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Bial’s test for Carbohydrates

Bial’s test for carbohydrates post we briefly summarises about: principle, reagents requirements, procedure, result, application and limitations of Bial’s test.

Bial’s test for Carbohydrates

Bial’s test is a chemical test for the presence of pentoses. It is named after Manfred Bial, a German physician. The components include orcinol, hydrochloric acid, and ferric chloride. A pentose, if present, will be dehydrated to form furfural which then reacts with the orcinol to generate a coloured substance.

Principle

Bial’s test is used to distinguish between pentose monosaccharide and hexose monosaccharide. Bial’s reagent contains concentrated HCl as a dehydrating acid, orcinol and ferric chloride as condensation reagent. Bial’s test reagent dehydrates pentoses to form fufural and dehydrates hexoses to form 5-hydroxymethyl fufural, fufural reacts with orcinol and ferric chloride to produce blue-green complex, while 5-hydroxymethyl fufural produce muddy-brown color complex.

bial’s test

Requirements

Chemicals/Reagents

Bial’s Reagent: 300 mg orcinol is dissolved in 5 mL ethanol. Add 3.5 ml of this mixture to 100ml of 0.1% solution of FeCL3.6H2O. The resulting reagent should be stored in a dark bottle and utilised within a few hours.

Ribose stock solution: Prepare a 200g ribose per mL distilled water ribose stock solution from the stocked solution. Note: If required, other carbs with the same concentration can be used as samples. When using RNA, 300 g/ml of RNA stock solution is added to the Tris-EDTA buffer.

Glasswares

  1. Test tubes
  2. Test tube stand
  3. Pipettes
  4. Beaker
  5. Wash bottle.

Equipment

  1. UV Spectrophotometer
  2. Vortex mixer
  3. Mantle heater/Water Bath

Procedure

  1. In clean dry test tube add 1 ml of 5% ribose solution (pentose). In the second test tube add 1 ml of 5% glucose solution (hexose).
  2. For each tube add 2.5 ml of Bial’s reagent and mix well. Keep both tubes in boiling water bath 10 minutes and allow the tubes to cool down to room temperature and measure the optical density of the solutions at 620 nm against a blank.
  3. Prepare a standard curve of absorbance against ribose concentration. Determine the amount of ribose in the unknown sample by plotting a standard curve of A620 on the Y-axis and concentration of Ribose on the X-axis.

Result

Bial's test for Carbohydrates
  • The presence of a blue-green complex in the sample shows the presence of pentoses. 
  • The concentration of ribose sugar in the sample can be determined using the graph. RNA detection can also be interpreted in the same way.

Applications

  1. Bial’s test is used to detect pentose and pentosans in a variety of samples. Bial’s test can also be used to determine the amount of RNA in a sample.

Limitations

  1. Glucoronates can produce a blue-green coloured precipitate when heated for a long time, which can lead to false-positive results.
  2. With different sugars, the colour generated may differ, and the concentration may not be proportionate to the intensity at higher levels.

Further Readings

Reference