Instrumentation and Analytical Principles

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Author:
ncrook
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203680
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Instrumentation and Analytical Principles
Updated:
2013-06-06 20:48:21
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Hemoglobin Electrophoresis Amperometry Coulometry Potentiometry
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Hemoglobin Electrophoresis, Amperometry, Coulometry, Potentiometry
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  1. What is hemoglobin composed of?
    • 4 Globin chains
    • 4 Heme groups
    • 4 Iron atoms
  2. What chains make up Hemoglobin A1?
    • 2 Alpha
    • 2 Beta
  3. What chains make up Hemoglobin A2?
    • 2 Alpha
    • 2 Delta
  4. What chains make up Hemoglobin F?
    • 2 Alpha
    • 2 Gamma
  5. What substitution occurs in Hemoglobin S?
    Substitution of valine for glutamic acid in position 6 of the beta chain
  6. What substitution occurs in Hemoglobin C?
    Substitution of lysine for glutamic acid in position 6 of the beta chain
  7. How can Hemoglobin F be differentiated from the majority of the rest?
    Alkali resistance
  8. At pH 8.6, on cellulose acetate, hemoglobins have a ______ charge and migrate toward the ______.
    Net negative charge and migrate toward the anode
  9. At pH 6.2 on agar gel, what is different and why is it done?
    • Hemoglobins exhibit different electrophoretic mobilities in comparison with hemoglobins electrophoresed at pH 8.6 on cellulose acetate
    • Usefule in differentiating hemoglobins that migrate with the same electrophoretic mobility
  10. What is Electrophoresis used for?
    Used clinically to separate and identify proteins, including serum, urine, and CSF proteins, lipoproteins, isoenzymes, and so on
  11. What is defined as the movement of charged molecules in a liquid medium when an electric field is applied?
    Electrophoresis
  12. What is defines as the movement of charged molecules in a porous supporting medium where the molecules separate as distinct zones?
    Zone electrophoresis
  13. What provides a matrix that allow molecules to separate in electrophoresis (like agarose gel, starch gel, polyacrylamide gel, and cellulose acetate membranes)?
    Support medium
  14. What does movement of charged particles though a medium depend upon?
    • Net charge
    • Size
    • Shape
    • Character of the buffer and supporting medium
    • Temp
    • Intensity of the electric field
  15. When dealing with the nature of charged particles, what is true about proteins?
    Proteins are amphoteric and may be charged positively or negatively depending on the pH of the buffer solution
  16. When the pH at which negative and positive charges are equal on a protein is called what?
    Protein's isoelectric point
  17. What is the pH of buffer solutions that are generally used for serum protein electrophoresis?
    Buffer solutions of pH 8.6
  18. Using agarose gel or cellulose acetate at an alkaline pH, serum proteins will take on what charge and will migrate to what side?
    Serum proteins take on a net negative charge and will migrate toward the anode (+)
  19. What colorimetric stains or fluorescent chemicals are used in electrophoresis and for what specimens?
    • Serum proteins - Amido black B, Ponceau S, and Coomassie brilliant blue stains
    • CSF - Silver nitrate
    • Lipoproteins - Fat red 7B and Oil red O
    • Lactate dehydrogenase isoenzymes - Nitrotetrazolium blue
  20. How is detection and quantification of the separated protein accomplished in electrophoresis?
    Using a densitometer
  21. Name some commonly encountered problems in electrophoresis
    • Holes in staining pattern - analyte present in too high a concentration
    • Very slow migration - voltage too low
    • Sample precipitates in support - pH too high or low; excessive heat production
  22. Describe Isoelectric focusing
    A type of zone electrophoresis in which protein separation is based on the isoelectric point (pI) of the proteins
  23. What is capillary electrophoresis based on?
    Electroosmotic flow (EOF)
  24. How does Amperometry work?
    Electrochemical technique that measures the amount of current produced throught the oxidation or reduction of the substance to be measured at an electrode held at a fixed potential
  25. What system does chloride coulometer employ?
    Coulometric system
  26. What law is the basis of the Coulometric system of a chloride coulometer?
    Faraday's law
  27. Describe Faraday's law
    States that in an electrochemical system, the number of equivalent weights of a reactant oxidized or reduced is directly proportional to the quantity of electricity used in the reaction
  28. If the current is constant, what is true with Coulometry?
    The number of equivalent weights of reactant oxidized or reduced depends only on the duration of the current
  29. How is the endpoint of the titration indicated in Coulometry?
    Indicated by the increase in conductivity of the solution
  30. What is used to measure the increase in conductivity of Coulometry?
    Amperometry
  31. What is a technique used to determine the concentration of a substance in solution employing and electrochemical cell that consists of two half-cells, where the potential difference between an indicator electrode and a reference electrode is measured?
    Potentiometry
  32. What is composed of single metallic conductor surrounded by solution of electrolyte?
    Half-cell
  33. What is also called an electrode?
    Half-cell
  34. In potentiometry, what type of connection is between two metallic conductors and between two electrolyte solutions?
    Salt bridge
  35. What is the universally accepted standard half-cell and at what potential?
    • Standard Hydrogen Electrode
    • Assigned a potential E degree of 0.000 volt
  36. What type of electrode is Calomel?
    Reference electrode
  37. What does the Calomel reference electrode consist of?
    Mercury covered by a layer of mercurous chloride in contact with saturated solution of potassium chloride
  38. Name some types of reference electrodes
    • Calomel electrode
    • Silver-silver chloride (Ag/AgCl) electrode
  39. What is another type of electrode used in potentiometry and how is it selected?
    • Indicator electrode
    • Selected on the basis of chage in its potential with change in concentration of analyte to be measured
  40. What does a pH/blood gas analyzer use to measure blood pH?
    pH sensitive glass electrode for measuring blood pH
  41. What does a pH/blood gas analyzer use to measure gases in blood?
    PCO2 and PO2 electrodes for measuring gases in blood
  42. What does a pH/blood gas analyzer use to measure pH?
    pH electrode (functioning glass electrode) that is dependent on properties of pH-sensitive glass
  43. Name the different parts of a pH/blood gas analyzer used in measurements
    • pH-sensitive glass electrode
    • PCO2 and PO2 electrode
  44. What does the PCO2 electrode specifically measure?
    Partial pressure of carbon dioxide (PCO2)
  45. What is the PCO2 electrode immersed in?
    Bicarbonate solution
  46. What type of potentiometric is the ion-exchange electrode?
    Ion-selective electrode
  47. How is K+ analyzed with the ion-exchange electrode?
    Antibiotic valinomycin, because of its ability to bind to K+, is used as a neutral carrier for K+ elective membrane
  48. How is NH4+ analyzed with the ion-exchange electrode?
    Antibiotics nonactin and monactin used in combination as neutral carrer for NH4+, selective membrane
  49. How is Sodium analyzed with the ion-exchange electrode?
    • Utilizes glass membrane electrodes with selective capability
    • Constructed form glass that consists of silicon dioxide, sodium oxide, and aluminum oxide

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