Nelson's Test for Equivalents of Reducing Sugar (Essay Sample)

Analysis of Glucose
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Nelson’s Test for Equivalents of Reducing Sugar
Introduction
Reducing sugars are those sugars containing aldehyde groups, oxidized to carbolic acids. The aldehyde group is the functional group that allows the sugars to be reducing sugars. The most common test reagents for the reducing sugars are Benedict’s reagent (CUSO4 citrate) and Fehling’s reagent (CUSO4 tartrate). The reducing sugars reduce the Cu2+ ions to Cu+ therefore forming a red precipitate of copper (I) oxide. Therefore, any sugars containing hemi-acetyl is a reducing sugar. A sugar is therefore seen as a reducing sugar, only if it is in form of an open chain with a free hemiacetel group or an aldehyde group (Ian, 2013).
Purpose: To determine the molar concentration of reducing sugars in a sample of guinea pig plasma
Procedure
Prepare standards in screw top test tubes containing 0.1, 0.2, 0.4 0.6, and 0.8 μmol of glucose with 2ml final volume. Dilute with water. Prepare sample using 30 ul of guinea pig serum.
Prepare an appropriate blank.
Mix the Nelson A reagent with Nelson B reagent at a ratio of 25: 1. Prepare 8ml of this mixture.
Add 1ml of Nelson A+B mixture to each screw top test tube.
Heat the tubes in a vigorously boiling water bath and heat for 20 minutes.
Transfer tubes into a beaker of cold water and incubate for 10minutes.
Add 1ml of Arsenomolybdate reagent to each tube.
Dilute tubes to 10ml with H2O.
Measure absorbance at 540nm. Construct a standard curve of A540 versus micromoles of Reducing Sugar (glucose).
Based on the A540 of your plasma sample and the volume of plasma used in the assay, determine the molar concentration of reducing sugar in your guinea pig plasma sample.
Glucose assay reagent
Introduction
Glucose is a monosaccharide having the chemical formula C6H12O6. They are classified according to two major properties, the type of functional group and the number of carbon atoms. Glucose has six carbon atoms and contains the aldehyde group (-CHO). Glucose is very important in that it is a major energy source, plays an important role in the synthesis of non-carbohydrates and is a component of other carbohydrates (American Association for Clinical Chemistry, 2013).
Purpose: To determine the molar concentration of glucose in a sample of guinea pig plasma.
Procedure
Prepare 13.0 ml of glucose assay reagent :
50 mM Tris, pH 7.5 1.5 mM NADP+ 1.0 mM ATP 1.0 units/ml hexokinase 1.0 units/ml glucose-6-phosphate dehydrogenase 5 mM MgCl2
Aliquot 2.0 ml of glucose assay reagent into 6 cuvettes.
Select four concentrations of glucose within the range of 35mM and 3mM inclusively for standard curve preparation (Maximize the range). Prepare 1ml of each standard.
Add 20μl of glucose standard to a cuvette containing glucose assay reagent.
Add 20μl water to a cuvette containing glucose assay reagent.
Add 20μl of hamster plasma to a cuvette containing glucose assay reagent.
Incubate the cuvettes at room temperature for 10min. It is important that the reaction be allowed to run to completion.
Add 1.0ml of H2O to each cuvette.
Take absorbancies at 340 nm. Construct a standard curve of A340 versus micromoles of glucose.
Based on the A340 of your plasma sample and the volume of plasma used in the assay, determine the concentration of glucose in your plasma sample.
Discussion
Discussion and Analysis
Determine concentration of component analyzed in the serum
In the first chart, the concentration component analyzed in the serum will follow the following
y = -2E-05x + 0.6589R² = 0.9675
From the above equation, one can suggest that there is a negative relationship existing between the absorption of glucose and its concentration.
In the second, chart, the concentration component analyzed in the serum will follow the following
y = 1.0489x - 0.0059R² = 0.9975
From the above equation, one can suggest that there is positive relationship existing between the absorption of glucose and its concent...