Tests for

Carbohydrates
Prepared by: Brent Jericko P. Narciso, RPh.

Qualitative Analysis of Carbohydrates Introduction Carbohydrates are one of the most important constituents in foods. It
may be present as isolated molecules, physically associated, or chemically bound to other molecules. These substances are
divided into monosaccharides, disaccharides, oligosaccharides, and polysaccharides. Whereas, polysaccharides serve as a
site for storing energy.

These substances are polyhydroxy aldehydes and ketones or a substance that hydrolyzes to yield polyhydroxy aldehydes
and ketones. The major groups of carbohydrates are aldehydes and ketones.

Tests for Carbohydrates

TEST BASED ON THE REDUCING PROPERTIES OF SUGARS

REDUCING SUGARS
• Reducing sugar comes under the category
of carbohydrate or natural sugar but it
consists of either a free aldehyde group or
a ketone group.
The sugar structure with a free aldehyde or
the ketone group is called the reducing end
of sugar.
The end of the molecule with the free
anomeric carbon is referred to as the
reducing end.
• Common dietary monosaccharides:
glucose, galactose and fructose
Benedict’s Test:
 It is a very sensitive test done under mildly alkaline conditions.
 Benedict’s reagent: composed of CuSO4, Na2CO3, and Sodium
citrate
 Positive result: change of color of benedict’s reagent into brick
red precipitate.
 It is used to test for the presence of glucose in urine

 As in Fehling’s test, free aldehyde or keto

group in the reducing sugars reduce cupric
hydroxide in alkaline medium to red
colored cuprous oxide. Depending on the
concentration of sugars, yellow to green
color is developed.

 All monosaccharides are reducing sugars

as they all have a free reactive carbonyl group. Some disaccharides, like maltose, have exposed carbonyl groups and
are also reducing sugars, but less reactive than monosaccharides

Principle: When a benedict’s solution and simple carbohydrates are heated, the solution changes to orange/brick red.
This reaction is caused by the reducing property of simple carbohydrates. The copper (II) ions in the
benedict’s solution is reduced to copper (I) ions, which causes the color change.
IMPORTANT NOTES

o Blue solution: color of benedict solution

(no other reagent added)
o Green/yellow precipitate: traces of
reducing sugar.
o Orange red precipitate: moderate
reducing sugar present.
o Brick red: large amount of reducing
sugar.

Barfoed’s Test
 Barfoed’s reagent – contains cupric acetate in dilute acetic acid.
 It oxidizes monosaccharides but not oligosaccharides.
 Disaccharides are less oxidized but are oxidized if they undergo prolonged heating which causes hydrolysis of the
disaccharides into monosaccharides and produces a positive result.
 It is used to distinguish between monosaccharides, disaccharides and oligosaccharides.
 Positive Result: brick red precipitate
 Unlike benedict’s test, the test is carried out under acidic rather than basic medium.
 Barfoed's test is used to detect the presence of monosaccharide (reducing) sugars in solution. Barfoed's reagent, a
mixture of ethanoic (acetic) acid and copper (II) acetate, is combined with the test solution and boiled.
 A red copper (II) oxide precipitate is formed will indicates the presence of reducing sugar.
 The reaction will be negative in the presence of disaccharide sugars because they are weaker reducing agents. This
test is specific for monosaccharides. Due to the weakly acidic nature of Barfoed's reagent, it is reduced only by
monosaccharides.
 • Faster result: monosaccharide is present
• Slower result: disaccharide is present
• Remain blue if carbohydrate is absent
• Positive result: brick red precipitate

Tollen’s Test
• Also known as “Silver mirror test”.
• Sugars with aldehyde groups are capable reducing Tollen’s reagent.
• Tollen’s reagent: ammoniacal solution of silver.
• Positive result: gray to black precipitate (silver mirror)
• Active component is the ammoniacal silver nitrate.

Fehling’s Test
 This forms the reduction test of carbohydrates. Fehling’s
solution contains blue alkaline cupric hydroxide solution, heated with
reducing sugars gets reduced to yellow or red cuprous oxide and is
precipitated.

 Hence, formation of the yellow or brownish-red colored

precipitate helps in the detection of reducing sugars in the test solution.
Osazone Test
 Osazone test is a chemical test used to detect reducing sugars. This test even allows the differentiation of different
reducing sugars on the basis of the time of appearance of the complex. This test is also termed Phenylhydrazine
test based on the reagent used for this test.

 The ketoses and aldoses react with phenylhydrazine to produce a phenylhydrazone which further reacts with
another two molecules of phenylhydrazine to yield osazone.

 Needle-shaped yellow osazone crystals are produced by glucose, fructose and mannose, whereas lactosazone
produces mushroom shaped crystals. Crystals of different shapes will be shown by different osazones. Flower-
shaped crystals are produced by maltose.
TESTS BASED ON THE FORMATION OF FURFURAL AND ITS DERIVATIVES

Seliwanoff’s Test
 It is a color reaction specific for ketoses.
When conc. HCl is added ketoses undergo
dehydration to yield furfural derivatives more
rapidly than aldoses. These derivatives form
complexes with resorcinol to yield deep red
color.
 The test reagent causes the dehydration of
ketohexoses to form 5-hydroxymethylfurfural.
 5-hydroxymethylfurfural reacts with
resorcinol present in the test reagent to
produce a red product within two minutes.
Aldohexoses reacts so more slowly to form
the same product.

Molisch’s Test
 This is a common test for all carbohydrates larger than tetroses.
The test is on the basis that pentoses and hexoses are dehydrated
by conc. Sulphuric acid to form furfural or hydroxymethylfurfural,
respectively.
 These products condense with α-naphthol to form purple
condensation product.
Bial’s Orcinol Test
✓ Used to determine the presence of pentoses and nucleotides that contain pentose sugars.
✓ When pentoses are treated with orcinol, furfurals are formed and they will yield a blue-green compound in the
presence of ferric ions.
✓ The reaction is not specific for pentoses because other compounds like trioses, uronic acids, and certain heptoses
will also give blue or green products.
✓ Hydroxymethyl furfural is formed from hexoses to give yellow-brown condensation products.
✓ 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 a muddy
brown color complex.
Source:

 Essential Algebra for Chemistry Students, 2nd Edition

 Essentials of Inorganic Chemistry: For Students of Pharmacy, Pharmaceutical Sciences and Medicinal Chemistry
 https://microbenotes.com/barfoeds-test/#procedure-of-barfoeds-test
 https://vlab.amrita.edu/?sub=3&brch=63&sim=631&cnt=1
 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/