Locker #3 Date Performed: 12/16/19

Members:
1. Javier, Andrew A.
2. San Pedro, Krizia Corrine C.
3. Sia, Aleeya Hannah M.

Activity 5
Carbohydrates: Common and Specific Reactions

Introduction:
Carbohydrates are prevalent in plants and animals as they have crucial roles in the
structural composition and metabolism of these organisms. Carbohydrates are considered
to be important biomolecules that facilitate or take part in biochemical reactions.
Concomitantly, these enable carbohydrates to fulfill a gamut of functions, including:
primary energy reservoir; structural component; and cell-cell interaction initiator. For
example, plants synthesize carbon dioxide and water to produce glucose, which is then
stored as starch or used to synthesize the cellulose of their cell walls. In animals, various
carbohydrates carry various functions, including: glycogen for storage; ribose and deoxy
ribose as backbone constituents in nucleic acids; or galactose as lactose synthesizer
(Murray, Bender, Botham, Kennelly, Rodwell & Veil, 2012).
Only specific carbohydrates are capable of fulfilling the outlined functions. The
foregoing is dependent on the structure, or classification, of the carbohydrates. The latter
being polyhydroxy ketones or aldehydes, or aldehyde or ketone derivatives of polyhydric
alcohols, are classified as monosaccharides, disaccharides and polysaccharides. The first
type of carbohydrate, monosaccharides, is further classified according to the number of
carbon atoms present as trioses, tetroses, pentoses, hexoses, or heptoses, and as aldoses
(with aldehyde functional group) or ketoses (with ketone functional group); all of which
are single units of sugars (e.g., erythrose, xylose, glucose) that are incapable of undergoing
hydrolysis into simpler carbohydrates. The second type of carbohydrate, disaccharides, is
a condensation product of two monosaccharide units, including lactose, maltose and
sucrose. The last type of carbohydrate, polysaccharides, is a condensation product of more
than ten monosaccharide units, including starch, glycogen and dextrin; all of which may
be linear, branched or highly-branched polymers (Murray et al., 2012).

Objectives:
1. To describe the effects of the various test solutions on carbohydrates.
2. To elaborate how each test induces carbohydrates to undergo common and specific
reactions.

Results and Discussion / Interpretation:

Table 1. Common Reactions of Carbohydrates
Test Solution Result
1% glucose A purple/violet ring gradually formed a layer
at the interface
Molisch’s Test 1% xylose A purple/violet ring gradually formed at the
interface
 1% lactose A purple/violet ring gradually formed at the
interface
1% sucrose A purple/violet ring gradually formed at the
interface
1% starch
Unknown sugar
1% glucose Brick-red/reddish-brown precipitate formed
1% xylose Brick-red/reddish-brown precipitate formed
1% fructose Brick-red/reddish-brown precipitate formed
Fehling’s Test 1% lactose Brick-red/reddish-brown precipitate formed
1% maltose Brick-red/reddish-brown precipitate formed
1% sucrose No reddish-brown/brick-red precipitate
formed; no change in hue
1% starch No reddish-brown/brick-red precipitate
formed; no change in hue
1% glycogen No reddish-brown/brick-red precipitate
formed; no change in hue
Unknown sugar
1% glucose Brick-red/reddish-brown precipitate formed
1% xylose Brick-red/reddish-brown precipitate formed
Benedict’s Test Brick-red/reddish-brown precipitate formed
1% fructose Brick-red/reddish-brown precipitate formed
1% lactose Brick-red/reddish-brown precipitate formed
1% sucrose No change in hue; no precipitate formed
1% starch Retained blue hue; no precipitate formed
Barfoed’s Test 1% glucose Brick-red precipitate formed
1% fructose Brick-red precipitate formed
1% xylose Brick-red precipitate formed
1% lactose No change in hue (remained blue); no
precipitate formed
1% maltose No change in hue (remained blue); no
precipitate formed
1% sucrose No change in hue (remained blue); no
precipitate formed
Unknown sugar
Picric Acid Test 1% glucose Immediately turned mahogany-red
1% fructose Immediately turned mahogany-red
1% xylose Immediately turned mahogany-red
1% lactose Slowly turned mahogany-red
1% maltose Slowly turned mahogany-red
Tollen’s Test 1% glucose Silver-colored mirror-like solution formed

Figure 1. Reaction of Carbohydrates in Molisch’s Test

 Molisch’s test is a general test for all carbohydrates. It is conducted to test for the
presence of carbohydrates in a given solution. In order to achieve the foregoing,
carbohydrates, particularly monosaccharides, are treated with concentrated H2SO4 (sulfuric
acid) or concentrated HCl (hydrochloric acid) for dehydration. Following the dehydration
process, an aldehyde in the form of furfural (i.e., a pentose) or hydroxymethyl furfural (i.e.,
a hexose) derivatives are formed, as seen in Figure 1. The more complex carbohydrates,
disaccharides and polysaccharides, are first converted to monosaccharides via hydrolysis
of the glycosidic bonds present. Subsequently, the resulting monosaccharides will undergo
the dehydration as mentioned prior. From here on, the furfural derivatives react with
sulfonated 1-naphthol or α-naphthol (with a molecular formula and density of C10H8O and
1.10g/cm3, respectively), present in Molisch’s reagent, to form a purple ring via a
condensation reaction. Hence, a positive test will show the formation of a purple ring along
the point of contact of the liquid carbohydrate solution and Molisch’s reagent (Kari, 2018,
May 1).
Table 1 summarizes the results of the reactions of various carbohydrates with
sulfonated α-naphthol. Monosaccharides, including 1% glucose and 1% xylose,
monosaccharides, including, 1% lactose and 1% sucrose, a polysaccharide, 1% starch, and
an unknown sugar all reacted with sulfonated α-naphthol to form a purple ring along the
point of contact between them and the reagent. Moreover, the reagent is denser than the
test solutions from which the formation of the purple ring layer along the interface of the
two solutions is observed. In lieu of the principle of Molisch’s test, these solutions contain
carbohydrates; hence, the purple ring.
Fehling’s test is a sensitive qualitative test for carbohydrates as it tests for the
presence of reducing sugars. The latter refers to carbohydrates that have an anomeric
carbon with a free potential or aldehyde group with the capability to reduce solutions
(Murray et al., 2012). This test, along with Benedict’s and Barfoed’s test are characterized
by their goal of testing for the presence of reducing sugars. As such, these tests are
collectively called reduction tests.
Moving on, Fehling’s reagents comprise of two distinct solutions: aqueous copper
sulfate (CuSO4) solution or Fehling’s A; and alkaline sodium potassium tartrate
(C4H4O6KNa·4H2O or C4H4KNaO6) or Fehling’s B. These two solutions are mixed prior
to the test. The Fehling’s B reagent, in particular, acts as a chelating agent, which means
that it reacts with metal ions to form a stable, water-soluble complex. A sugar solution will
be added into the mixed Fehling’s reagent solution and heated subsequently. When a brick-
red or reddish-brown precipitate of cuprous oxide (as seen in Figure 2) gradually formed,
the added sugar solution is a reducing sugar; otherwise, the sugar has no reducing
properties. This precipitate is formed due to the reduction of the Cu2+ ions to Cu+ ions
(Karki, 2018, April 27).

Figure 2. Reaction of Carbohydrates in Fehling’s Test

Benedict’s test, as mentioned prior, is also a reduction test. As such, it is conducted

specifically to test for the presence of reducing sugars. Moving on, Benedict’s reagent is
composed of a bluish copper sulfate solution. Upon addition and mixing of a sugar solution
and heating in a water bath, changes in color will vary depending on the reducing properties
of the sugar. Reducing sugars will gradually produce a brick-red cuprous oxide precipitate,
whereas non-reducing sugars will not produce said precipitate and the boiled solution will
remain perfectly clean. Cuprous oxide is formed due to the oxidation of the carbohydrate
and reduction of the Cu2+ ions to Cu+ ions (as seen in Figure 3) and is insoluble in water;
thus, precipitated out of the solution (Biology Discussion, 2015, October 14). The color
and amount of the precipitate is dependent on the volume of the reducing sugar present –
the greater the amount of free carbonyl groups present, the greater the amount and intensity
of the brick-red precipitate.

Figure 3. Reaction of Carbohydrates in Benedict’s Test

Barfoed’s test is another reduction test but differs from the Fehling’s and Benedict’s
test because it is monosaccharide-specific. Mixing a batch Barfoed’s reagent, which is
comprises of cupric acetate and acetic acid in solution, with various carbohydrates and
heating them in a water bath will produce varying results. Reducing monosaccharides, in
particular, will be oxidized by the copper ion solution in order to form carboxylic acid and
a brick-red precipitate made of cupric oxide (CuO) or cuprous oxide (Cu 2O), as seen in
Figure 4, no more than two minutes after the heating initiated. However, more complex
carbohydrates will have a slower rate of precipitate formation, which is the case for
reducing disaccharides as they must first undergo hydrolysis (Carpio, n. d.; Biology
Discussion, 2015, October 14). Hence, Barfoed’s test serves as a mean to distinguish
monosaccharides from disaccharides, especially those with reducing capabilities.

Figure 4. Reaction of Carbohydrates in Barfoed’s Test

The Picric Acid Test is similar to Fehling’s, Benedict’s and Barfoed’s tests as they
test for the presence of reducing sugars but is a more sensitive test. The reducing sugars
react with picric cid (toxic yellow crystalline solid) also chemically known as 2,4,6-
trinitrophenol (TNP) to form a red colored picramic acid, as depicted in Figure 5. As
ascertained in the earlier discussion, all monosaccharides and disaccharides containing the
potentially free aldehyde or ketone group possess reducing properties; hence, reducing
sugars. These sugars are capable of reducing some organic acids in alkaline solution. Thus,
reducing sugars reduce picric acid to picramic acid with solution colors of yellow and
mahogany-red, respectively (Bioscience Notes: Online Biological Notes for Students,
2018, November 4).

Figure 5. Reaction of Carbohydrates in Picric Acid Test

Tollens’ test is a qualitative laboratory test specifically used to distinguish between

an aldehyde and a ketone. This test, also called the silver-mirror test, ascertains that
aldehydes are readily oxidized, whereas ketones are not. Tollens’ test uses a reagent known
as Tollens’ reagent, which is a colorless, basic, aqueous solution containing silver ions
coordinated to ammonia [Ag(NH3)2+]. In order to prepare this reagent, a two-step
methodology is observed.
Step 1: Aqueous silver nitrate is mixed with aqueous sodium hydroxide.
AgNO3(aq) + NaOH2(aq) → AgOH + NHO3
2AgOH → Ag2O + H2O
Step 2: Aqueous ammonia is added drop-wise until the precipitated silver oxide
completely dissolves.
Ag2O(aq) + 4NH3(aq) + H2O → 2Ag(NH3)12+ + 2OH−

Tollens’ reagent oxidizes an aldehyde into the corresponding carboxylic acid, as

seen in Figure 6. The aforementioned oxidation reaction is accompanied by the reduction
of silver ions present in Tollens’ reagent [Ag(NH3)2+] into metallic silver (Ag) from which
a silver-colored mirror can be observed in the test tube – if it is clear to begin with. Ketones,
however, are not oxidized by Tollens’ reagent, so the treatment of a ketone with Tollens’
reagent will not yield a silver mirror (LibreTexts, 2019, June 16). The result of the
experiment yielded a murky black mixture, which defied the aforementioned principle of
Tollen’s test.

Figure 6. Reaction of Carbohydrates in Tollen’s Test

Table 2. Specific Reactions of Carbohydrates

Test Solution Result
1% fructose Cherry-red complex
attained after 1 minute and
Seliwanoff’s Test 10 seconds
1% glucose
1% sucrose No change in hue
1% maltose No change in hue
Unknown sugar
1% xylose Green-yellow in hue after
Bial’s Orcinol Test 1 minute and 40 seconds
1% glucose Muddy color formed
1% lactose Muddy color formed
Unknown sugar
1% starch Stained deep blue-black in
Iodine Test hue
1% glycogen
1% glucose

Seliwanoff’s test is conducted to distinguish ketoses from aldoses. It specifically

identifies ketoses as they undergo the reaction at a much faster rate. Seliwanoff’s reagent
consists of a non-oxidizing acid (e.g., HCl) and resorcinol [C6H4(OH)2 or 1,3-C₆H₄(OH)₂].
As seen in Figure 7b, Seliwanoff’s reagent dehydrates ketohexoses, forming 5-
hydroxymethylfurfural as a product. The latter further reacts with the reagent’s resorcinol
in a condensation reaction (as seen in Figure 7a), producing a solution with a cherry-red
complex hue within approximately one to two minutes. Nevertheless, aldohexoses undergo
the same reactions to produce the same results albeit comparatively slower (Carpio, n. d.).
However, this is test may lose its specificity for testing for the presence of ketoses as time
elapses; and it does not differentiate the specific ketose present as it only proves the
presence of a ketose (Bioscience Notes: Online Biological Notes for Students, 2018,
November 4).

Figure 7. Reactions of Carbohydrates in Seliwanoff’s Test

(a) (b)

Bial’s Orcinol test is conducted to identify the presence of pentoses and distinguish
them from hexoses. Bial’s reagent is composed of 0.4g orcinol [CH₃C₆H₃(OH)₂], 200mL
of concentrated hydrochloric acid (HCl) and 0.5mL of a 10% solution of ferric chloride
(FeCl3). When Bial’s reagent is mixed with a variety of sugar and then heated in a water
bath, changes in color will be observed relative to the type of sugar used. As seen in Figure
8, the pentose sugar is hydrolyzed by HCl, forming a furfural derivative. Subsequently, the
latter reacts with CH₃C₆H₃(OH)₂, forming a blue-green or green-yellow complex via
condensation reaction in the presence of Fe3+ or ferric ion (Biology Discussion, 2015,
October 14; Bioscience Notes: Online Biological Notes for Students, 2018, November 4).
The formation of the color complex will occur at a quick rate for pentoses but opposite for
pentosans as they must undergo hydrolysis in order to break the glycosidic bonds.
Contrastingly, hexoses will form a complex of red to brown hue.

Figure 8. Reaction of Carbohydrates in Bial’s Orcinol Test

Iodine test is specifically conducted for starch. The latter, is a homopolymer of

glucose forming an α-glucosidic chain, hence a glucosan. This polysaccharide is composed
two main constituents: helical, non-branching amylose (13-20%); and branched
amylopectin (80-87%). Starch is composed of an interval of 24-30 glucoside residues with
α-1,4-glucosidic linkages in the chains an α-1,6-glucosidic linkage at the branch points
(Murray et al., 2012). Figure 9a shows the coiled structure of starch.
The amylose in starch is responsible for the formation of a blue-black color in the
presence of iodine. As depicted in Table 2, out of the three sugars used for this test, starch
formed the deepest and darkest color, which is correlated with the amount of amylose
present and due to it being a polysaccharide. The iodine molecule diffuses into the amylose
 coil (Figure 9a) and causes this reaction. The reagent used for this test is the Iodine - KI
reagent, which is made by dissolving iodine in water in the presence of potassium iodide
because iodine has low solubility in water. As a result, a linear triiodide ion complex
(Figure 9b) is produced (LibreTexts, 2019, June 6).

Figure 9. Reactions of Carbohydrates (i.e., starch) in Iodine Test

(a) (b)

Guide Questions:
1. Why do all sugars give a positive result for Molisch’s test?
2. What compounds other than α-naphthol can be used as a reagent for the test?
3. Why is it important to use freshly prepared Fehling’s reagent?
4. Compare the action of a strong and a weak alkali on a reducing sugar.
5. How are the conditions for Barfoed’s test different from those for Benedict’s test?
6. Write the chemical equation involved in the reaction between concentrated nitric acid
(HNO3) and glucose.
7. What is responsible for the intense blue color formed in the iodine test for starch?
8. What is the purpose of grinding with sand TCA in preparing glycogen?

Bibliography:
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Carbohydrates. Retrieved December 31, 2019, from Biology Discussion:
http://www.biologydiscussion.com/carbohydrates/test/qualitative-and-
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6IACN3-hUoQLJATfQOqPSwcaM
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Carpio, P. T. (n. d.). Reactions of Carbohydrates. Dasmariñas, Cavite, PH: De La Salle
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