Harr MLS Review Chemistry 5.1 Instrumentation

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?

10.0 mg 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.

Which statement regarding reflectometry is true?

100% reflectance is set with an opaque film called a white reference 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.

A solution that has a transmittance of 1.0 %T would have an absorbance of:

2.0

Given the following real-time PCR amplification curve, what is the threshold cycle? [PICTURE]

25 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.

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

450 nm 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.

Which monochromator specification is required in order to measure the true absorbance of a compound having a natural absorption bandwidth of 30 nm?

5-nm bandpass 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.

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

525 nm 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.

All of the following are required when measuring magnesium by atomic absorption spectrophotometry except:

A 285-nm reference beam to correct for background absorption 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.

A flameless atomic absorption spectrophotometer dehydrates and atomizes a sample using:

A graphite capillary surface 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 type of monochromator produces the purest monochromatic light in the UV range?

A prism and a variable exit slit 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.

Which instrument requires a highly regulated DC power supply?

A spectrophotometer with a photomultiplier tube 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.

Select the chemical that is used in most HPLC procedures to decrease solvent polarity.

Acetonitrile 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.

Which reagent is used in thin-layer chromatography (TLC) to extract cocaine metabolites from urine?

Alkali and organic solvent 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.

The response of a sodium electrode to a 10-fold increase in sodium concentration should be:

An increase in potential of approximately 60 mV

The method for measuring iron or lead by plating the metal then oxidizing it is called:

Anodic stripping voltometry 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.

Ion selective analyzers using undiluted samples have what advantage over analyzers that use a diluted sample?

Are not subject to pseudohyponatremia caused by high lipids 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.

In real-time PCR, what value is needed in order to determine the threshold?

Background signal 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.

Persistent noise from an ion-selective electrode is most often caused by:

Blocked junction at the salt bridge 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.

In gas chromatography, the elution order of volatiles is usually based upon the:

Boiling point 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?

Bromide 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.

The fragments typically produced and analyzed in methods employing mass spectroscopy are typically

Cations caused by electron loss or proton attachment 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 electrodes below is a current-producing (amperometric) rather than a voltage-producing (potentiometric) electrode?

Clark electrode 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:

Concentration of the potassium chloride filling solution 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 × 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 = E° - RT/nF × 2.3 log10[Cl-] = E° - 59.2 mV × log[Cl-] (at room temperature)

Which of the following statements accurately characterizes the coulometric titration of chloride?

Constant current must be present across the generator electrodes 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.

The purpose of the nebulizer in an atomic absorption spectrophotometer that uses a flame is to:

Convert ions to atoms 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.

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

In polarography, the voltage needed to cause depolarization of the cathode is called the:

Decomposition potential 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 condition is a common cause of stray light?

Dispersion from second-order spectra 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.

What component is used in a GC-MS but not used in an LC-MS?

Electron source 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?

Electrospray ionization 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.

Which method is the most useful when screening for errors of amino and organic acid metabolism?

Electrospray ionization tandem-mass spectroscopy 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.

Which of the following situations is likely to cause an error when weighing with an electronic analytical balance?

Failure to close the doors of the balance before reading the weight 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:

Flame ionization 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.

Which instrument requires a primary and secondary monochromator?

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

What is the confirmatory method for measuring drugs of abuse?

Gas chromatography with mass spectroscopy (CG-MS) 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)?

Glassy carbon electrode 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.

In addition to velocity, what variable is also needed to calculate the relative centrifugal force (g force) of a centrifuge?

Head radius 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 = s^2 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.

Which of the following materials is best suited for verifying the wavelength calibration of a spectrophotometer?

Holmium oxide glass 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 wide- bandpass filter made by placing a thin layer of colored gelatin between two glass plates and is unsuitable for spectrophotometric calibration.

What method is used to introduce the sample into a mass spectrometer for analysis of a trace element?

Inductively charged plasma (ICP) ionization 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 outer- shell electrons, forming cations of the element. ICP-MS is used to measure any trace element that readily forms cations.

The freezing point osmometer differs from the vapor pressure osmometer in that only the freezing point osmometer:

Is sensitive to ethanol 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?

It prevents collision between fragments 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.

When measuring calcium by atomic absorption spectrophotometry, which is required?

Lanthanum oxide to chelate phosphates 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.

All of the following compounds contribute to the osmolality of plasma except:

Lipids 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.

Interference in atomic absorption spectrophotometry caused by differences in viscosity is called:

Matrix effect 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.

Which type of filter is best for measuring stray light?

Matrix interference 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.

The term isocratic is used in high-performance liquid chromatography (HPLC) to mean the:

Mobile phase consists of a constant solvent composition 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.

The term reverse phase is used in HPLC to indicate that the mobile phase is:

More polar than the stationary phase 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.

Select the equation describing the potential that develops at the surface of an ion-selective electrode.

Nernst equation 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.

One mole per kilogram H2O of any solute will cause all of the following except:

Raise vapor pressure by 0.3 mmHg 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.

Which element is reduced at the cathode of a Clark polarographic electrode?

Oxygen 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

What is the primary means of solute separation in HPLC using a C18 column?

Partitioning 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.

Which component is required in a spectrophotometer in order to produce a spectral absorbance curve?

Photodiode array 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.

Which photodetector is most sensitive to low levels of light?

Photomultiplier tube 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 component is needed for a thermal cycler to amplify DNA?

Programmable heating and cooling unit 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 of the following would cause a "response" error from an ion-selective electrode for sodium when measuring serum but not the calibrator?

Protein coating the ion-selective membrane 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.

Light scattering when the wavelength is greater than 10 times the particle diameter is described by:

Rayleigh's law 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.

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

Replace the source lamp 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.

In thin-layer chromatography (TLC), the distance the solute migrates divided by the distance the solvent migrates is the:

Rf 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.

Which substance is used to generate the light signal in electrochemiluminescence?

Ruthenium 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.

Which measurement principle is employed in a vapor pressure osmometer?

Seebeck 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.

The term RT/nF in the Nernst equation defines the:

Slope of the electrode 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 of the following components is not needed in a chemiluminescent immunoassay analyzer?

Source lamp 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.

A linearity study is performed on a visible spectrophotometer at 650 nm and the following absorbance readings are obtained: Concentration of standard; Absorbance 10.0 mg/dL; 0.20 20.0 mg/dL; 0.41 30.0 mg/dL; 0.62 40.0 mg/dL; 0.79 50.0 mg/dL; 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?

Stray light 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.

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?

Substitute saline for the reagent 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.

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. These results indicate:

The carrier gas in contaminated 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:

The chromatography column 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.

Bichromatic measurement of absorbance can correct for interfering substances if:

The contribution of the interferent to absorbance is the same at both wavelengths 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).

Which statement regarding nephelometry is true?

The detector response is directly proportional to concentration 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.

In the coulometric chloride titration:

The endpoint is detected by amperometry 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:

The peak with the most abundance 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.

What component of a freezing point osmometer measures the sample temperature?

Thermistor 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?

To compensate for variation in extraction and injection 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.

Why do many optical systems in chemistry analyzers utilize a reference light path?

To minimize error caused by source lamp fluctuation 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.

When measuring lead in whole blood using atomic absorption spectrophotometry, what reagent is required to obtain the needed sensitivity and precision?

Triton X-100 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 of the following statements about fluorometry is accurate?

Unsaturated cyclic molecules are often fluorescent 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:

Valinomycin gel 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.

What type of measuring circuit is used in a freezing point osmometer?

Wheatstone bridge 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.

In absorption spectrophotometry:

absorbance is directly proportional to concentration 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.

The reagent blank corrects for absorbance caused by:

the color of reagents 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.

The half-band width of a monochromator is defined by:

the range of wavelengths passed at 50% maximum transmittance 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.