Instrumentation and Analytical Principles
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What is hemoglobin composed of?
- 4 Globin chains
- 4 Heme groups
- 4 Iron atoms
What chains make up Hemoglobin A1?
What chains make up Hemoglobin A2?
What chains make up Hemoglobin F?
What substitution occurs in Hemoglobin S?
Substitution of valine for glutamic acid in position 6 of the beta chain
What substitution occurs in Hemoglobin C?
Substitution of lysine for glutamic acid in position 6 of the beta chain
How can Hemoglobin F be differentiated from the majority of the rest?
At pH 8.6, on cellulose acetate, hemoglobins have a ______ charge and migrate toward the ______.
Net negative charge and migrate toward the anode
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
What is Electrophoresis used for?
Used clinically to separate and identify proteins, including serum, urine, and CSF proteins, lipoproteins, isoenzymes, and so on
What is defined as the movement of charged molecules in a liquid medium when an electric field is applied?
What is defines as the movement of charged molecules in a porous supporting medium where the molecules separate as distinct zones?
What provides a matrix that allow molecules to separate in electrophoresis (like agarose gel, starch gel, polyacrylamide gel, and cellulose acetate membranes)?
What does movement of charged particles though a medium depend upon?
- Net charge
- Character of the buffer and supporting medium
- Intensity of the electric field
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
When the pH at which negative and positive charges are equal on a protein is called what?
Protein's isoelectric point
What is the pH of buffer solutions that are generally used for serum protein electrophoresis?
Buffer solutions of pH 8.6
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 (+)
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
How is detection and quantification of the separated protein accomplished in electrophoresis?
Using a densitometer
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
Describe Isoelectric focusing
A type of zone electrophoresis in which protein separation is based on the isoelectric point (pI) of the proteins
What is capillary electrophoresis based on?
Electroosmotic flow (EOF)
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
What system does chloride coulometer employ?
What law is the basis of the Coulometric system of a chloride coulometer?
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
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
How is the endpoint of the titration indicated in Coulometry?
Indicated by the increase in conductivity of the solution
What is used to measure the increase in conductivity of Coulometry?
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?
What is composed of single metallic conductor surrounded by solution of electrolyte?
What is also called an electrode?
In potentiometry, what type of connection is between two metallic conductors and between two electrolyte solutions?
What is the universally accepted standard half-cell and at what potential?
- Standard Hydrogen Electrode
- Assigned a potential E degree of 0.000 volt
What type of electrode is Calomel?
What does the Calomel reference electrode consist of?
Mercury covered by a layer of mercurous chloride in contact with saturated solution of potassium chloride
Name some types of reference electrodes
- Calomel electrode
- Silver-silver chloride (Ag/AgCl) electrode
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
What does a pH/blood gas analyzer use to measure blood pH?
pH sensitive glass electrode for measuring blood pH
What does a pH/blood gas analyzer use to measure gases in blood?
PCO2 and PO2 electrodes for measuring gases in blood
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
Name the different parts of a pH/blood gas analyzer used in measurements
- pH-sensitive glass electrode
- PCO2 and PO2 electrode
What does the PCO2 electrode specifically measure?
Partial pressure of carbon dioxide (PCO2)
What is the PCO2 electrode immersed in?
What type of potentiometric is the ion-exchange electrode?
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
How is NH4+ analyzed with the ion-exchange electrode?
Antibiotics nonactin and monactin used in combination as neutral carrer for NH4+, selective membrane
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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