1.

) The Molisch Test

Molisch's Test is a sensitive chemical test for all carbohydrates and some compounds
containing carbohydrates in a combined form. It is based on the dehydration of the
carbohydrate by sulfuric acid to produce an aldehyde (either furfural or a derivative), which
then condenses with the phenolic structure to produce a red or purple compound.
Molisch's test is a chemical test used to detect the presence of carbohydrates in a given
analyte. This test is named after Czech-Austrian botanist Hans Molisch, who is credited with
discovering it. Molisch's test involves adding Molisch's reagent (a solution of -naphthol in
ethanol) to the analyte and then adding a few drops of concentrated H2SO4 (sulphuric acid) to
the mixture.

The formation of a purple or purplish-red ring at the point of contact between the H2SO4 and
the analyte + Molisch's reagent mixture confirms the presence of carbohydrates in the analyte.
The formation of a purple ring is a positive indicator for Molisch's test.

Molisch's Test procedure

2-3 drops of Molisch's reagent must be mixed thoroughly with a small amount of analyte in a
test tube. To facilitate the formation of a layer and avoid mixing, a few drops of concentrated
sulphuric acid must be added drop-wise along the walls of the test tube. The formation of a
purple ring at the concentrated acid layer is a positive indicator for Molisch's test. If no purple
or reddish-purple color appears, the analyte contains no carbohydrate.

…….
Molisch Test Applications
The Molisch test is used to determine the presence of carbohydrates in various samples.
It can be used to detect and distinguish the formation of carbohydrates as a by-product in
various reactions
Because trioses and tetroses lack the required five carbon atoms for furfural formation, they do
not produce a positive result in this reaction.
The Molisch test is not a carbohydrate-specific test. Some organic acids, such as citric acids,
lactic acid, oxalic acid, formic acid, and others, can produce furfurals or furfural-producing
substances.
Fehling's test
Fehling's test is a chemical method for distinguishing between reducing and non-reducing
sugars. This test can also be used to differentiate between carbohydrates with ketone
functional groups and carbohydrates with water-soluble functional groups. The test developed
by German chemist H.C. Von Fehling 
Fehling's Test procedure
Hermann von Fehling, a German chemist, performed the first Fehling's test in 1849. This test
involves heating aldehyde with Fehling's Reagent/solution. This process will eventually result in
the formation of a reddish-brown precipitate. This is because the solution oxidizes the
aldehyde, resulting in the formation of carboxylate anion.
Despite this, the aromatic aldehydes show no reaction to Fehling's Test. Ketones do not react
either. Using Fehling's reagents, we can easily distinguish between ketones and aldehydes with
such properties.
To perform Fehling's test,
place the sample to be tested in a dry test tube (preferably 1ml).
To serve as a control, distilled water should be placed in another test tube.
These test tubes are filled with Fehling's solutions (1ml of each solution A and B).
The tubes are then immersed in a bath of boiling water.
Observe and record any signs of the formation of the red precipitate.
If there is any formation of reddish-brown precipitate, the result is positive; if there is no
indication of such change, the result is negative.
…….
Fehling's Test Restrictions
This test is incapable of detecting aromatic aldehydes.
Only in an alkaline environment does this reaction occur. In an acidic environment, the copper
(II) ions would be stabilized and difficult to oxidize, resulting in the reaction failing.
Barfoed’s Test:
 
 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.

A positive test is indicated by:

The formation of a reddish precipitate within three minutes.

How to perform the test:

One ml of a sample solution is placed in a test tube. Three ml of Barfoed's reagent (a solution of
cupric acetate and acetic acid) is added. The solution is then heated for three minutes in a
boiling water bath.
Within three minutes, the copper ion in solution oxidizes reducing monosaccharides to form a
carboxylic acid and a reddish precipitate of copper (I) oxide. Reducing disaccharides undergo
the same reaction as reducing monoccharides, but at a slower rate.
…
Glucose and galactose were positive in the test since they are monosachharide and a positive
test shows for the reducing monosaccharides. The saccharin is also a monosaccharide and
tested positive but I doubted it at somepoint but base on what I’ve read online mono
means ‘single’ and saccharin means ‘sugars’, hence these are termed as the simplest form of
carbohydrates. They consist of one sugar unit which cannot be further hydrolyzed or broken
down into simpler ones so I wrote here positive in and monosaccharide in the saccharin table.
While sucrose tested negative since it is a disaccharide. Starch and cellulose are polysaccharides
and since poly specifies a number higher than one so I opted to write negative.
Reducing monosaccharides are oxidized by the copper ion in solution to form a carboxylic acid
and a reddish precipitate of copper (I) oxide within three minutes. Reducing disaccharides
undergo the same reaction but the delay in the development of the color
Benedict's Test
Benedict's Test is used to determine the presence of simple carbohydrates. The Benedict's test
identifies reducing sugars (mono- and disaccharides) with free ketone or aldehyde functional
groups. The presence of glucose in urine can be detected using Benedict's solution. When
reducing sugars are heated in the presence of Benedicts reagent, a reduction reaction occurs,
causing the Benedicts reagent to change color. Depending on the amount and type of sugar,
the color can range from green to dark red (brick) or rusty-brown.
Procedure of Benedict’s Test

1. Approximately 1 ml of sample is placed into a clean test tube.

2. 2 ml (10 drops) of Benedict’s reagent (CuSO4) is placed in the test tube.
3. The solution is then heated in a boiling water bath for 3-5 minutes.
4. Observe for color change in the solution of test tubes or precipitate formation.

Brick red with heavy precipitate =  large amount of reducing sugar is present
Yellow with precipitate =  small amount of reducing sugar is present
Blue color or cloudy = No reducing sugar is present

All monosaccharides are reducing sugars. Some disaccharides are reducing sugars, and some
are not.
Benedict's solution turns orange when it reacts with individual glucose molecules. Glucose is a
simple molecule made of one sugar unit. Sucrose, or "table sugar," is made of two different
sugar units, glucose and fructose, bonded together. Starch is made up of hundreds of glucose
sugar units, bonded together in long chains. It does not react with the bonded glucose
molecules in sucrose or starch. They have no reducing sugar present in them and are blue in
terms of color.
Glucose is called reducing sugar because it is capable of transferring hydrogen to other
compounds. Such a process is known as reduction or redox reaction.
starch is unable to be formed the open aldehyde and as a result unable to be oxidized and
reduced other sugars
Polysaccharides do not test positive for reducing sugars unless they undergo a hydrolysis
reaction (by heating or digestion) during which the polysaccharides are broken down to form
monosaccharides. Since starch and cellulose are polysaccharide they do not give a positive
result in Benedict’s test unless they are broken down through heating.
Tollens' test 
Tollens' test is a chemical test used to distinguish between reducing and non-reducing sugars. 
Because of the end result of this test, it is also known as the silver mirror test.
Procedure of Tollens’ test
1. Take two clean, dry test tubes and add 1 ml of the test sample in one test tube and 1 ml of
distilled water in another as blank.
2. Add 2 ml of Tollen’s reagent to both the test tubes.
3. Keep both test tubes in a water bath for 1 min.
4. Observe the formation of color and note it down.

 The formation of a dark grey precipitate or silver mirror on the bottom and sides of the test
tube indicates a positive result, which means that the given sample contains reducing
sugars/ aldoses.
 The absence of such precipitate indicates a negative result, which means that the test
sample doesn’t have reducing sugars/ aldoses/ α-hydroxy ketoses.

When the carboxylate ion is acidified, the corresponding carboxylic acid is formed. Because the
reaction takes place in an alkaline environment, the carboxylic acid is not formed directly. This
means that glucose and galactose are the only substances that produce a dark grey precipitate
or a silver mirror end product in this test.
Because ketones cannot be easily oxidized, they produce a negative result. A ketone does not
have an available hydrogen atom attached to the carbonyl carbon, making it more difficult to
oxidize than an aldehyde, which does. In this test, sucrose, starch, saccharin, and cellulose all
showed a negative result.
Seliwanoff's test 

Seliwanoff's test is a biochemical test developed in 1887 by Russian chemist Theodore Seliwano
ff. 
The primary goal of the test is to distinguish between Aldoses and ketose sugars. 
If the sugar contains a ketone group, it is called a ketose; if it contains an aldehyde group, it is c
alled an Aldose.

The test is based on the idea that when exposed to acid, ketoses dehydrate faster than aldoses. 
Acid hydrolysis of polysaccharide and oligosaccharide ketoses produces simpler sugars, which a
re then followed by furfural. 
In a series of condensation reactions, the dehydrated ketose reacts with two equivalents of res
orcinol to produce a molecule with a deep cherry red color.
When Seliwanoff reagent is added to a ketose-containing solution, a red color forms quickly,
indicating a positive test. When added to an Aldoses-containing solution, a slower-forming light
pink is observed.

Procedure of Seliwanoff’s test

1. Take two clean, dry test tubes and add 1 ml of the test sample in one test tube and 1 ml of
distilled water in another as blank.
2. Add 2 ml of Seliwanoffs’ reagent to both the test tubes.
3. Keep both the test tubes in a water bath for 1 min.
4. Observe the formation of color and note it down.

 The formation of the cherry red-colored complex indicates a positive result which means
that the given sample contains ketoses.
 The absence of such color or the appearance of the color after a prolonged period of time
indicates a negative result which means that the test sample doesn’t have ketoses.

Sucrose, starch, saccharin and cellulose are ketoses and a ketose reacted with this reagent, it
becomes dehydrated and a cherry-red complex forms (not a precipitate). Aldoses which are
glucose and galactose also react with this reagent, but much more slowly than ketoses.
IODINE TEST
This test is specific for polysaccharides and is used to distinguish polysaccharides from other
carbohydrates. Starch and glycogen contribute positively. It can also be used to distinguish
glycogen, starch, and cellulose.
The absorptive properties of large polysaccharide molecules are used in the iodine test. In most
polysaccharides, the glucose chains are organized to form helices. Small iodine molecules can
be held in the space between the helix's turns. This is demonstrated by amylase chains found in
starch. These iodine molecules can also be absorbed by glycogen and amylopectin on their
surfaces. When polysaccharides are heated, their absorptive property decreases.
For the Procedure

1. 2 ml of the given solution will be placed in a test tube

2. And Add 1-2 drops of iodine reagent in the above test tube
3. Wait for some time and observe for the color
4. And this procedure will be performed using those six carbohydrate solutions

In this test a
Dark blue color indicates a positive result
And a yellow or brown color appears to be negative

It was said that we will be able to distinguish starch from glucose (and other carbohydrates)
using this iodine solution test. Glucose in iodine test doesn’t show any positive test but instead
Benedict’s reagent can be used to test for glucose and will give a positive result.
A chemical test for starch is to add an iodine solution and because of the presence of starch,
iodine turns into a blue/black colour.
The sucrose saccharin and galactose turned negative in result. And for the cellulose it turned
negative even though it is a polysaccharide for this case Cellulose doesn't contain complex
helices and cannot hold Iodine so theres no reaction takes place.
Answers for Glycolysis and Gluconeogenesis

1.     The BMI is calculated as the weight of the person (in kg) divided by the height
squared (in meters). Thus, for this patient, the BMI is equal to 45.85 divided by (1.67)2, which
is 16.44. The BMI stands for body mass index and can be used to estimate body fat content.
A value of <18.5 is considered underweight, values between 18.5 and 24.9 are considered in
the normal range, values of 25 through 29.9 are considered overweight, and values of 30 or
greater are considered obese. Values of 40 or more are considered morbidly obese, whereas
values between 35 and 40 are considered obese.

2.     Providing substrate for glyceraldehyde-3-phosphate dehydrogenase. Under anaerobic

conditions, the NADH generated by the glyceraldehyde-3- phosphate dehydrogenase step
accumulates. Normally, the NADH would transfer its electrons to mitochondrial NAD+, and
the electrons would be donated to the electron transfer chain. However, in the absence of
oxygen, the electron transfer chain is not functioning. Thus, as NADH accumulates in the
cytoplasm, the levels of NAD+ decrease to the point that there would be insufficient NAD+
available to allow the glyceraldehyde-3- phosphate dehydrogenase reaction to proceed,
thereby inhibiting glycolysis. To prevent glycolytic inhibition, lactate dehydrogenase will
convert pyruvate to lactate, regenerating NAD+ for use in glycolysis, specifically as a
substrate for the glyceraldehyde-3-phosphate dehydrogenase reaction. While hexokinase is
inhibited by its product glucose-6-phosphate, this allosteric effect does not explain lactate
formation under anaerobic conditions. Similarly, while phosphoglyceromutase does require
2,3-bisphosphoglycerate, anaerobiosis does not increase 2,3-bisphosphoglycerate levels,
nor does it alleviate the lack of NAD+ under these conditions. Pyruvate kinase is not
inhibited by pyruvate (ATP and alanine are the allosteric inhibitors of this enzyme). AMP is
an activator of phosphofructokinase-1; however, this activation does not relate to lactate
formation under anaerobic conditions.

3.     Three moles of ATP. When glycogen produces glucose via the action of glycogen
phosphorylase, glucose-1-phosphate is produced. As this is converted to two molecules of
pyruvate, four moles of ATP is generated and one is utilized at the PFK-1 step for the net
production of three moles of ATP. Two moles of NADH are also produced, but those are
utilized by lactate dehydrogenase to reduce pyruvate to lactate (anaerobic conditions) such
that NAD+ can be regenerated for the glyceraldehyde-3-phosphate dehydrogenase step. A
small amount of free glucose will be released from glycogen by the debranching enzyme
(about 5% of the total); for that glucose, the net yield is two moles of ATP (since hexokinase
has to phosphorylate the free glucose to glucose-6-phosphate), but since the majority of
glucose released is in the form of glucose-1-phosphate, three moles of ATP is the better
answer.
4.     Phosphoglycerate kinase. In gluconeogenesis, phosphoglycerate kinase catalyzes the
phosphorylation of 3-phosphoglycerate to 1,3-bisphosphoglycerate, a step that requires
ATP. The other two steps requiring a high-energy phosphate bond in the conversion of
pyruvate to glucose are pyruvate carboxylase and phosphoenolpyruvate carboxykinase.
Fructose- 1,6-bisphosphatase and glucose-6-phosphatase are enzymes that remove
phosphates from substrates, releasing the phosphates as inorganic phosphate. They do not
require, nor generate, ATP. Pyruvate kinase is not utilized for gluconeogenesis and triose
phosphate

5.     Pyruvate carboxylase. The body’s major energy source for gluconeogenesis is fatty

acids, which are oxidized to acetyl-CoA, at which point acetyl- CoA enters the TCA cycle to
produce ATP. Acetyl-CoA activates pyruvate carboxylase (and inhibits pyruvate
dehydrogenase), a key gluconeogenic enzyme. Acetyl- CoA does not regulate any of the
other enzymes listed as potential answers (PEPCK is transcriptionally regulated by CREB;
Fructose-1,6 bisphosphatase is inhibited by fructose-2,6-bisphosphate; glucose-6-
phosphatase is regulated by a regulatory protein; and pyruvate kinase has both allosteric
and covalent controls in the liver, but none involve acetyl-CoA).

6.     2-phosphoglycerate. Fluoride inhibits the glycolytic enzyme enolase, which catalyzes

the dehydration of 2-phosphoglycerate to phosphoenolpyruvate. Thus, 2-phosphoglycerate
accumulates under these conditions.

7.     Na+, K+ ATPase. Most monosaccharides are transported with sodium from the

intestinal lumen into the enterocyte. The energy for active transport of the carbohydrate is
derived from the sodium gradient that is established by the Na+, K+ ATPase, which pumps
sodium out of the cell (three atoms of sodium) in exchange for potassium (two atoms of
potassium). This creates both a sodium gradient (outside concentration higher) and a
charge gradient (outside positive as compared to inside the cell) across the plasma
membrane. Due to these gradients, the entry of sodium into the cell is energetically
favorable, and the monosaccharide piggybacks with the sodium for transport into the cell.
The Na /H+ exchanger is not operative in intestinal epithelial cells and none of the other
enzymes (glucose-6-phosphate dehydrogenase, hexokinase, and chloride transporter) will
create the necessary sodium gradient for monosaccharide transport.

8.     Enhanced production of fructose-2, 6-bisphosphate. When phosphorylated, heart PFK-

2 is activated to produce more fructose-2,6-bisphosphate to stimulate heart PFK-1 and to
increase the glycolytic rate of the heart. Phosphorylation of heart PFK-2 can be
accomplished through the AMP-activated protein kinase (when the heart is having trouble
generating energy) or in response to insulin (indicating that high levels of glucose are
available for use). Phosphorylation of heart PFK-2 does not affect its transcription or
turnover rate, and also does not affect the degradation of fructose-1, 6-bisphosphate.
Answers

1.     a-ketoglutarate dehydrogenase. The alcoholic has become deficient in vitamin B1,

thiamine, which is converted to thiamine pyrophosphate for use as a coenzyme. One of the
symptoms of B1 deficiency is neurological, due to insufficient energy generation within the
nervous system. B1 is required for a small number of enzymes, including transketolase,
pyruvate dehydrogenase, and α-ketoglutarate dehydrogenase. By reducing the activity of
the latter two enzymes, glucose oxidation to generate energy is impaired, and the nervous
system suffers because of it.

2.  When acetyl-CoA enters the TCA cycle and is converted to two molecules of carbon
dioxide and oxaloacetate is regenerated, three molecules of NADH are produced, along with
one molecule of FADH2 and one substrate-level phosphorylation resulting in the generation
of GTP. As each NADH can give rise to 2.5 ATP, and each FADH2 to 1.5 ATP via oxidative
phosphorylation, the net yield of high-energy bonds from one acetyl-CoA being oxidized by
the cycle is 10 (7.5 from NADH, 1.5 from FADH2, and 1 from GTP).

3.     Ethanol’s carbons are lost as carbon dioxide before a gluconeogenic precursor can be
generated. Ethanol is converted to acetaldehyde, which is further oxidized to acetic acid and
is then activated to acetyl-CoA. The acetyl-CoA enters the TCA cycle to generate energy,
and two carbons are lost for each turn of the cycle as CO2. Thus, ethanol cannot provide
carbons for the net synthesis of glucose. Ethanol is not converted to acetone, nor is it
directly lost in the urine. Ethanol is primarily oxidized in the liver, and its carbons cannot be
used for the biosynthesis of lysine, which is an essential amino acid for humans.

4.     Cytoplasmic malate dehydrogenase. The cytoplasmic malate dehydrogenase is

required in the liver as part of the malate/aspartate shuttle in transferring reducing
equivalents across the inner mitochondrial membrane. In the absence of such an activity,
NADH levels will build up in the cytoplasm (since the electrons cannot be transferred to the
mitochondrial matrix) and will lead to the reduction of pyruvate to lactate to regenerate
NAD+ for other cytoplasmic reactions. A defect in glucokinase will block glycolysis, with no
pyruvate or lactate formation from glucose. The same is true for an inactivating mutation in
PFK-1. If pyruvate kinase were defective, PEP would accumulate, which cannot be converted
to lactate without forming pyruvate first. A defect in glycerol-3-phosphate dehydrogenase
will prevent the glycerol-3-phosphate shuttle from transferring electrons to the
mitochondrial matrix, but the liver uses primarily the malate/aspartate shuttle for this
activity.

5.     Fasting blood glucose. A pyruvate carboxylase deficiency will impair gluconeogenesis

from lactate and pyruvate, thereby leading to fasting hypoglycemia more easily than a
pyruvate dehydrogenase deficiency (which will primarily affect the ability to generate energy
from carbohydrates). Alanine aminotransferase activity in the blood is a measure of liver
damage, which would not distinguish between the two possibilities. Free fatty acid levels
would be the same under both conditions, during fasting conditions, as would insulin and
glucagon levels.

6.     Adenylate cyclase. If adenylate cyclase is defective, glucagon cannot initiate the

activation of glycogenolysis and inhibition of glycolysis in the liver (cAMP levels will not
increase, and PKA will stay inactive). Under such conditions, only the allosteric effectors in
the liver will be active, and there is no activator of glycogen phosphorylase b. When the
hypoglycemia is severe enough, epinephrine release, working through its α-receptors, will
activate phospholipase C, leading to calcium release. The increased calcium can activate
phosphorylase kinase, which will activate phosphorylase, but fasting hypoglycemia will still
occur. Defects in liver PFK-1 or glucokinase will not affect glycogenolysis or
gluconeogenesis. Defects in the liver galactokinase or fructokinase will not allow for the
metabolism of galactose or fructose but do not affect the ability of the liver to degrade
glycogen or perform gluconeogenesis from other precursors.

7.     One high-energy bond. For a molecule of glucose-6-phosphate (G6P) to be

incorporated into glycogen, the following pathway must be utilized: G6P is converted to
glucose-1-phosphate (G1P) via phosphoglucomutase, the G1P reacts with UTP to form
UDPglucose via glucose-1-phosphate uridyl transferase, releasing pyrophosphate. The
resultant pyrophosphate is hydrolyzed to two inorganic phosphates, with the loss of one
high-energy bond. The UDP-glucose then reacts with glycogen to produce a glycogen chain
with one additional sugar, and UDP is released. The overall equation for these steps is: G6P
+ UTP + glycogenn  yields UDP + 2Pi + (glycogen)n+1.

Laboratory

Lipids are organic substances insoluble in water but soluble in organic solvents, adopting
the principle of “like dissolves like”. The physical and chemical properties of lipids are due to
the presence of carboxyl groups, a number of double bonds, and hydroxyl groups, and the
length of the carbon atoms chain. In this experiment, the focus is to characterize lipids.

The following are the tests to be performed, with their corresponding underlying principles:

1.     Test for unsaturation. Palmitic acid is a saturated fatty acid. Oleic acid is a  monoenoic
fatty acid. When unsaturated fatty acids are treated with halogens like iodine, they add
across the double bond. The color of the halogen disappears.
 

2.     Acrolein formation. When lipids are heated in the presence of KHSO4, a  characteristic
odor of acrolein is produced.

3.     Saponification. Coconut oil is a triacylglycerol. On treatment with alkali, it

is  hydrolyzed to glycerol and salts of fatty acid. When NaCl is added, sodium salts of fatty
acids precipitate. On acidification, few fatty acids separate and float.

4.     Liebermann-Burchardt reaction. A chloroform (carbon tetrachloride) solution

of  sterol. When treated with acetic anhydride and concentrated sulfuric acid, it gives a
characteristic color.

5.     Salkowski test.  Sterols undergo a dehydration process in the presence of sulfuric

acid  to give 3,5-cholestadiene, which dimerizes to bis-cholestadiene.