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Questions and Answers
How long can glucose oxidase reagent maintain its activity if refrigerated?
How long can glucose oxidase reagent maintain its activity if refrigerated?
What must be done to the concentration of glucose after preparing a protein-free supernatant from serum?
What must be done to the concentration of glucose after preparing a protein-free supernatant from serum?
Which reagent is used for protein precipitation and removal from serum?
Which reagent is used for protein precipitation and removal from serum?
What is the role of Cu2+ in the determination of blood glucose?
What is the role of Cu2+ in the determination of blood glucose?
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What is the function of 1N-H2SO4 in the blood glucose determination method?
What is the function of 1N-H2SO4 in the blood glucose determination method?
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What is the stability condition of standard glucose solutions at room temperature?
What is the stability condition of standard glucose solutions at room temperature?
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What does the formation of molybdenum blue indicate in colorimetric analysis?
What does the formation of molybdenum blue indicate in colorimetric analysis?
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What is the purpose of adding 0.5 ml of glucose oxidase in the preparation of the glucose oxidase reagent?
What is the purpose of adding 0.5 ml of glucose oxidase in the preparation of the glucose oxidase reagent?
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At what wavelength is colorimetric analysis performed in glucose detection?
At what wavelength is colorimetric analysis performed in glucose detection?
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What is the purpose of adding Na2SO4 in the process described?
What is the purpose of adding Na2SO4 in the process described?
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How is the acetate buffer prepared for use in the glucose oxidase reagent?
How is the acetate buffer prepared for use in the glucose oxidase reagent?
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What is the purpose of protein precipitation in the glucose determination method?
What is the purpose of protein precipitation in the glucose determination method?
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Which intermediate is formed during the oxidation process of D-Glucose?
Which intermediate is formed during the oxidation process of D-Glucose?
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Which solution contains a potential carcinogen and requires special care during handling?
Which solution contains a potential carcinogen and requires special care during handling?
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Which solution is essential in determining the calibration curve for detecting blood glucose?
Which solution is essential in determining the calibration curve for detecting blood glucose?
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What is the primary purpose of adding Na2WO4 and H2SO4 during the protein precipitation process?
What is the primary purpose of adding Na2WO4 and H2SO4 during the protein precipitation process?
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What is the first step in the process of protein precipitation from serum?
What is the first step in the process of protein precipitation from serum?
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Which solution is used as a blank in the blood glucose determination?
Which solution is used as a blank in the blood glucose determination?
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During spectrophotometric analysis, what is the wavelength utilized for reading absorbance?
During spectrophotometric analysis, what is the wavelength utilized for reading absorbance?
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What is the concentration of glucose in Working Standard 2 after dilution?
What is the concentration of glucose in Working Standard 2 after dilution?
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Why is it important to cool the test tubes after boiling for 10 minutes?
Why is it important to cool the test tubes after boiling for 10 minutes?
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What role does Harding's solution play in the assay?
What role does Harding's solution play in the assay?
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What is the first step when preparing the serum for glucose determination?
What is the first step when preparing the serum for glucose determination?
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What is the final volume to which test solutions are diluted after the addition of distilled water?
What is the final volume to which test solutions are diluted after the addition of distilled water?
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Study Notes
Reagents for Glucose Determination
-
Glucose Oxidase Reagent:
- Contains 0.5 ml glucose oxidase, 5 ml peroxidase solution, and 1 ml of 0.5% o-dianisidine-HCl in acetate buffer.
- Stores well in the refrigerator for at least two weeks.
-
Standard Glucose Solution:
- Prepared by dissolving 100 mg of glucose in 100 ml of 0.1% benzoic acid.
- This standard solution is equivalent to 100 mg glucose/100 ml.
- Glucose solutions are stable indefinitely at room temperature.
-
Protein Precipitation & Removal Reagents:
- Section A: Zinc Sulphate, Sodium Hydroxide, Distilled Water.
- Section B: Isotonic Sodium Sulphate, Sodium Tungstate, 2/3 N Sulphuric Acid.
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Glucose Determination Reagents:
- Harding's Solution: Alkaline Anticoagulant Medium.
- Nelson's Solution: Contains Arseno-molybdenate (toxic).
- Copper Sulphate Solution: (CuSO4).
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Standard Glucose Solutions (WS):
- Low Standard (WS1): Diluted 5.0ml of stock Standard Solution to 200ml with CuSO4 - NaSO4 solution (equivalent to 100mg/100ml of blood).
- High Standard (WS2): Diluted 10.0ml of stock Standard solution to 200ml with CuSO4-NaSO4 solution (equivalent to 200mg/100ml of blood).
Safety Precautions
-
Glucose Oxidase Reagent:
- Contains γ-dianisidine hydrochloride which is a potential carcinogen. Do not pipette or spill.
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Standard Glucose Solutions:
- Contains Benzoic Acid, a skin irritant. Flush exposed areas with water if spilled.
-
Zinc Sulphate:
- Can cause skin irritation. Flush exposed areas with water if spilled.
Glucose Determination Method (Section B)
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Method Overview:
- Uses a colorimetric method with Nelson's reagent to measure glucose concentration.
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Steps:
- Label 4 test tubes: B, S1, S2, and T (Blank, Standards 1 & 2, and Test).
- Add Harding's solution, CuSO4 solution, supernatant, and appropriate standard solutions as per the table.
- Place tubes in a boiling water bath for 10 minutes.
- Allow tubes to cool.
- Add 2 ml Nelson's arseno-molybdenate solution to each tube.
- Dilute to 25 ml in a volumetric flask with distilled water.
- Let the tubes stand for 5 minutes.
- Read absorbance in a spectrophotometer at a wavelength of 650 nm.
Glucose Oxidation Reaction
- Glucose is oxidized to gluconic acid.
- The oxidation reaction involves the following steps:
- Glucose is converted to a 1-2 endiol intermediate.
- Cupric ions (Cu2+) are reduced to cuprous ions (Cu+).
- Cu2+ (Cupric ions) react with the endiol intermediate to form Cu2O (Cuprous Oxide) and gluconic acid.
- Sodium sulfate (Na2SO4) is added to prevent reoxidation of Cu2O, ensuring that the Cu2+ ions are used solely in the oxidation reaction.
- Arseno-molybdenate (Nelson's reagent) reacts with the cuprous oxide (Cu2O) forming a green-blue "molybdenum blue" which is measured spectrophotometrically at 650nm.
Additional Notes
-
Protein precipitation and removal:
- Performed using different reagents in Sections A and B.
- Aims to remove proteins from the serum to obtain a clear supernatant for the glucose determination.
-
Blood Dilution:
- In Section A, the blood is diluted 25 times during protein precipitation.
- Remember to multiply the final glucose concentration by 25 to get the actual blood glucose concentration.
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Description
This quiz explores various reagents used in the determination of glucose levels, including glucose oxidase, standard glucose solutions, and protein precipitation agents. Additionally, it covers the preparation and stabilization of these reagents for effective laboratory use. Test your knowledge on this essential aspect of biochemical analysis.
