EXPERIMENT NO: 11

Quantitative estimation o f pentose

sugar present in the given solution
of
carbohydrates
Presented To: Dr. Habib
by Bial's test.
Presented By: Hafiz. Muhammad Fawad

Date of Submission: 19-Dec-2023

Objective:
• To detect the presence of carbohydrates.

• To distinguish the pentose's and pentosans from other derivatives of carbohydrates like the

hexoses.

• This test is used to distinguish between pentose and hexose monosaccharides.

R equirements

A. Equipment: C. Reagents:
• UV Spectrophotometer • Bial’s Reagent: 300 mg of orcinol is dissolved
• Water Bath. in 5 ml ethanol. Add 3.5 ml of this mixture to
• Test tubes, 100ml of 0.1% solution of FeCL3.6H2O. The
• Test tube stand reagent thus formed is to be stored in a dark
• Beaker. bottle and used within a couple of hours.

B. Chemicals:
• Bial’s Reagent
• Ribose sugar
• Sample
Procedure

• Two ml of a sample solution is placed in a test tube. Two ml of Bial's reagent (a solution of

orcinol, HCl and ferric chloride) is added. The solution is then heated gently in a hot water

bath. If the color is not obvious, more sample can be added to the tube.
 Principle:
Bial's Test

• Conc. HCl acts on pentose's to foam furfurals.

• This condenses with orcinol to give green color compounds.

• B i a l ’ s t e s t i s u s e f u l i n d i s t i n g u i s h i n g p e n t o s e ' s s u g a r f r o m h e x o s e s s u g a r.

Pentose's(Such as Ribose sugar)form furfural in acidic medium which condense with

orcinol in the presence of ferric ion to give blue green colored complex.

• The concentration of the sugars is determined by measuring the absorbance of 620 nm

w a v e l e n g t h i n a s p e c t r o p h o t o m e t e r o r i n a r e d f i l t e r c o l o r i m e t e r.
Reactions
The test reagent dehydrates pentose's to form furfural. Furfural further reacts with orcinol and the iron
ion present in the test reagent to produce a bluish product.
R esu lt
The presence of a blue-green complex indicates the presence of pentose's in the sample.

Bial's Test
Positive result: Formation of
purple-blue or green color
indicates the presence of
pentose sugars.
Application

This test is used to detect the This test can additionally be used for
presence of pentose in different the quantification of RNA in a
samples. sample.
.
Limitations
1 Specificity
Bial's test is specific for pentose
sugars and may not detect other
types of carbohydrates.
2 Sample Compatibility
Not suitable for all types of carbohydrate solutions, and
additional tests or modifications may be necessary
depending on the sample characteristics.
P recau tio n s

• Follow rules
• Don’t drink
• Maintain distance
• Wear lab coat
References and Sources
• Tiwari A. (2015). Practical Biochemistry. LAP Lambert Academic Publishing.

• https://www.iitg.ac.in/biotech/MTechLabProtocols/Carbohydrate%20estimation%20by%20Anthrone.pdf

• https://en.wikipedia.org/wiki/Biuret_test

• https://www.slideshare.net/memijecruz/qualitative-analysis-of-carbohydrates

• https://www.graphpad.com/support/faq/prism-3-calculating-unknown-concentrations-using-a-standard-curve/

• https://msu.edu/course/lbs/145/luckie/Lab1.html

• https://en.wikipedia.org/wiki/Bial%27s_test

• https://www.enzolifesciences.com/science-center/technotes/2017/march/what-are-the-differences-between-pcr-rt-pcr-qpcr-and-rt-qpcr?/
Thank You