What is Bial’s test?

Bial’s Test is a type of chemical test which is used to determine the existence of pentoses. It was named in honor of Manfred Bial, a German doctor. The ingredients are orcinol, hydrochloric acid and ferric chloride. If Pentose is present it will dehydrated to form furfural that is then reacted with orcinol, resulting in a colored substance. The solution turns blue and a precipitate can develop. The solution has two absorption bands. One in red between Fraunhofer Lines B and C, and one close to that D line.

Objectives of Bial’s Test

• To identify whether carbohydrates are present.
• To distinguish the pentoses as well as pentosans from the other derivatives of carbohydrates such as the hexoses.

Principle of Bial’s test

This test can be used to differentiate between pentose monosaccharide as well as hexose monosaccharide. Bial’s reagent has concentrated HCl as dehydrating acid ferric chloride, orcinol as a condensation reagent. The test reagent dehydrates pentoses in order to make fufural and dehydrates the hexoses, resulting in 5-hydroxymethyl fufural Fufural reacts with ferric chloride and orcinol, to create a blue-green compound, while 5-hydroxymethyl fufural creates an muddy brown color complex.

Pentose + H+ = Fufural

Fufural + Orcinol + FeCl3 = Blue-green complex

hexose + H+ = 5-hydroxymethyl fufural

5-hydroxymethyl fufural + Orcinol + FeCl3 = muddy-brown complex

Requirements for Bial’s test

Chemicals/Reagents

• Bial’s Reagent: 300 mg orcinol dissolving in 5mL of alcohol. Add 3.5 milliliters of the mixture to 100ml of 0.1 percent solution of FeCL3.6H2O. The resulting reagent needs to be kept in a dark bottle , and used within a couple of hours.
• Ribose stock solution: Make 200g ribose for each 1mL of distilled water ribose stock solution using the stock solution. Be aware that if you require other carbs having similar concentrations may be used as sample. If you are using RNA, 300 grams per ml of stock solution containing RNA can be added to Tris-EDTA.

Glasswares

• Test tubes
• Test tube stand
• Pipettes
• Beaker
• Wash bottle.

Equipment

• UV Spectrophotometer
• Vortex mixer
• Mantle heater/Water Bath

Procedure of Bial’s test

1. In a clean, dry test tube, add 1ml of 5% ribose (pentose).
2. In the second test tube , add 1 milliliter of 5% glucose solution (hexose).
3. Each tube should be filled with 2.5 milliliters of Bial’s Reagent and mix thoroughly.
4. Keep the tubes in a the boiling water bath for 10 minutes, then allow for the tube to cool to room temperature. Then, measure how dense the solution’s optical properties are at 620nm against the blank.
5. Create an absorption standard curve against the concentration of ribose. Find the amount of ribose in the unknown by drawing a standard curve of A620 along the Y-axis as well as the level in Ribose along the X-axis.

Bial’s test results

• Positive Bial’s test: formation of blue color ( eg. Ribose sugar)
• Negative Bial’s test: Formation in any of the other colors signifies that the test was negative. Hexose sugar ( fructose, glucose) generally produces brown, red, or green color products.

Applications

• Bial’s test can be used to identify pentose as well as pentosans within a range of samples. 
• Bial’s test may also be used to detect what amount of RNA contained in an individual sample.

Limitations

• Glucoronates can create an emerald-blue-green-colored precipitate when they are heated for long periods of time that can cause false-positive results.
• When different sugars are used the colour produced can vary, and the intensity could not be proportional to the intensity at higher levels.

FAQ

References

• http://dept.harpercollege.edu/chemistry/chm/100/dgodambe/thedisk/carbo/bial/bials.htm
• https://www.chemeurope.com/en/encyclopedia/Bial%27s_Test.html
• https://biomedres.us/fulltexts/BJSTR.MS.ID.004515.php
• https://allmedtests.com/bials-test/
• https://biocheminsider.com/bials-test-objective-principle-procedure-reagent-and-results-interpretation/