Acree-Rosenheim Test Definition, Principle, Procedure, Result, Uses

The Acree-Rosenheim Test, named after biochemists Solomon Farley Acree and Sigmund Otto Rosenheim, is a biochemical assay utilized to identify tryptophan molecules within a protein sample. This test holds significant importance in confirming the presence of formaldehyde in milk, as unscrupulous vendors may employ formaldehyde as a preservative. By employing this test, the quality of a milk sample can be assessed, ensuring that any excess milk is not tainted. A more straightforward method to ascertain the presence of formaldehyde in milk involves heating the sample with a few drops of concentrated hydrochloric acid (HCl), commonly referred to as the aldehyde test due to its reliance on detecting aldehyde molecules within the sample.

Objectives of Acree-Rosenheim test

1. Detection of Tryptophan: One of the primary purposes of the test is to identify the presence of tryptophan molecules within a protein sample. Tryptophan is an essential amino acid and its presence can provide valuable information about the composition of the protein.
2. Confirmation of Formaldehyde in Milk: Another crucial objective is to confirm the presence of formaldehyde in milk. This is significant as some unscrupulous vendors may use formaldehyde as a preservative, potentially compromising the quality and safety of the milk.
3. Quality Assessment of Milk: The test helps in assessing the condition of the milk sample. This is particularly important in ensuring that surplus milk is not spoiled or adulterated, which could pose health risks to consumers.
4. Simplicity and Accessibility: The Acree-Rosenheim Test provides a relatively simple and accessible method for detecting formaldehyde in milk. This allows for quick and practical on-site testing without the need for complex laboratory equipment.
5. Alternative to Formaldehyde Testing: The test serves as an alternative to more sophisticated and time-consuming laboratory techniques for detecting formaldehyde in milk. This makes it a valuable tool for routine quality control assessments.
6. Based on Aldehyde Detection: The test relies on the detection of aldehyde molecules in the sample. This specificity is essential for accurately identifying formaldehyde, which is an aldehyde compound.

Principle of Acree-Rosenheim test

1. Reagent Selection: The primary reagent used in the Acree-Rosenheim test is formaldehyde. This compound is introduced into the protein solution to initiate the reaction.
2. Detection of Tryptophan: The test is designed to identify the presence of tryptophan, an essential amino acid, within the protein solution.
3. Condensation Reaction: Formaldehyde undergoes a condensation reaction with two molecules of tryptophan. This reaction leads to the formation of a distinct compound.
4. Acidic Conditions: Sulfuric acid (H2SO4) is added to create an acidic environment. This acidic condition is crucial for promoting the condensation reaction between formaldehyde and tryptophan.
5. Phase Separation: The addition of sulfuric acid results in the formation of two distinct layers within the test tube. This separation aids in the visual identification of the reaction product.
6. Color Change: The condensation product formed as a result of the reaction between tryptophan and formaldehyde exhibits a violet color. This coloration serves as an indicator of the presence of tryptophan molecules in the sample.

Reaction:

Tryptophan + Formaldehyde + Tryptophan → Coloured Condensation Product + H2O

Requirements:

Reagents:

• Protein sample (with at least 1% tryptophan or other amino acids)
• Dilute Formaldehyde (CH2O)
• Sulfuric acid (H2SO4)

Materials:

• Test tubes
• Test tube stand
• Pipettes

Procedure of Acree-Rosenheim test

Materials Needed:

• Protein sample (containing at least 1% tryptophan or other amino acids)
• Dilute Formaldehyde (CH2O)
• Sulfuric acid (H2SO4)
• Test tubes
• Test tube stand
• Pipettes

Procedure:

1. Preparation of Protein Sample:
   • Take a known amount of the protein sample that is suspected to contain tryptophan. Ensure that it has a minimum of 1% tryptophan content.
2. Addition of Formaldehyde:
   • Add a few drops of dilute formaldehyde (CH2O) to the protein sample. The formaldehyde serves as the reagent that initiates the reaction.
3. Creation of Acidic Environment:
   • Introduce sulfuric acid (H2SO4) to the mixture. This creates an acidic environment, which is essential for the condensation reaction between formaldehyde and tryptophan.
4. Mixing and Reaction:
   • Gently mix the contents of the test tube to ensure thorough distribution of the reagents. Allow the reaction to proceed for a specific period of time (usually a few minutes).
5. Phase Separation:
   • Due to the addition of sulfuric acid, two distinct layers will form within the test tube. The upper layer may be clear or milky, while the lower layer may be more dense and possibly colored.
6. Observation:
   • Carefully examine the test tube for any color change. The condensation product resulting from the reaction between tryptophan and formaldehyde will typically exhibit a violet color.

Interpretation:

• If a violet-colored condensation product is observed, it indicates the presence of tryptophan in the protein sample.

Note:

• It’s important to handle formaldehyde with care, as it is a potentially hazardous chemical. Proper lab safety protocols should be followed.

Result and Interpretation of Acree-Rosenheim test

If a violet-colored condensation product forms after conducting the Acree-Rosenheim test, it indicates the presence of tryptophan in the protein sample.

Interpretation:

• The formation of a violet color indicates that the formaldehyde has reacted with tryptophan molecules present in the protein sample. This reaction is characteristic of the Acree-Rosenheim test.
• Tryptophan is an essential amino acid, and its presence in the protein sample can be confirmed through this test.
• In the context of milk analysis, if the test is positive for the presence of tryptophan, it suggests the potential presence of formaldehyde in the milk. This is important information for quality control, as formaldehyde should not be present in milk due to its potential health risks.
• It’s worth noting that a negative result (i.e., no violet coloration) does not necessarily imply the absence of tryptophan in the sample. It may be due to factors such as low tryptophan concentration, insufficient reaction time, or other experimental conditions.

Uses of Acree-Rosenheim test

1. Detection of Tryptophan in Proteins: The primary use of the Acree-Rosenheim Test is to identify the presence of tryptophan molecules within a protein sample. Tryptophan is an essential amino acid and its presence can provide valuable information about the composition of the protein.
2. Confirmation of Formaldehyde in Milk: This test is particularly significant in the dairy industry. It helps confirm the presence of formaldehyde in milk. Formaldehyde is sometimes used as a preservative by unscrupulous vendors, and its presence can be harmful to consumers.
3. Quality Assessment of Milk: The test aids in assessing the condition of milk samples. This is crucial for ensuring that surplus milk is not spoiled or adulterated, which could pose health risks to consumers.
4. Routine Quality Control Measures: The Acree-Rosenheim Test provides a relatively simple and accessible method for detecting formaldehyde in milk. This allows for quick and practical on-site testing without the need for complex laboratory equipment, making it an invaluable tool for routine quality control assessments.
5. Alternative to Sophisticated Laboratory Techniques: It serves as an alternative to more sophisticated and time-consuming laboratory techniques for detecting formaldehyde in milk. This makes it particularly useful in situations where immediate, on-site testing is required.
6. Food Safety and Regulatory Compliance: The test can play a role in ensuring compliance with food safety regulations. It helps identify and mitigate potential health risks associated with formaldehyde-contaminated milk products.
7. Research and Educational Purposes: The Acree-Rosenheim Test is also used in research settings and educational laboratories to teach principles of biochemistry and protein analysis.

Limitations of Acree–Rosenheim test

1. Specificity: The test specifically detects tryptophan, but it may not be as sensitive to other amino acids. Therefore, it may not be suitable for samples with low tryptophan content or high concentrations of other amino acids.
2. False Negatives: Factors such as low tryptophan concentration, incomplete reaction, or improper experimental conditions can lead to false negatives, where the test may not detect tryptophan even if it is present.
3. Interference from Other Compounds: Some compounds in the sample may interfere with the reaction, leading to inaccurate results. This can be a challenge when dealing with complex mixtures or samples with high chemical diversity.
4. Handling of Formaldehyde: Formaldehyde is a hazardous chemical and should be handled with care. Proper safety measures must be taken to ensure the safety of the operator.
5. Potential for Variability: The test results may vary depending on factors such as the skill of the operator, the quality of reagents used, and the consistency of experimental conditions. This can introduce some degree of subjectivity into the interpretation of results.
6. Limited to Amino Acid Analysis: The Acree-Rosenheim Test is specific to tryptophan detection and does not provide information about other amino acids or compounds present in the sample.
7. Not Quantitative: The test provides a qualitative result (positive or negative) indicating the presence of tryptophan, but it does not quantify the amount present. For quantitative analysis, additional methods would be required.
8. Not Suitable for High-Throughput Screening: The Acree-Rosenheim Test is a manual, time-consuming process that may not be suitable for high-throughput screening of large numbers of samples.

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