RESULTS AND DISCUSSION

A. Positive Reactions for Carbohydrate Tests

Each tests indicates the qualitative analysis of carbohydrates. A certain color represents positive
results according to the tests.
Table 1. Reactions of Carbohydrates

Samples Molisch Iodine Benedict Barfoed Seliwanoff 2,4-DNP

Glucose positive - positive positive - positive
Galactose positive - positive positive - positive
Ribose positive - positive - - positive
Fructose positive - positive positive positive positive
Lactose positive - positive - - positive
Sucrose positive - - - positive positive
Starch positive positive - - - -
Unknown 1 positive - positive positive positive positive
Unknown 2 positive - positive - - positive
Identity of Unknown 1 Fructose
Identity of Unknown 2 Lactose

Molisch’s test yielded a positive result when the carbohydrates were treated with H2SO4. All kinds
of carbohydrates including monosaccharide, disaccharide and polysaccharide gave positive results to this
test. This happened because polysaccharide and disaccharide hydrolyzed by the acid to form
monosaccharides. The monosaccharides then dehydrated because of the removal of the –OH group in the
form of water. When the reaction happened, pentoses dehydrated to form furfural, or when the sugar was
hexoses, it dehydrated to form hydroxymethyl furfural. The derivatives then reacted with sulphonated α-
naphthol in the reagent to give a deep purple color complex. All samples including unknown one and two
gave positive results. This implied that unknown samples were carbohydrates (1).

Figure 1. Reaction of Pentose to Furfural

 Figure 2. Reaction of Hexose to Hydroxymethyl Furfural

Iodine test marked the presence of starch distinguishing the polysaccharide (starch) from
monosaccharides and disaccharides. This happened because solution of iodine-potassium iodide (I2KI)
interacted with amylose, which is the coiled polymers of starch, and became bluish black in color. The helix
shaped amylose gave starch an ability to trap iodine. The trapped iodine formed charge transfer complexes
and became excited. When the reaction happened, light was absorbed by the compounds and showed
dark blue color. Carbohydrates that are non-coiled did not react with the solution. Only starch contained
coiled glucose polymers, so the test gave negative results for all sample except starch. Thus, a blue-black
color indicated positive result for starch, while yellow-brown color indicated negative results (2). As an
answer to guide question, when the starch is hydrolyzed by enzyme in body, the starch breaks down and
is converted to sugars. As the process happens, there will be lesser starch to react with iodine. Thus, the
residue of starch hydrolysis reduces its reaction with iodine (3).
Benedict’s test identified the reducing sugars which contains free ketone or aldehyde functional
groups. The sugars included all monosaccharides and some disaccharides. When the benedict’s solution
was mixed with the carbohydrates, hydrogens from free ketone and aldehyde were transferred to cupric
ions with the help of heat. This caused cupric ions to be reduced and caused cupric ion to become cuprous
ion. The reaction gave positive result which showed formation of a rust colored precipitate. On the other
hand, a negative result yielded was met with no color change, meaning there is an absence in reducing
sugars (4). Benedict’s test can also be used to identify sugar concentration in urine of a diabetic patient.
Normal urines do not contain glucose in it. However, when there is excessive amount of sugar in body,
glucose starts to appear in one’s urine. When urine are heated with the reagent, copper (II) sulphate
(CuSO4) in the reagent is reduced by glucose in urine and causes a color change. The change in color of
urine depends on the concentration of glucose. Blue indicates absence of sugar, green indicates 0.5 to 1%
of sugar concentration, yellow indicates 1 to 2 % sugar concentration, and brick red color indicates 2 % or
more sugar concentration (5).

Figure 3. Benedict’s Reaction

Barfoed’s test gave positive results only to monosaccharides. This occurred because the low pH of
Barfoed’s reagent could only be reduced by monosaccharides, which were strong sugar reducing agent.
Monosaccharides can reduce sugar fast compared to disaccharide and polysaccharide because of
presence of free aldehydes. (6) The monosaccharides’ reducing reaction causes acetic acid and copper(II)
acetate to become red copper(II) oxide precipitate. Glucose, galactose, fructose, and unknown 1 gave
positive results to the test.
Seliwanoff test was specific test for aldolases containing aldehyde groups and ketoses having
ketone group. When heated, ketose in carbohydrates dehydrated faster than aldolase to form 5-
hydroxymethylfurfural courtesy of the HCL in the reagent. Thereafter, the product condensates with
resorcinol in the reagent to give a red complex (7). Aldohexoses reacted significantly slower to form the
same products. Fructose, sucrose, and unknown 1 samples gave positive results. Sucrose yielded positive
result since it is a disaccharide that possessed fructose and glucose.

Figure 4. Seliwanoff’s Reaction

2,4-DNP (2,4-Dinitrophenylhydrazine) test is used to identify the presence of aldehydes and

ketones that reacted with the reagent to form a yellow precipitate. The formation of precipitate indicated a
positive result in the test since it indicated the presence of aldehyde and ketones. The reaction of propanone
to 2,4 DNP undergoes a condensation reaction in which one in two molecules join together with the loss of
a small molecule in the process. The small molecule in the reaction is the water, which is often omitted
since it is a byproduct (8).

Figure 5. 2,4 DNP Reaction

To conclude, carbohydrates tests were utilized to identify the presence, class, and groups of
carbohydrates to distinguish it from one another. Each tests had different reaction mechanisms in order to
differentiate and produce a derivative of the sample and reagent. The unknown samples were identified
through cross-analyzing the given carbohydrates. It was found out that unknown sample 1 is fructose
base from the same reaction as the sample fructose. Likewise, unknown sample 2 was identified to be
lactose since its tests were the same with the sample lactose. Identification of carbohydrates were not
only applied, but it was found that the tests served a higher purpose in the medical field. Certain tests
such as the Benedict’s test were employed to check the sugar concentration of urine in a diabetic person.
References

(1) Harper College. The Molisch Test Retrieved 29 April 2019 from
http://dept.harpercollege.edu/chemistry/chm/100/dgodambe/thedisk/carbo/molisch/molisch.htm

(2) Goedecke, C. Why Does Iodine Turn Starch Blue? 2016 Retrieved 29 April 2019 from
https://www.chemistryviews.org/details/education/10128441/Why_Does_Iodine_Turn_Starch_Blue.html

(3) LibreTexts. Starch Hydrolysis. 2019. Retrieved 29 April 2019 from

https://bio.libretexts.org/Ancillary_Materials/Experiments/Microbiology_Labs_I/28%3A_Starch_Hydrolysis

(4) Michigan State University. Testing Organic Compounds. Retrieved 29 April 2019 from
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(5) Nedungadi P., Raman R. & McGregor M. Detection of Sugar in Urine. 2013. Retrieved 29 April 2019
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https://vlab.amrita.edu/?sub=3&brch=63&sim=631&cnt=1
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seliwanoffs-test-bials-test-and-iodine-test/
(8) ChemGuide. ADDITION-ELIMINATION REACTIONS OF ALDEHYDES AND KETONES. 2015.
Retrieved 29 April 2019 from https://www.chemguide.co.uk/organicprops/carbonyls/addelim.html