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Questions and Answers
What aspect of a biochemical assay ensures that color development remains accurate for absorbance readings?
What aspect of a biochemical assay ensures that color development remains accurate for absorbance readings?
Why is it important to select an appropriate range of standards during a biochemical assay?
Why is it important to select an appropriate range of standards during a biochemical assay?
What is the consequence of choosing a sample volume that produces too low an absorbance reading in the assay?
What is the consequence of choosing a sample volume that produces too low an absorbance reading in the assay?
How many points should typically be included on the standard curve during a biochemical assay?
How many points should typically be included on the standard curve during a biochemical assay?
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In the context of a biochemical assay, what is the effect of selecting a volume of unknown solution that corresponds to an amount of a compound outside the range of the standards?
In the context of a biochemical assay, what is the effect of selecting a volume of unknown solution that corresponds to an amount of a compound outside the range of the standards?
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What is gravimetric determination primarily used for in biochemical assays?
What is gravimetric determination primarily used for in biochemical assays?
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Why are indirect assay procedures commonly used in biochemical analyses?
Why are indirect assay procedures commonly used in biochemical analyses?
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What role does a reagent blank play in an assay?
What role does a reagent blank play in an assay?
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Which of the following is a desired property of a biochemical assay?
Which of the following is a desired property of a biochemical assay?
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What does Beer’s Lambert Law relate in the context of standard curves?
What does Beer’s Lambert Law relate in the context of standard curves?
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In the context of biochemical assays, what is meant by specificity?
In the context of biochemical assays, what is meant by specificity?
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Which method is primarily used to calculate the amount of a specific compound in a sample?
Which method is primarily used to calculate the amount of a specific compound in a sample?
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What is the importance of measuring absorbance relative to a reference solution?
What is the importance of measuring absorbance relative to a reference solution?
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Study Notes
Biochemical Assays
- An assay determines the concentration of a given substance in a sample.
- Direct assays are used to weigh the amount of a particular compound or class of compounds in a given sample.
- This is called a gravimetric determination.
- Indirect assay procedures are used since compounds usually cannot be assayed directly in biological samples.
- This is because the compound of interest is usually part of a complex mixture of biomolecules, making it impossible to simply weigh a particular compound separately.
Indirect Assays
- Indirect assays involve a series of steps:
- Selecting a chemical reaction specific for the compound of interest.
- Reacting known amounts of that compound with the appropriate reagents to produce measurable products (standards).
- Measuring the response produced by a known volume of sample using the same procedure used for the standards.
- The absorbance of the colored product can be measured relative to water (absolute absorbance) or relative to a reference solution (reagent blank).
- A reagent blank contains zero amount of the compound being assayed. This means the sample volume is replaced with solvent alone (e.g., water or buffer) and all assay steps are followed.
- The blank is used to set the spectrophotometer to zero absorbance, ensuring the standard curve passes through zero.
- The purpose of the reagent blank is to correct for any absorbance contribution made by the reagents used in the color development.
Nature of Standard Curves
- Standard curves are often linear.
- Beer–Lambert Law states that A = kcl, where A is absorbance, k is a constant, c is concentration, and l is path length.
- Desired properties of an assay include:
- High sensitivity: the assay should detect very small amounts of the compound of interest.
- Specificity: the reaction should only be specific for the compound of interest, unaffected by other substances.
- Stability: the color development should be stable for accurate absorbance readings.
- Proportionality: the color change produced should be proportional to the amount of compound present.
Steps Involved in Performing Biochemical Assays
- Choose the assay.
- Choose the standards.
- Decide on an appropriate range for each assay.
- The range should be characteristic of the assay's sensitivity.
- Determine the number of points on the standard curve (usually 5 to 6) and their values.
- Set up standards:
- Use a known concentration of the standard solution appropriate for the assay.
- Calculate the volumes of the standard solution required to achieve the desired amounts of the compound.
- Choose the appropriate volumes of the unknown solution to assay.
- Aim to choose a volume of unknown solution that will absorb approximately in the middle, linear section of the standard curve.
- A chosen volume that results in an amount of unknown substance that is too small might lead to unreliable results or a too low absorbance reading.
- A volume that results in an amount of unknown substance that is too large might lead to an absorbance reading outside the range covered by the standards.
- Carry out the assay procedure:
- It is recommended to assay unknowns and standards simultaneously.
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Description
Explore the fascinating world of biochemical assays, focusing on both direct and indirect methods for determining the concentration of substances in biological samples. Learn about gravimetric determination and the complexities involved in measuring compounds within mixtures. This quiz offers insights into the chemical reactions and measurement techniques utilized in these assays.
