A Study on Bial’s Test and its objectives, principles & more

Bial’s test is known to be a Biochemical test which is used for detecting the presence of pentoses (A five-carbon monosaccharide) and pentosans (derivatives of pentoses). The classification of Monosaccharides is done according to the number of carbon atoms in a Monosaccharides. Specifically ,Pentose is a monosaccharide which has five carbon atoms. A monosaccharide that contains an aldehyde group (-CHO) at position 1 is known as Aldose ,while on the contrary a Ketose is the one Monosaccharide that contains a ketone group (C=O) at position 2 or 3. Thus, a Pentose containing aldehyde group is called aldopentose. On the other hand , a pentose sugar with a ketone functional group located in position 2 or 3 is known as ketopentose.

Ribose has a chemical formula C5H10O5 and on the other hand Deoxy-ribose is the constituent of nucleotides and nucleic acid.

Bial’s test has been named after Manfred Bial, who was a German physician. The components of Bial’s reagent consists of orcinol, hydrochloric acid, and ferric chloride. If there is the presence of pentose sugar in the solution , it will be dehydrated by the dehydrating reagent to form furfural which further then reacts with the orcinol and ferric chloride to generate a colored substance.

The Bial’s test can also be performed colorimetrically as a quantitative analysis test using a spectrophotometer. There have been some procedures published by Fernell and King for simultaneous determination of pentoses and hexoses by taking measurements at two wavelengths in the Spectrophotometer. This test is also called the orcinol test when it is used for a quick chemical determination of RNA .

Objectives of Bial’s Test

• It is used for detecting and determining the presence of carbohydrates ,that is a Qualitative test for carbohydrates.
• It also helps in differentiating the pentoses and pentosans from various other derivatives of carbohydrates like the hexoses.

Principle of Bial’s Test

The basis of the principle of this test is that when put under hydrolysis , Pentosans are hydrolyzed into pentoses. After that ,further , the pentoses are dehydrated by the test reagent to yield the furfural. Furfural ,which in turn undergoes the condensation reaction with the orcinol and ferric chloride(condensation reagent) to form a blue-green coloured precipitate.

In contrast ,if hexoses are present in solution , 5-hydroxyfurfural is formed on hydrolyzation (HCl acts as a dehydrating reagent)instead of furfural product ,which further upon performing the condensation reaction with orcinol and ferric chloride yields a precipitate which is muddy brown colored. The magnitude or intensity of the precipitate Formed is directly proportional to the concentration of the pentoses in the sample. The precipitate formed and the intensity of the color of these precipitates yielded is dependent on the concentration of reagent that are – HCl, ferric chloride, orcinol, and on the the duration of boiling the test solution with reagent. Then using a spectrophotometer or a red filter colorimeter the absorbance of wavelength at 620 nm is measured and the concentration of the sugars is estimated.

Insert Image of reaction

Requirements

Materials required

• Equipment:
  • A UV Spectrophotometer
  • A Vortex mixer
  • Water bath
  • 1 Magnetic stirrer with hotplate
  • 1 Thermometer, -10…+150 °C/1 K..

• Chemicals/Reagents:
  • Bial’s Reagent
  • Ribose sugar
  • Other carbohydrates ( if desired)
  • Test Sample

• Glasswares and other equipment:
  • 1 Glass stirring rod
  • 1 Spatula, micro double ended
  • 1 Dropping pipette, 7 x 150mm
  • Dry Test tubes, 30 x 200 mm
  • 1Test tube stand
  • 1 Measuring cylinder 100 ml
  • 1 Beaker, Boro 3.3, 600 ml
  • 1 Boiling stones 250
  • Test tube caps
  • Tissue paper
  • Wash bottle
  • Ice

Reagents Preparations

• Bial’s Reagent: it is prepared by weighing 300 mg of orcinol and is dissolved in 5 ml ethanol. Next , 3.5 ml of this mixture is added to 100ml of 0.1% solution of FeCL₃.6H2O.
• Ribose stock solution: here , 200µg ribose per mL distilled water stock solution of ribose is to be prepared from the stocked solution.

Note: if desired ,Other carbohydrates of the same concentration can be used as test samples. In case , If RNA is used, 300 µg/ml of RNA stock solution is to be added to the Tris-EDTA buffer.

Procedure of Bial’s Test

1. Take cleaned and dried test tubes for the test.
2. Pour different volumes (50 µl, 100 µl, and so on up to 500µl) of ribose solution from the supplied stock solution of ribose ( conc. 200µg /ml) by using a pipette into a number of series of test tubes that are labelled accordingly and then make up the volume to 1 mL by adding appropriate volume of distilled water.
3. Now, a tube labeled as one is taken as a blank or control for the test which contains 1ml of just distilled water and the remaining test tubes are labeled 2 to 9 for the measurement ,calculation and construction of a standard curve for the Bial’s test Assay for carbohydrates. And the Tubes numbered 10-15 are taken for the unknown samples.
4. In this step , 5 ml of the bial’s reagent is added to each test tube containing the test sample and mixed well by vortexing.
5. Cool the test tubes.
6. The test Tubes are Covered with caps on top and then are incubated at 90°C for 17 minutes or boiling water bath for 10 minutes
7. At room temperature bring the test tubes to Cool and then measure the optical density of the test solutions at 620 nm wavelength against a blank test tube.
8. After collecting the Optical density ,calculate and Prepare a standard curve of absorbance against the concentration of Ribose sugar.
9. Estimate the amount of ribose sugar present in the unknown sample by plotting a standard curve on a graph that is Absorbsnce at 620 nm on the Y-axis and concentration of Ribose on the X-axis.

Result and Interpretation of Bial’s Test

Positive Bial’s test result: the formation of blue color precipitate in the reaction ( eg. Ribose sugar)
Negative Bial’s test result: the formation of any other color indicates the negative test results.

Hence, The presence of a blue-green precipitate complex in the test tubes indicates the presence of pentoses in the test sample. Following those readings that are noted down for the absorbance, a standard curve is prepared by using the graph, thus, the concentration of ribose sugar can be calculated and determined. A very similar interpretation and determination can also be performed for the Detection of RNA as well.

Precautions:

• While heating the test tube in the water bath ,be careful and keep your distance from the water bath.
• Use dropping pipette or a dropper to put the reagent in the corresponding test tubes.
• Keep the Bial’s reagent in a dark bottle after preparing it ,and use it within a couple hours.
• While the test tubes are in a water bath there should be no prolonged heating , because the glucoronates might also give a blue-green colored precipitate which might result in false-positive results.
• Perform the test with precision and accuracy because sometimes the color produced in the test might be different with the different sugars, and the concentration obtained might not be proportional to the intensity at the higher levels.

REFERENCES

https://en.wikipedia.org/wiki/Bial%27s_test
https://www.academia.edu/35559469/Bials_Test