UNIVERSITY OF RWANDA

COLLEGE OF SCIENCE AND TECHNOLOGY

SCHOOL OF SCIENCE
DEPARTMENT OF CHEMSTRY
BIOORGANIC OPTION
MODULE: CHEMICAL ANALYSIS AND INSTRUMENTION
LEVEL 3
Group members:

 Félicien SIBOMANA 220004223

 Ericka BIHOYIKI 220004309

LECTURER: NKURANGA Jean Bosco

ASSIGNMENT1: HOW TO ANALYSE CARBOHYDRATE, PROTEIN AND LIPID EITHER

BY QUALITATIVE OR QUANTITATIVE.

On 29.9.2022
QUALITATIVE ANALYSIS: Is analysis which help us to detect the presence and absence of a
given molecule in a sample. There are different ways through which this could be made as seen
below in carbohydrates, fats, and proteins.
QUANTITATIVE ANALYIS this analysis which help us to identify the amount of substance in
the sample by using a specific technique with method. We usually need to make a calibration
curve. You have also to prepare sample depending to the method to be used.
1. CARBOHYDRATE ANALYSIS
1.1 QUALITATIVE ANALYSIS OF CARBOHYDRATE: Carbohydrates are
macronutrients and they comprise carbon, hydrogen and oxygen at their chemical level. They
are essential nutrients which include sugars, fibers and starches. They are found in grains,
vegetables, fruits and in milk and other dairy products. Owing to their crucial role in the body
they are found in many commercial product and we can analyze it by those methods:
 Molisch’s test: it is a chemical test for all carbohydrates by which one drop of molisch
reagent (ethanol and ἀ-napthol) is added to carbohydrate and also 2 drops of sulfuric acid
and produce aldehyde with in red or purple color which confirm the presence of
carbohydrates.
 Benedict test: it is chemical test for reducing sugar and aldehyde by which blue copper
sulphate solution is added and appearance of red-brick precipitate due to reduction of
copper(II) to copper(I) confirm appearance of reducing sugar as carbohydrate.
 Iodide test: this is another qualitative test for starch by which iodine solution 1-2 drops is
added to liberate blue black precipitate to confirm the presence of starch.
 Bial’s test: this specific to 5c sugar like pentose , through which The components of this
reagent are resorcinol, HCl, and ferric chloride. In this test, the pentose is dehydrated to
form furfural and the solution turns bluish and a precipitate may form. 5 mL of Bial’s
reagent, add 2-3 drops of sugar solution and boil.
 Other tests include: action of alkalis on sugar, Fehling reagent test. Seliwanoff’s Test
1.2 QUANTITATIVE ANALYSIS OF CARBOHYDRATE
I.2.1. gravimetric method: it is one of the method that is used to determine the concentration of
reducing sugar in the sample, where copper sulfate is added to the sample in presence of alkaline
and heat to produce precipitate which are directly related to the concentration of reducing sugar
in the initial sample. The concentration of the precipitate is determined by filtration, drying ,
and weighing.
I.2.2 calorimetric method: this method is determination carbohydrate in the sample whereby,
aqueous solution of carbohydrate is placed in test tube then phenol and sulfuric acid are also
added resulting in yellow orange color as the result of interaction between carbohydrate and
phenol then the absorbance at 420nm is directly proportion to the concentration of carbohydrate
within the sample
I.2.3 Polarimetry: this is based on use of polarimetry device that is used to identify the angle of
rotation in sample with known length which are determined by using monochromatic light The
extent of polarization is related to the concentration of the optically active molecules in solution
by the equation a = [a]lc, where a is the measured angle of rotation, [a] is the optical activity
(which is a constant for each type of molecule), l is the pathlength and c is the concentration. The
concentration of carbohydrate in an unknown sample is then determined by measuring its angle
of rotation and comparing it with the calibration curve.

I.2.4: Density: The density of a material is its mass divided by its volume. The density of
aqueous solutions increases as the carbohydrate concentration increases. Thus the carbohydrate
concentration can be determined by measuring density, e.g., using density bottles or
hydrometers. This technique is routinely used in industry for determination of carbohydrate
concentrations of juices and beverages.

I.2.5 Infrared: material absorbs infrared due to vibration or rotation of molecular groups.
Carbohydrates contain molecular groups that absorb infrared radiation at wavelengths where
none of the other major food constituents absorb consequently their concentration can be
determined by measuring the infrared absorbance at these wavelengths. 
2. FATS ANALYSIS

2.1 QUALITAYIVE ANALYSIS OF FATS

2.1.1 Solubility Test: It is the preliminary test to detect lipid solubility in various solvents to
check whether it is miscible or immiscible in polar or non-polar solvents. Lipids are readily
miscible in non-polar solvents like chloroform, partially soluble in a polar solvent like ethanol
and immiscible in a polar solvent like water. Method:
1. Take the lipid sample in three different test tubes by labelling it as A, B and C.
2. Then, add different solvents like water, ethanol and chloroform in each test tubes A, B and C.
3. Shake the tubes and allow it to stand for 1 minute.
4. Check the solution for whether lipid is soluble or insoluble.
Result:  Lipids are soluble in a non-polar solvent, i.e. chloroform and partially soluble in
ethanol which can solubilize upon heating.

2.1.2 Translucent Spot Test this is also a preliminary test which help us to determine the
presence of lipid using filter paper whreby the sample containing lipids after being poured on
filter paper they will produce translucent and greasy spot.

2.1.3 Saponification Test: In this in which the triglycerides of lipid react with an alkali NaOH
to produce soap and glycerol in the presence of ethanol. Method:
1. Take a sample of lipid in a test tube.
2. Then, add strong alkali NaOH.
3. Then, boil the solution in a water bath for 5 minutes.
4. At last, add ethanol.
5. Observe the test tube for the appearance of froth.
Result: Froth appears in the test tube
2.1.4 Acrolein Test, Method
1. Take 1 ml of the lipid sample in a test tube.
2. Add crystals of potassium hydrogen sulphate.
3. Heat the solution for a few minutes.
4. Smell the test tube for the pungent smell.
Result : If glycerol present in the sample, it will give a pungent smell.
2.1.5 Sudan IV Test: Sudan IV is a non-polar stain, the lipid will bind with it and retain the
stain’s colour by giving a red-orange colour. Method:

1. Take 1 ml of the lipid sample in a test tube.

2. Then add 1-2 drops of Sudan IV to the solution.
3. Observe the tubes for the appearance of red-orange colour in the solution.
Result:  result: Gives red-orange colour to the solution

2.2 QUANTITATIVE ANALYSIS OF FATS

2.2.1 Detergent Method:  A sample is mixed with a combination of surfactants in a Babcock
bottle. The surfactants displace the fat globule membrane which surrounds the emulsion droplets
in milk and causes them to coalesce and separate. The sample is centrifuged which allows the fat
to move into the graduated neck of the bottle, where its concentration can then be determined.
2.2.2 Nuclear Magnetic Resonance: NMR spectroscopy is routinely used to determine the total
lipid  concentration of foods. The lipid content is determined by measuring the area under a peak
in an NMR chemical shift spectra that corresponds to the lipid fraction. Lipid contents can often
be determined in a few seconds without the need for any sample preparation using commercially
available instruments.
2.2.3Babcock Method A specified amount of milk is accurately pipetted into a specially
designed flask (the Babcock bottle). Sulfuric acid is mixed with the milk, which digests the
protein, generates heat, and breaks down the fat globule membrane that surrounds the droplets,
thereby releasing the fat. The sample is then centrifuged while it is hot (55-60oC) which causes
the liquid fat to rise into the neck of the Babcock bottle. The neck is graduated to give the
amount of milk fat present in wt%. It does not determine phospholipids because thaey are
boundary between the lipid and aqueous phases.
2.2.3 Ultrasonic velocity: The speed at which an ultrasonic wave travels through a material
depends on the concentration of fat in a food. Thus the lipid content can be determined by
measuring its ultrasonic velocity. This technique is capable of rapid, nondestructive on-line
measurements of lipid content.
2.2.3 Light scattering: The concentration of oil droplets in dilute food emulsions can be
determined using light scattering techniques because the turbidity of an emulsion is directly
proportional to the concentration of oil droplets present.

3. ANALYIS OF PROTEINS

3.1 QUALITATIVE& QUANTITATIVE ANALYSISOF PROTEINS

 3.1.1Kjeldahl Method: Sample preparation- Solid foods are ground to pass a 20 mesh
screen. 1Digestion- • Sample is accurately weighed in a Kjeldahl flask. • Acid and
catalyst is added and digestion is continued until solution becomes clear to get complete
breakdown of all organic matter. • Nonvolatile ammonium sulfate is formed from the
reaction of nitrogen and sulfuric acid.2Neutralization and Distillation- • The digest is
diluted with water. • Alkali containing sodium thiosulfate is added to neutralize the
sulfuric acid. • The ammonia formed is distilled into a boric acid solution containing the
indicators methylene blue and methylene red. (NH4 )2SO4+ 2NaOH>>>>>2NH3
+Na2SO4 +2H2O. 3Titration- Borate anion is titrated HCl. 4Calculations- Moles of HCl
= Moles of NH3 = moles of N in the sample A reagent blank is run to subtract reagent
nitrogen from the sample. %N=NHClxCorected acid volume gofsample x 14gN mole x10
Where, N HCl = normality of HCl in moles/1000ml. Corrected acid value = (ml std. acid
for sample) - (ml std. acid for blank) 14 = atomic weight of nitrogen. A factor is used to
convert % N to % crude protein. Most proteins contain 16% N, so the conversion factor is
6.25 (100/16 = 6.25). % N x 6.25 = % protein
3.1.2 Infrared:  Infrared techniques can be used to determine the concentration of
proteins in food samples. Proteins absorb IR naturally due to characteristic vibrations
(stretching and bending) of certain chemical groups along the polypeptide backbone.
Measurements of the absorbance of radiation at certain wavelengths can thus be used to
quantify the concentration of protein in the sample. IR is particularly useful for rapid on-
line analysis of protein content.
3.1.3 Turbimetric method Protein molecules which are normally soluble in solution can
be made to precipitate by the addition of certain chemicals, e.g., trichloroacetic acid.
Protein precipitation causes the solution to become turbid. Thus the concentration of
protein can be determined by measuring the degree of turbidity. 3.1.3
3.1.4 Biuret Method: A violet-purplish color is produced when cupric ions (Cu ) 2+

interact with peptide bonds under alkaline conditions. • 5ml of Biuret reagent is mixed

with 1ml of protein solution. The reagent includes copper sulfate, NaOH and potassium
sodium tartrate, which is used to stabilize the cupric ion in the alkaline solution. • The
reaction mixture is allowed to stand at room temperature for 15-30 mins. • Filteration or
centrifugation is done if the reaction mixture is not clear. • The absorbance is read at
540nm against a reagent blank. • A standard curve of concentration vs absorbance is
constructed using bovine serum albumin (BSA).
3.1.5 Ultrasonic scattering: The concentration of protein aggregates can also be
 determined using ultrasonic scattering techniques because the ultrasonic velocity and
absorption of ultrasound are related to the concentration of protein aggregates present.

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