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

Acree-Rosenheim Test is a biochemical test that detects the presence of tryptophan molecules in a protein sample. Tryptophan is one of the 20 amino acids that make up proteins and has a unique structure with an indole ring. The test is based on the reaction of tryptophan with formaldehyde, which produces a violet-colored compound under acidic conditions. The test can also be used to detect formaldehyde in milk samples, as some milk vendors may use formaldehyde as a preservative to prevent spoilage.

The test is named after Solomon Farley Acree and Sigmund Otto Rosenheim, two biochemists who developed the test in the early 20th century. Acree was an American chemist who studied the chemistry of proteins and amino acids, while Rosenheim was a German-British biochemist who worked on the metabolism of purines and pyrimidines. They collaborated on several research projects and published their findings on the Acree-Rosenheim test in 1912.

The Acree-Rosenheim test is also known as the aldehyde test, as it relies on the presence of aldehyde groups in formaldehyde and tryptophan. The test is simple, quick, and inexpensive to perform and can be used to identify proteins that contain tryptophan or to check the quality of milk samples. However, the test has some limitations, such as being specific only for tryptophan and being affected by the shaking or mixing of the test tube. Therefore, the test should be used with caution and in conjunction with other methods to confirm the results.

The main objectives of the Acree-Rosenheim test are:

• To detect the presence of tryptophan in a protein sample. Tryptophan is one of the essential amino acids that cannot be synthesized by the human body and must be obtained from dietary sources. Tryptophan is also a precursor for the synthesis of serotonin, melatonin, and niacin in the body. Therefore, it is important to identify the proteins that contain tryptophan and their concentration in food and biological samples.
• To detect the presence of formaldehyde in a sample. Formaldehyde is a toxic and carcinogenic compound that can be used as a preservative or adulterant in some food products, especially milk. Formaldehyde can cause various health problems such as irritation, allergy, asthma, and cancer. Therefore, it is essential to check the quality and safety of food products by testing for the presence of formaldehyde. The Acree-Rosenheim test can be used as a simple and rapid method to detect formaldehyde in milk samples.

The principle of the Acree-Rosenheim test is based on the reaction of formaldehyde with tryptophan molecules in a protein sample. Formaldehyde is a simple aldehyde that can form a condensation product with two tryptophan molecules in acidic conditions. Tryptophan is an aromatic amino acid that has an indole ring in its side chain. The indole ring can react with formaldehyde to form a violet-colored compound.

The reaction can be represented as follows:

\ce{2 Trp + CH2O -> (Trp-CH2-Trp) + H2O}

Where Trp stands for tryptophan and (Trp-CH2-Trp) is the violet-colored condensation product.

The test is performed by adding a few drops of dilute formaldehyde and concentrated sulfuric acid to the protein solution. The sulfuric acid acts as a catalyst and also separates the solution into two layers: an upper aqueous layer and a lower acid layer. The violet-colored compound forms at the junction of the two layers, indicating the presence of tryptophan in the protein sample.

The test can also be used to detect formaldehyde in a milk sample, as formaldehyde is sometimes added as a preservative to prevent spoilage. In this case, the milk sample is heated with a few drops of concentrated sulfuric acid, which converts the lactose (milk sugar) into formic acid and formaldehyde. The formaldehyde then reacts with any tryptophan present in the milk proteins to form the violet-colored compound.

The principle of the Acree-Rosenheim test is simple and effective, but it has some limitations. It can only detect tryptophan and not other amino acids, so a negative result does not rule out the presence of other proteins. Also, some proteins, such as gelatin, are deficient in tryptophan and may give a false-negative result. Moreover, the test requires careful handling of the reagents and avoiding shaking of the test tube, as this may cause mixing of the two layers and interfere with the result.

The Acree-Rosenheim test requires the following reagents and materials:

• Reagents
  • Protein sample (1% tryptophan or other amino acids)
  • Dilute formaldehyde (CH2O)
  • Sulfuric acid (H2SO4)
• Materials
  • Test tubes
  • Test tube stand
  • Pipettes

The protein sample can be any solution that contains tryptophan or other amino acids. For example, egg albumin can be used as a protein source, but it should be prepared as a 5-10% solution. Alternatively, milk can be used as a sample to test for the presence of formaldehyde as a preservative.

The dilute formaldehyde is the main reagent that reacts with tryptophan molecules to form a colored condensation product. The concentration of formaldehyde should be low enough to avoid excess reaction and interference with other amino acids.

The sulfuric acid is used to create acidic conditions and to separate two distinct layers in the test tube. The concentration of sulfuric acid should be high enough to prevent the mixing of the layers and to enhance the color of the condensation product.

The test tubes are used to hold the sample and the reagents. They should be clean and dry before use. The test tube stand is used to support the test tubes in a vertical position. The pipettes are used to transfer the reagents into the test tubes. They should be rinsed with distilled water before and after use.

The procedure of Acree-Rosenheim test varies depending on the type of sample being tested. For protein samples, the following steps are followed:

• About 2-3 ml of protein solution is added in a test tube. If egg albumin is to be used, 5-10% solution is to be prepared.
• To the test tube, a few drops of dilute formaldehyde are added.
• Thereafter, few drops of concentrated sulfuric acid are added from the side of the test tubes.
• The shaking of the test tube should be avoided to prevent the mixing of the two layers.
• The formation of the violet-colored ring at the junction of two layers is observed.

For milk samples with preservatives, the following steps are followed:

• In the case of milk samples, a few drops of concentrated acid (H2SO4) are added to the milk sample.
• The mixture is then heated to obtain a purple-colored solution.

The procedure of Acree-Rosenheim test is simple and quick, and can be performed in a laboratory or at home. However, some precautions should be taken while performing the test, such as:

• The reagents used for the test are corrosive and toxic, so they should be handled with care and disposed of properly.
• The test tubes should be clean and dry before adding the reagents.
• The test should be performed in a well-ventilated area to avoid inhaling the fumes from the reaction.

The result of the Acree-Rosenheim test depends on the appearance of a purple ring at the junction of two layers in the test tube. The purple ring is formed by the condensation product of tryptophan and formaldehyde in acidic conditions. The presence or absence of the purple ring indicates the presence or absence of tryptophan in the protein sample.

• Positive result: A positive result of the Acree-Rosenheim test is represented by the appearance of a purple ring at the junction of two layers, indicating the presence of tryptophan in the protein sample. A positive result can also be obtained by heating a milk sample with sulfuric acid and formaldehyde, which produces a purple-colored solution. A positive result indicates that the milk sample contains formaldehyde as a preservative.
• Negative result: A negative result of the Acree-Rosenheim test is represented by the absence of a purple ring at the junction of two layers, indicating the absence of tryptophan in the protein sample. A negative result can also be obtained by heating a milk sample with sulfuric acid and formaldehyde, which produces no color change. A negative result indicates that the milk sample does not contain formaldehyde as a preservative.

The Acree-Rosenheim test is a simple and quick method to detect tryptophan and formaldehyde in samples. However, it has some limitations that should be considered while interpreting the results. For example, some proteins or amino acids might interfere with the reaction and produce false-positive or false-negative results. Therefore, it is advisable to use other confirmatory tests along with the Acree-Rosenheim test for accurate identification of tryptophan and formaldehyde in samples.

The Acree-Rosenheim test has several applications in biochemistry and food science. Some of the uses of the test are:

• The test is used to detect the presence of distinct proteins and amino acids in a sample. By comparing the results of the test with different protein solutions, one can identify the protein that contains tryptophan. For example, if the test is positive for egg albumin and negative for gelatin, it means that egg albumin contains tryptophan while gelatin does not.
• The test is the standard test for the detection of tryptophan in a sample. Tryptophan is one of the essential amino acids that humans cannot synthesize and must obtain from their diet. Tryptophan is also a precursor for serotonin, melatonin, and niacin, which are important neurotransmitters and vitamins. Therefore, detecting tryptophan in food or biological samples can help assess the nutritional value and physiological functions of the sample.
• A modified version of this test is also used for the detection of formaldehyde in a milk sample. Formaldehyde is a toxic chemical that can cause irritation, allergy, cancer, and death if ingested. Some milk vendors use formaldehyde as a preservative to prevent spoilage and increase shelf life. However, this practice is illegal and harmful to consumers. By heating the milk sample with a few drops of concentrated sulfuric acid, one can observe the formation of a purple-colored solution if formaldehyde is present. This method is simpler and faster than the original Acree-Rosenheim test and can help detect adulteration in milk.

• The test can only be used for the detection of tryptophan, so a negative result doesn’t rule out the presence of other amino acids. Some proteins may contain other amino acids that can react with formaldehyde and produce different colors, such as tyrosine (yellow), phenylalanine (green), or histidine (red). Therefore, the test is not specific for tryptophan and may give false-positive results in some cases.
• Gelatin might give a negative Acree-Rosenheim test as it is deficient in tryptophan. Gelatin is a protein derived from collagen, which lacks tryptophan. Therefore, gelatin samples will not show the purple ring in the Acree-Rosenheim test, even though they contain protein. This may lead to false-negative results and misinterpretation of the protein content of gelatin samples.
• The shaking of the test tube might result in the mixing of the two layers, which might result in false-negative results. The Acree-Rosenheim test relies on the formation of a distinct purple ring at the junction of two layers: the protein-formaldehyde layer and the sulfuric acid layer. If the test tube is shaken or disturbed, the two layers may mix and dilute the color intensity of the ring. This may make it difficult to observe the ring or cause it to disappear altogether. Therefore, it is important to avoid shaking or moving the test tube during the test.

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