrange from high concentration D. Improper calibration

B. Blocked junction at the salt bridge B Electrode noise most often results from an unstable junction potential. Most reference electrodes contain a high concentration of KCl internal solution used to produce the reference potential. This forms a salt bridge with the measuring half cell by contacting sample, but is kept from equilibrating via a barrier called a junction. When this junction becomes blocked by salt crystals, the reference potential will be unstable, resulting in fluctuation in the analyzer readout.

The fragments typically produced and analyzed in methods employing mass spectroscopy are typically: A. Of low molecular size ranging from 10-100 daltons B. Cations caused by electron loss or proton attachment C. Anions caused by bombarding the molecule with an electron source D. Neutral species formed after excited molecules form a stable resonance structure

B. Cations caused by electron loss or proton attachment B In almost all MS applications, cations of the molecule are measured. Cations can be formed by various methods, the most common of which is electron bombardment (electron ionization). The energy transferred to the molecule causes ejection of an outer shell electron. MS can analyze sizes from trace metals through macromolecules. Proteins are measured following conversion to cations by ionization procedures such as matrix-assisted laser desorption ionization (MALDI) in which energy from a nitrogen laser causes transfer of a proton from the matrix (an acid) to the protein.

Which of the following statements accurately characterizes the coulometric titration of chloride? A. The indicator electrodes generate voltage B. Constant current must be present across the generator electrodes C. Silver ions are formed at the generator cathode D. Chloride concentration is inversely proportional to titration time

B. Constant current must be present across the generator electrodes B The Cotlove chloridometer is based upon the principle of coulometric titration with amperometric detection. Charge in the form of silver ions is generated by oxidation of silver wire at the generator anode. Silver ions react with chloride ions, forming insoluble silver chloride (AgCl). When all of the chloride is titrated, free silver ions are detected by reduction back to elemental silver, which causes an increase in current across the indicator electrodes (a pair of silver electrodes with a voltage difference of about 1.0 V DC). Charge or titration time is directly proportional to chloride concentration as long as the rate of oxidation remains constant at the generator anode.

Which of the following situations is likely to cause an error when weighing with an electronic analytical balance? A. Failure to keep the knife edge clean B. Failure to close the doors of the balance before reading the weight C. Oxidation on the surface of the substitution weights D. Using the balance without allowing it to warm up for at least 10 minutes

B. Failure to close the doors of the balance before reading the weight B Electronic balances do not use substitution weights or knife edges to balance the weight on the pan. Instead, they measure the displacement of the pan by the weight on it using electromagnetic force to return it to its reference position. Regardless of the type of balance used, all need to be located on a firm weighing table free of vibration. Doors must be closed to prevent air currents from influencing the weighing, and the pan and platform must be clean and free of dust and chemical residue

The most commonly used detector for clinical gas-liquid chromatography (GLC) is based upon: A. Ultraviolet light absorbance at 254 nm B. Flame ionization C. Refractive index D. Thermal conductance

B. Flame ionization B Volatile solutes can be detected in GLC using flame ionization, thermal conductivity, electron capture, and mass spectroscopy. In flame ionization, energy from a flame is used to excite the analytes as they elute from the column. The flame is made by igniting a mixture of hydrogen, carrier gas, and air. Current is produced when an outer shell electron is ejected from the excited analyte

The freezing point osmometer differs from the vapor pressure osmometer in that only the freezing point osmometer: A. Cools the sample B. Is sensitive to ethanol C. Requires a thermoelectric module D. Requires calibration with aqueous standards

B. Is sensitive to ethanol B Alcohol enters the vapor phase so rapidly that it evaporates before the dew point of the sample is reached. Therefore, ethanol does not contribute to osmolality as measured using the vapor pressure osmometer. Freezing-point osmometers measure alcohol and can be used in emergency department settings to estimate ethanol toxicity.

Why is vacuum necessary in the mass filter of a mass spectrometer? A. Ionization does not occur at atmospheric pressure B. It prevents collision between fragments C. It removes electrons from the ion source D. It prevents contamination

B. It prevents collision between fragments B Vacuum is needed in the mass filter of the MS to prevent random collisions between ions that would alter their trajectory or time of flight. It is also needed in CG-MS instruments that use electron ionization. The vacuum prevents collision between the carrier gas molecules and the ions. In spectrometers that use electrospray ionization, chemical ionization, and laser desorption ionization (MALDI and SELDI TOF), the ion source is not under vacuum.

Which element is reduced at the cathode of a Clark polarographic electrode? A. Silver B. Oxygen C. Chloride D. Potassium

B. Oxygen B The Clark electrode is designed to measure oxygen. O2 diffuses through a gas-permeable membrane covering the electrode. It is reduced at the cathode, which is made of platinum or other inert metal. Electrons are supplied by the anode, which is made of silver. The net reaction is: 4 KCl + 2 H2O + O2 + 4 Ag° →4 AgCl + 4 KOH

Which of the following would cause a "response" error from an ion-selective electrode for sodium when measuring serum but not the calibrator? A. Interference from other electrolytes B. Protein coating the ion-selective membrane C. An overrange in sodium concentration D. Protein binding to sodium ions

B. Protein coating the ion-selective membrane B Response is the time required for an electrode to reach maximum potential. Ion-selective analyzers use a microprocessor to monitor electrode response, slope, drift, and noise. When an electrode gives an acceptable response time when measuring an aqueous calibrator, but not when measuring serum, the cause is often protein buildup on the membrane.

One mole per kilogram H2O of any solute will cause all of the following except: A. Lower the freezing point by 1.86°C B. Raise vapor pressure by 0.3 mm Hg C. Raise the boiling point by 0.52°C D. Raise osmotic pressure by 22.4 atm

B. Raise vapor pressure by 0.3 mm Hg B Both freezing point and vapor pressure are lowered by increasing solute concentration. Boiling point and osmotic pressure are raised. Increasing solute concentration of a solution opposes a change in its physical state and lowers the concentration of H2O molecules.

SITUATION: A GC-MS analysis using nitrogen as the carrier gas shows an extensively noisy baseline. A sample of the solvent used for the extraction procedure, ethyl acetate, was injected and showed the same noise. Results of an Autotune test showed the appearance of a base peak at 16 with two smaller peaks at 17 and 18. Tese results indicate: A. The solvent is contaminated B. The carrier gas is contaminated C. There is electrical noise in the detector D. The ion source is dirty

B. The carrier gas is contaminated B All of these situations are sources of baseline noise in GC-MS. However, the peak at 16 indicates the presence of oxygen in the carrier gas. Oxygen in the atmosphere also contains small quantities of two isotopes with molecular weights of 17 and 18 owing to one and two extra neutrons, respectively.

In tandem-mass spectroscopy, the first mass filter performs the same function as: A. The ion source B. The chromatography column C. Extraction D. The vacuum system

B. The chromatography column B A tandem mass spectrometer uses two or more mass filters in sequence. The first filter functions as an ion trap. Once the sample is ionized, the filter selects molecular or parent ions of interest by excluding ions outside a specified size range. Therefore, it effectively separates the analyte(s) of interest from unwanted compounds. Tandem MS uses ESI to introduce the sample into the first mass filter, usually a quadrapole. The RF and DC voltages of the quadrapole are set to optimize the trajectory of the parent ions of interest and cause ejection of unwanted ions. The parent ions are drawn into a second mass filter where they are bombarded by argon atoms. The collisions result in the formation of mass fragments called daughter ions. This process is called collision-induced dissociation and the second filter is called a collision chamber. The process can be repeated in a third mass filter that generates granddaughter ions. A total-ion chromatogram is produced from these, enabling the compound of interest to be identified and quantified. Tandem MS is used to screen for inborn errors of fatty acid, amino acid, and organic acid metabolism.

In the coulometric chloride titration: A. Acetic acid in the titrating solution furnishes the counter ion for reduction B. The endpoint is detected by amperometry C. The titrating reagent contains a phosphate buffer to keep pH constant D. Nitric acid (HNO3) is used to lower the solubility of AgCl

B. The endpoint is detected by amperometry B Reduction of Ag+ back to Ag° generates the current, which signals the endpoint. The titrating reagent contains HNO3, acetic acid, H2O, and either gelatin or polyvinyl alcohol. The HNO3 furnishes nitrate, which is reduced at the generator cathode, forming ammonium ions. The ammonium becomes oxidized back to nitrate at the indicator anode. Gelatin or polyvinyl alcohol is needed to prevent pitting of the generator anode. Acetic acid lowers the solubility of AgCl, preventing dissociation back to Ag+

In mass spectroscopy, the term base peak typically refers to: A. The peak with the lowest mass B. The peak with the most abundance C. A natural isotope of the molecular ion D. The first peak to reach the mass detector

B. The peak with the most abundance B The base peak is typically the "molecular ion" or parent ion, meaning that it is the initial fragment made by releasing an electron. The cation thus formed has a charge of +1, and therefore, its m/z ratio is equal to its mass. The base peak is used for selective ion monitoring (SIM). It is the most abundant and most stable ion, and gives the best sensitivity for quantitative analysis.

In thin-layer chromatography (TLC), the distance the solute migrates divided by the distance the solvent migrates is the: A. tR B. Kd C Rf D. pK

C Rf C Rf is the distance migrated by the solute divided by the distance migrated by the solvent. The tR refers to the retention time of the solute in HPLC or gas chromatography (GC). The Kd is the partition coefficient, and is a measure of the relative affinity of solutes for the stationary phase. The solute with the greater Kd will be retained longer. The pK is the negative logarithm of K, the ionization constant, and is a measure of ionization.

Given the following real-time PCR amplification curve, what is the threshold cycle? A. 15 B. 20 C. 25 D. 30

C. 25 C The maximum curvature of the plot approximates the threshold cycle. A line is drawn from the threshold value on the y-axis through the curve, and a perpendicular dropped to the x-axis. The Ct is determined by the intersection point on the x-axis. The threshold is usually determined by an algorithm but can be calculated manually as 10 times the average standard deviation of the RFUs for cycles 2-10.

The method for measuring iron or lead by plating the metal and then oxidizing it is called: A. Polarography B. Coulometry C. Anodic stripping voltometry D. Amperometry

C. Anodic stripping voltometry C Anodic stripping voltometry is used to measure lead and iron. The cation of the metal is plated onto a mercury cathode by applying a negative charge. The voltage of this electrode is reversed until the plated metal is oxidized back to a cation. Current produced by oxidation of the metal is proportional to concentration.

In polarography, the voltage needed to cause depolarization of the cathode is called the: A. Half-wave potential B. Isopotential point C. Decomposition potential D. Polarization potentia

C. Decomposition potential C In polarography, a minimum negative voltage must be applied to the cathode to cause reduction of metal ions (or O2) in solution. This is called the decomposition potential. It is concentration dependent (dilute solutions require greater negative voltage), and can be determined using the Nernst equation.

Which method is the most useful when screening for errors of amino and organic acid metabolism? A. Two-dimensional thin-layer chromatography B. Gas chromatography C. Electrospray ionization tandem-mass spectroscopy D. Inductively charged coupled-mass spectroscopy

C. Electrospray ionization tandem-mass spectroscopy C While two-dimensional thin-layer chromatography can separate both amino and organic acids, it is not sufficiently sensitive for newborn screening. Electrospray ionization allows a small alcohol-extracted whole-blood sample to be analyzed by two mass spectrometers without prior separation by liquid or gas chromatography. Disorders of both organic and fatty acid metabolism are identified by the specific pattern of acylcarnitine ions produced. Amino acids are detected as amino species that have lost a carboxyl group during ionization, a process called neutral loss.

What is the confirmatory method for measuring drugs of abuse? A. HPLC B. Enzyme-multiplied immunoassay technique (EMIT) C. Gas chromatography with mass spectroscopy (GC-MS) D. TLC

C. Gas chromatography with mass spectroscopy (GC-MS) C GC-MS determines the mass spectrum of the compounds eluting from the analytic column. Each substance has a unique and characteristic spectrum of mass fragments. This spectrum is compared to spectra in a library of standards to determine the percent match. A match of greater than 95% is considered confirmatory.

What type of detector is used in high-performance liquid chromatography with electrochemical detection (HPLC-ECD)? A. Calomel electrode B. Conductivity electrode C. Glassy carbon electrode D. Polarographic electrode

C. Glassy carbon electrode C HPLC-ECD uses a glassy carbon measuring electrode and a silver-silver chloride reference. The analyte is oxidized or reduced by holding the glassy carbon electrode at a positive voltage (oxidization) or negative voltage (reduction). The resulting current flow is directly proportional to concentration. Phenolic groups such as catecholamines can be measured by HPLC-ECD.

What method is used to introduce the sample into a mass spectrometer for analysis of a trace element? A. Electrospray ionization B. Laser desorption C. Inductively charged plasma (ICP) ionization D. Direct injection

C. Inductively charged plasma (ICP) ionization C Mass spectrometers can be used to measure trace metals, but the atoms need to be vaporized and ionized like molecules before they enter the mass filter. This is done by introducing the sample into a very hot plasma (6,000-10,000°K) called a torch. The torch is made by circulating argon through inner and outer quartz tubes. The tubes are wrapped with a coil of wire that receives a radio frequency. This creates current flow through the wire and a magnetic field at the torch end. Argon atoms are excited by the current and magnetic field and ionize. When the argon is ignited by a spark, it forms the plasma. The sample is mixed with argon at the other end to create an aerosol. When it reaches the torch, the solvent is evaporated and the energy from the torch and collisions with argon ions cause ejection of outershell electrons, forming cations of the element. ICP-MS is used to measure any trace element that readily forms cations.

The term isocratic is used in high-performance liquid chromatography (HPLC) to mean the: A. Mobile phase is at constant temperature B. Stationary phase is equilibrated with the mobile phase C. Mobile phase consists of a constant solvent composition D. Flow rate of the mobile phase is regulated

C. Mobile phase consists of a constant solvent composition C An isocratic separation uses a single mobile phase of constant composition, pH, and polarity, and requires a single pump. Some HPLC separations use a gradient mobile phase to increase distance between peaks. Gradients are made by mixing two or more solvents using a controller to change the proportions of solvent components.

What is the primary means of solute separation in HPLC using a C18 column? A. Anion exchange B. Size exclusion C. Partitioning D. Cation exchange

C. Partitioning C Stationary phases (column packings) used in HPLC separate solutes by multiple means, but in reverse-phase HPLC the relative solubility between the mobile phase and stationary phase is most important and depends upon solvent polarity, pH, and ionic strength.

What type of measuring circuit is used in a freezing point osmometer? A. Electrometer B. Potentiometer C. Wheatstone bridge D. Thermal conductivity bridge

C. Wheatstone bridge C The resistance of the thermistor is measured using a network of resistors called a Wheatstone bridge. When the sample is frozen, the bridge is balanced using a calibrated variable resistor, so that no current flows to the readout. The resistance required to balance the meter is equal to the resistance of the thermistor.

When calibrating a semiautomatic pipet that has a fixed delivery of 10.0 µL using a gravimetric method, what should be the average weight of deionized water transferred? A. 10.0 µg B. 100.0 µg C. 1.0 mg D. 10.0 mg

D. 10.0 mg D Gravimetric and spectrophotometric analysis are the two methods used to verify pipet volume accuracy and precision. Since spectrophotometric analysis involves dilution, gravimetric analysis is associated with greater certainty. At 20°C, the density of pure water is 0.99821 g/mL. Therefore, each microliter weighs almost exactly 1.0 mg.

Select the chemical that is used in most HPLC procedures to decrease solvent polarity. A. Hexane B. Nonane C. Chloroform D. Acetonitrile

D. Acetonitrile D All of the compounds mentioned have nonpolar properties. Because most HPLC is reverse phase (a polar solvent is used), hexane and nonane are too nonpolar. Acetonitrile is more polar and less toxic than chloroform and along with methanol is a common polarity modifier for HPLC.