Chromotography and Electrophoesis
Considering serum protein electrophoresis using an agarose gel medium, the main purpose of the medium is: a. to provide a site to bind the protein to, once separation is complete b. to allow electrophoretic migration of proteins to occur c. to prevent other macromolecules, such as lipids, from interfering with serum protein electrophoresis d. to bind the stain for visualization of the separated proteins
to provide a site to bind the protein to, once separation is complete Response Feedback: Following sample application, the applied electrical field causes proteins to move through the solvent that exists in the pores of the agarose medium. Once electrophoresis is complete, the individual proteins must be fixed in position to prevent continued diffusion. Fixation involves denaturing the protein (i.e., precipitating the protein, causing it to bind to the gel matrix). Fixation will hold the protein in place for evaluation.
A toxicology screen is set up using thin layer chromatographic plates. Three positive controls/standards are used with the following distance of migration from application point results: Std 1 = 5cm ; Std 2 = 10 cm; Std 3 = 17cm . The acetone solvent front moved 35 cm. After processing an unknown sample, a solute had a migration distance of 16 cm. What is the Rf of the unknown solute? Answer Question 36 answers a. 16 b. 0.23 c. 1.94 d. 0.45
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AGE of serum proteins and qantitation by densitometry is used to detect clinical disorders such as monoclonal gammopathy. What is Immunofixationelectrophoresis (IFE)? Explain the process of IFE and how it is used to confirm the identity of the type of monoclonal gammopathy.
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Chromatographic assessment of glycated hemoglobin commonly uses which type of chromatography? Answer Question 33 answers a. Adsorption b. Partition c. Ion exchange d. Affinity
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Chromatographic methods may be characterized according to: a. the physical apparatus associated with the stationary phase b. the mechanism of separation c. the physical state of the mobile phase d. all of the above
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Chromatography is best characterized as a separation technique in which the components of a mixture are separated based on: Answer Question 24 answers a. the chromophoric nature of the components b. how well the components dissolve in the mobile phase c. how the components distribute between the mobile phase and stationary phase d. the varying concentrations of the components
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Considering the chromatographic separation of two solutes that are present in a mixture at the same concentration and produce the same detector response (i.e., have the same molar absorptivity), the solute that has a higher affinity for the stationary phase will have a ______ retention time and a ______ detector response than the solute that has a higher affinity for the mobile phase. Answer Question 25 answers a. greater, greater b. lesser, greater c. greater, lesser d. lesser, lesser
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Describe the EP separation of serum proteins on AgaroseGel Electrophoresis (AGE) -use diagrams to support your answer..
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Describe the electrophoretic separation of serum proteins using AGE. Describe the zone separation pattern, and explain how the proteins are detected and quantified. Use diagrams and /or figures to support your answer.
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Explain the process of quantitation of serum proteins by densitometry
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Given that adsorption chromatography is being utilized with a polar adsorbent such as silica, predict the order of elution of the following three compounds with the first to elute being listed first. Compound A - nonpolar compound with minimal potential for hydrogen bonding Compound B - polar compound with intermediate potential for hydrogen bonding Compound C - polar compound with high potential for hydrogen bonding Question 29 answers a. compound A, compound C, compound B b. compound B, compound C, compound A c. compound A, compound B, compound C d. compound C, compound B, compound A
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In a gas-liquid partition chromatography, what constitutes the components of the stationary phase? Answer Question 37 answers a. Stainless steel columns packed with graphitized carbon b. Packed columns containing uncoated support particles c. The inert gas is the stationary phase. d. Methyl silicone polymers coated on capillary tubes
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In normal-phase chromatography, the mobile phase is relatively ______, and the stationary phase is relatively ______. _____ solute molecules are retained the longest in this type of chromatography. Answer Question 31 answers a. nonpolar, polar, polar b. nonpolar, polar, nonpolar c. polar, nonpolar, nonpolar d. polar, nonpolar, polar
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In the thin layer chromatography procedure for drug screening, the retention factor (Rf) value for a compound is given by the Answer Question 35 answers a. rate of movement of the mobile phase through the adsorbent compared with standards and controls. b. measurement in centimeters of the distance the solute moved in the mobile phase of from the point of application. c. distance moved by the mobile phase front from the point of application compared with a control. d. ratio of distance moved by the unknown solute to distance moved by the solvent in the mobile phase.
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Question 30 text The type of chromatography in which separation occurs by differential distribution of solutes between two immiscible liquids is known as: Answer Question 30 answers a. adsorption chromatography b. gel-permeation chromatography c. ion-exchange chromatography d. partition chromatography
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The chromatographic method based on biologically related interactions by immobilizing one of a pair of interacting biomolecules onto a solid support and placing it in a column is known as: Answer Question 42 answers a. affinity chromatography b. ion-suppression chromatography c. gel-permeation chromatography d. ion-pair chromatography
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The three types of suppportmedia used for electrophoreticseparations are agarosegel (AGE), Polyacrylamidegel (PAGE) and capillaries and buffer (CE). Compare the different media, describing what they are, the advantages and disadvantages of using them and a clinical application for each
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The type of chromatography that involves use of immunological principles is Answer Question 34 answers a. ion exchange. b. affinity. c. partition. d. adsorption.
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What is the correct interpretation of column selectivity (α) = 1.0? Question 26 answers a. The solutes are adequately separated by the chromatographic system. b. The solutes are partially separated by the chromatographic system. c. The separation of the solutes has been optimized, and no further improvement can be made in chromatographic conditions. d. The solutes are not separated by the chromatographic system.
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Which of the following molecular characteristics forms the basis for chromatographic retention in adsorption and partition chromatography? Answer Question 28 answers a. molar absorptivity b. molecular size and shape c. all of the above d. polarity
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Which of the following steps could be taken to improve column selectivity (α)? a. Change the composition of the stationary phase. b. All of the above. c. Change the volume of the sample analyzed. d. Change the mobile phase flow rate.
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Which of the following types of chromatography should be selected to separate relatively large molecules (100,000 D or greater)? Answer Question 41 answers a. gel-exclusion chromatography b. ion-exchange chromatography c. adsorption chromatography d. partition chromatography
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You are preparing placental tissue for separation and isolation of a specific enzyme. The enzyme has a negative charge, so you want to use chromatography to perform your task. Following homogenization, the tissue is reduced to a large volume of liquid. Because of budget cuts, you cannot purchase the correct packing material for your experiment. You have found, however, some cross-linked dextran in the laboratory that might work based on a different principle of separation. Which one of the following separation techniques could be used instead if you were looking to separate the molecule based on molecular weight? Answer Question 40 answers a. Adsorption b. Size exclusion c. Affinity d. Partition
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You are preparing placental tissue for separation and isolation of a specific enzyme. The enzyme has a negative charge, so you want to use chromatography to perform your task. Following homogenization, the tissue is reduced to a large volume of liquid. Which one of the basic types of chromatography would be most useful for your situation? Answer Question 38 answers a. Reverse b. Gas c. Planar d. Column
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You are preparing placental tissue for separation and isolation of a specific enzyme. The enzyme has a negative charge, so you want to use chromatography to perform your task. Following homogenization, the tissue is reduced to a large volume of liquid. Which one of the following separation mechanisms would be most useful? Answer Question 39 answers a. Ion-exchange b. Affinity c. Adsorption d. Partition
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n reversed-phase chromatography, the mobile phase is relatively ______, and the stationary phase is relatively ______. ______ solute molecules are least retained in this type of chromatography. Answer Question 32 answers A. polar, nonpolar, polar B. nonpolar, polar, nonpolar C. polar, nonpolar, nonpolar D. nonpolar, polar, polar
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List the factors that affect migration
1.Buffers 2.Electric Field Strength 3.Supporting medium 4.Temperature 5.Endosmosis 6.Net charge of the molecule 7.Size of the molecule 8.Volume of sample
What stains are used to stain a serum protein electrophoresis gel? a. Comassie Blue and Ethidium Bromide b. Crystal Violet and Acid Blue c. Oil Red O and Amido Black d. Amido Black and Acid Blue
Amido Black and Acid Blue Response Feedback: Most commercial methods for serum protein electrophoresis use Amido Black B or members of the Coomassie Brilliant Blue series of dyes such as Acid Blue for staining.
List the factors that affect migration and describe how each affects migration of molecules in electrophoresis.
Buffers - sets the pH of proteins and the higher the ionic strength, the lower the migration rate. pH is best 7-9. Barbital buffer is 8.6, minimal affect on proteins Electronic field - A weak electrical field will produce little or no migration.Increased voltage & increased current = increased migration rate -But heat generation?
What technique allows us to scan electrophoretic separation on a support medium and perform quantitation? a. Chromatography b. Densitometry c. Nephelometry d. Spectrophotometry
Densitometry Response Feedback: It is possible to quantify the individual protein fractions either as a percentage of the total or as absolute concentration by densitometry. In a densitometer, the gel (or other medium) moves past an optical system and the absorbance of each fraction is displayed on a recorder chart or an electronic display.
Electro-osmosis is the migration of supporting medium counterions and solvent toward the appropriate electrode. This net flow of counterions and solvent is in the opposite direction to the migration of the macromolecules of interest. Which of the following statements best describes the effect of electro-osmosis? Answer Question 4 answers a. Electro-osmosis only occurs in capillary electrophoresis and is the mechanism of separation in the relatively new technique. b. Electro-osmosis will only affect the final positions of the macromolecules but not their separation. c. Electro-osmosis will prevent macromolecule separation from occurring. d. Electro-osmosis is the mechanism by which electrophoretic separation of macromolecules occurs
Electro-osmosis will only affect the final positions of the macromolecules but not their separation. Response Feedback: Electro-osmosis creates an opposing flow to the macromolecules being separated by electrophoresis. This opposing flow is relatively constant and is much like rowing a boat against the flow of a stream. The macromolecules will separate in the electrical field applied. However, each macromolecule will encounter the same opposing flow, affecting the final position of each.
Explain the general principles of electrophoretic separations -distinguish between EP & IEF.
Electrophoresis is the migration of charged particles under the influence of an electric current. The process involves ionisation of the molecules and their drift towards the opposing charge electrode IEF stands for isoelectric focusing. A pH gradient is predictably created, through the use of ampholytes in the gel. The ampholytes pull proteins, until the proteins reach the pH matching their isoelectric point. Separation is based on PI differences in proteins.
How does the net charge of the proteins in a sample affect the electrophoretic mobility of protein fractions? a. There is no relationship between net charge and electrophoretic mobility. b. It is equal to the electrophoretic mobility. c. It is directly proportional to electrophoretic mobility. d. It is inversely proportional to electrophoretic mobility.
It is directly proportional to electrophoretic mobility. Response Feedback: Electrophoretic mobility (µ) is defined as the rate of migration (cm/s) per unit field strength (volts/cm). With the formula µ = net charge divided by viscosity, the mobility is seen as being directly proportional to net charge of the ions in solution.
Describe three types of support media, explaining what they are, their advantages and an application
Supporting media matrix for separation -buffer, insoluble gels, membranes
How does Isoelectric focussing differ from standard zone electrophoresis and what is the advantage of using IEF? Give an example of a clinical application of IEF
The advantage of IEF is that Proteins with pI values <0.1 pH units can be distinguished.
You have just set up the morning protein electrophoresis run and are going off for a coffee break. When you return 45 minutes later, you stain the gel and notice that the bands did not migrate very far from the point of application. What is the likely explanation? a. The electrophoresis chamber was not connected to a power supply. b. The current or voltage on the power supply was set too low. c. Too much sample had been applied to the gel. d. A chemical reaction occurred between the buffer and all samples.
The current or voltage on the power supply was set too low. Response Feedback: Rate of migration is dependent upon the strength of the electrical field. Electrophoretic mobility is defined as the rate of migration (cm/s) per unit field strength (volts/cm). A weak electrical field will produce little or no migration.
In routine clinical electrophoresis, the most common means used to provide quantitative information about the zones that have separated involves the use of a. densitometry b. chromatography c. nephelometry d. mass spectrometry
a. densitometry Response Feedback: Densitometry is the most common technique used to analyze electrophoretic patterns in routine clinical electrophoresis. Several companies have developed highly automated densitometers for this purpose
The isoelectric point (pI) of a protein is the pH at which a. the protein has a positive charge. b. the protein has a negative charge. c. a protein migrates best in electrophoresis. d. the protein has no net charge.
a. the protein has no net charge. Response Feedback: The isoelectric point of a molecule is the pH at which it has no net charge and will not move in an electric field.
Which support medium is most used in electrophoresis of larger serum proteins? Answer Question 6 answers a. agar b. polyacrylamide gel c. agarose
agarose Response Feedback: Agarose has much larger pore sizes than polyacrylamide. Specifically when it comes to electrophoresis of larger proteins, larger pore sizes allow migration freely through the gel and separation is based on charge.
If an electrophoretic buffer were diluted before use instead of using it at normal strength, how would electrophoresis be affected? a. No change in the rate of migration of protein b. Decreased migration rate of protein c. Increased migration rate of protein
b. Increased migration rate of protein Response Feedback: With increasing buffer concentration, the ionic cloud increases in size, and the molecule becomes hindered in its movement; therefore when buffer concentration is decreased as in a dilution, the migration rate of the molecule will increase
If the viscosity of the support medium used in a protein electrophoresis system is too high (thick), the migration rate will Answer Question 22 answers a. not be affected. b. decrease. c. increase.
b. decrease. Response Feedback: Electrophoretic mobility (µ) is defined as the rate of migration (cm/s) per unit field strength (volts/cm). With the formula µ = net charge divided by viscosity, the mobility is seen as being inversely proportional to the viscosity; therefore if viscosity increases, migration rate decreases.
Which of the following chemicals is used for fixation following serum protein electrophoresis? Answer Question 9 answers a. dilute acetic acid b. sodium dodecyl sulfate c. Ponceau S d. saturated sodium hydroxide
b. dilute acetic acid Response Feedback: Dilute acids such as acetic acid, trichloroacetic acid, and sulfosalicylic acid may all be used to denature proteins.
Serum used as the sample for electrophoretic analysis in clinical laboratories contains many groups of macromolecules. Examples include amino acids, serum proteins, lipoproteins, glycoproteins, and others. Suppose an analyst is interested in performing serum protein electrophoresis. How does the analyst prevent other groups of macromolecules from interfering with the analysis of serum proteins? Answer Question 10 answers a. by selecting an electrophoresis time that will allow all other macromolecules to migrate completely off the medium and leave only serum proteins b. by selecting a medium that will only allow the electrophoretic migration of serum proteins c. by selecting an electrophoresis voltage that will only cause migration of serum proteins d. by selecting a stain that will only visualize the desired serum proteins
by selecting a stain that will only visualize the desired serum proteins Response Feedback: Electrophoresis of a serum sample will result in migration of all macromolecules in the sample that contain a net charge. The most common way to analyze the particular group of interest is to select a stain that is specific for that group.
An increase in the ionic strength of the electrophoretic buffer while operating at constant current will cause the voltage to ________, the electrophoretic migration of macromolecules to ________, and the separation of macromolecules to _______. Answer Question 3 answers a. decrease, decrease, decrease b. decrease, decrease, increase c. increase, increase, decrease d. increase, increase, increase
c. decrease, decrease, decrease Response Feedback: An increase in ionic strength when operating an electrophoresis system under constant current conditions will cause the voltage to decrease. As a result, the electrical force to move the macromolecules will be decreased. The result of decreased electrophoretic migration is more time for macromolecules to freely diffuse, which decreases the resolution between the macromolecules.
The discontinuous system used to enhance electrophoretic resolution in which movement comes to a stop because of a lack of net charge is: Answer Question 5 answers a. isotachophoresis b. gel electrophoresis c. isoelectric focusing d. disk electrophoresis
c. isoelectric focusing Response Feedback: In isoelectric focusing, analytes migrate in an electrical field across a pH gradient. In this gradient, each analyte will migrate until it reaches the region where the pH is equal to the isoelectric point of the analyte. At this point, the compound will no longer have net charge, and its electrophoretic mobility will stop.
In an electrophoresis system, the _____ is the negative electrode. Answer Question 19 answers a. optode b. diode c. anode d. cathode
cathode Response Feedback: Chemical species that take on electrical charge by becoming ionized move toward either the cathode (negative electrode) or the anode (positive electrode) depending on the kind of charge they carry.
Following electrophoresis, it was determined that the desired results were not obtained. All of the expected zones showed very slow migration. Which of the following would be a likely solution for this slow migration? a. change the pH of the buffer to a value closer to the isoelectric point of the analytes b. decrease the voltage used for the analysis c. decrease the ionic strength of the buffer d. change to a support medium with a smaller pore size
d. decrease the ionic strength of the buffer Response Feedback: All of the responses to the situation would have an influence on electrophoretic migration. The only change that would result in an increase in migration would be to lower the ionic strength of the buffer to reduce the number of buffer ions competing with sample ions in the electrical field.
Considering only the effect of time on electrophoretic separation, the separation of bands ________ with time, and diffusion of bands _______ with time. Answer Question 7 answers a. decreases, decreases b. decreases, increases c. increases, decreases d. increases, increases
increases, increases Response Feedback: In electrophoresis, the separation of bands increases linearly with time. Diffusion of bands also increases with time. Diffusion reduces resolution. Thus it is always best to complete electrophoresis as quickly as possible
The main advantage of capillary electrophoresis over standard gel electrophoresis is a. more efficient heat dissipation for better separation. b. the increased amount of time required for separation. c. the ability to use a variety of buffers in the system. d. the small sample size required.
more efficient heat dissipation for better separation. Response Feedback: Capillary tubes have an inherent ability to dissipate heat quickly, allowing higher voltages to be used in this electrophoretic system. This results in better separation of the protein fractions in less time.
Which of the following proteins will show the greatest net negative charge in a pH 8.6 buffer? Answer Question 2 answers a. protein C with an isoelectric point of 8.6 b. protein A with an isoelectric point of 7.5 c. protein D with an isoelectric point of 9.6 d. protein B with an isoelectric point of 8.0
protein A with an isoelectric point of 7.5 Response Feedback: The isoelectric point (pI) is the pH of the buffer at which the protein has no net (overall) charge. Protein D, with a pI above the pH of the buffer, will have a net positive charge. Protein C, with a pI at the pH of the buffer, will have no net charge. Proteins A and B will both have a negative charge at the pH of the buffer. However, the net negative charge of protein A would be greater because the pI is further away from the pH of the buffer and that for protein B.
On a serum protein electrophoresis, if the albumin band appears distorted and large, the likely cause is a. inappropriate support material used. b. pH of electrophoretic buffer incorrect. c. instrument not connected. d. sample overload.
sample overload. Response Feedback: Too much sample applied to the gel can produce distorted protein zones. Because albumin in serum is about 10 times more concentrated than the alpha1-globulins ( the next band), the amount of serum applied should avoid overloading with albumin.
An increase in which of the following would increase the movement of particles through a solution in electrophoresis? Answer Question 1 answers a. size and symmetry of the particles b. all of the above c. viscosity of the solvent d. voltage applied to the electrophoretic system
voltage applied to the electrophoretic system Response Feedback: An increase in the voltage applied to the electrophoretic system will increase the force exerted on particles, increasing movement through the solvent. As the viscosity of the solvent increases, the resistance increases to particle movement. Likewise, as the size of the particles increase and as the particles become more asymmetrical, movement through the solvent will slow.
