Carbohydrates are one of the three essential nutrients; the other two are proteins and fats. Molisch’s, Benedict’s, Fehling’s, Tollen’s, Iodine and Bial’s test are the common tests for determining the presence of carbohydrates in the analytes.
Bial’s test helps separate pentose and pentose-derived compounds (pentosans) from other carbohydrates. Pentoses are simple sugars with five carbon molecules as the backbone. The most common examples of pentose and its derivatives are xylose, ribose, arabinose, hemicellulose, DNA (deoxyribonucleic acid), and RNA (ribonucleic acid).
Principle of Bial’s Test
Bial’s test aims to determine the presence of pentose and pentose-derived compounds in analytes. It also helps in the differentiation of pentose monosaccharides from other carbohydrates.
This test depends on the principle that when reagent reacts with a sample containing pentose or derivatives of pentose, it gives a blue-green complex. Here, derivatives of pentose degrades to pentose. Then, the dehydration of pentose occurs due to concentrated HCl (hydrochloric acid) in the Bial’s reagent forms furfural.
Pentose/pentose derived compounds + H+ (from concentrated HCl) → Furfural
Furfural + FeCl3 + Orcinol → Blue-green complex
The furfural reacts with orcinol in the presence of ferric ions to form a blue-green complex. The hexose monosaccharides in the sample react with conc. HCl forms a 5-hydroxymethyl furfural that reacts with orcinol in the presence of ferric ions, giving a muddy-brown complex.
Hexose/hexose derived compounds + H+ → 5-hydroxymethyl furfural
5-hydroxymethyl furfural + FeCl3 + orcinol → Muddy-brown complex
There are different materials required for performing this test. Reagents and equipment needed for the tests are Bial’s reagent, test tubes, dropper, and water bath.
Bial’s reagent, distilled water, xylose solution, and sample suspension are required reagents for performing this experiment. The reagent consists of orcinol, concentrated hydrochloric acid, and ferric chloride. The reagent must be fresh, i.e., prepared before performing the test.
Preparation of Bial’s reagent
- Prepare 10% ferric chloride by dissolving 10 g of FeCl3 in 100 ml distilled water.
- Dissolve 1.5 g of orcinol in 500 ml of concentrated HCl.
- Then, add 1 ml or 20 drops of the 10% FeCl3 solution.
- Finally, store the sample in a dark brown bottle before use. The reagent must be used within a couple of hours.
The equipment used and their purpose in this test are as follows:
- Test tube: It is a cylindrical tube made of glass. Its use is to mix the sample solution with the Bial’s reagent.
- Water Bath: It is the laboratory equipment that consists of hot water. It is used for heating the test tubes over constant temperature.
- Reagent bottle: The reagent bottle used in this test should be dark colored to preserve the reagent from degradation.
- Dropper: It helps to transfer the reagent from the bottle to the test tubes while performing the Bial test.
Procedure of Bial’s Test
The steps for performing Bial’s test are as follows:
- Label the first test tube as a positive control, the second as a negative control, and the third as a test.
- Then, in the tube labeled as a positive control, add 1 ml xylose solution; in the negative control, add 1 ml distilled water; and in the tube labeled as the test, add 1 ml of the sample solution.
- After that, add 1 ml of Bial’s reagent in all the tubes.
- Place all the tubes in a water bath for 3-5 minutes.
- Finally, note the change in color in all the tubes.
Result Interpretation of Bial’s Test
The result from the Bial’s test are interpreted as follows:
|Positive control (test tube with xylose)||Color changes from light green to blue-green||Positive Bial’s test|
|Negative control (test tube with water)||No change in color||Negative Bial’s test|
|Test (tube with samples)||1. Color changes to blue-green|
2. Color changes to muddy brown
|1. Positive Bial’s test (Presence of pentose or pentose derivatives)|
2. Negative Bial’s test (Absence of pentose or pentose derivatives)
The Bial’s test is used for analyzing carbohydrates in the samples. Following are the application of Bial’s test:
- The test helps differentiate pentose and pentose-derived carbohydrates from other carbohydrates.
- The Bial orcinol test, a modified version of this test, helps compute RNA concentrations.
Like other tests for determining carbohydrates, this test has various advantages. Some of them are as follows:
- The test procedure is simple and easy to perform.
- This test is less time-consuming, i.e., analyzing a single analyte takes less than an hour.
Like all procedures, this test also has some cons. Some of the shortcomings of this test are as follows:
- Prolonged heating can lead to false positive results due to the formation of gluconates. Hence care must be given to incubation time. Positive control also helps in mitigating this limitation.
- Different pentose sugars may give different colors; the intensity of the color does not correlate to the concentration of the sample.
- Mustansiriyah University. (2020). Bial`s Test. https://uomustansiriyah.edu.iq/media/lectures/6/6_2020_04_10!01_26_32_PM.pdf
- UNIVERSITY OF ANBAR. (n.d.). Bial’s test. https://www.uoanbar.edu.iq/eStoreImages/Bank/2063.pdf
- Tiwari, PhD, Anand. (2015). Practical Biochemistry: A Student Companion.