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Chemistry Project


Academic year: 2021

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Vinay A



Vinay A




Chemistry Project Vinay A Iyer

Help is but one of the basic necessities of life. Akin to an infant who cannot set forth to meet the

world unless with his hand held by someone else, no endeavor can seek completion without

support and encouragement meted out by many hands. So, it becomes my fond duty to thank

those who made this project possible.

After offering our humblest gratitude to the Lord Almighty we wish to offer our gracious thanks

to the CBSE for providing a means of practically applying what is learnt in school and hence

understand the subject better.

We are also indebted to our Principal, Mrs. Deepa Sridhar and the staff of our institution who

have never gone back in providing any kind of assistance and support to us.

Also, we are eternally grateful to our Chemistry teacher, Mrs.Shubha as also the exceedingly

helpful lab assistants led by Mr. Prakash for enriching the experience of working on this project

by their expertise and skill.

Lastly our acknowledgements are due to our kin and friends for all the help that they have

selflessly provided.


Chemistry Project Vinay A Iyer

This is to certify that

Vinay A Iyer

of class


has successfully completed the


Estimation of free amino acids


food samples



as prescribed by the CBSE board

for the year 2009-10 in

the laboratory of

Sri Kumaran Children’s Home


Chemistry Project Vinay A Iyer

Signature of the external examiner Signature of internal examiner




Chemistry Project Vinay A Iyer

Sl No


Page No





Amino acids



Free amino acids







Function &













Observations &










Chemistry Project Vinay A Iyer

Proteins are large molecules composed of one or more chains of amino acids in a specific order

that is determined by the base sequence of nucleotides in the coding for the protein. Proteins are

very useful structurally and metabolically and each protein has unique functions. They result due

to the formation of peptide linkages between free amino acid molecules via a dehydration

reaction of –COOH and –NH



The words protein, polypeptide, and peptide are a little ambiguous and can overlap in meaning.

Protein is generally used to refer to the complete biological molecule in a stable conformation,

whereas peptide is generally reserved for a short amino acid oligomer often lacking a stable

three-dimensional structure.

Amino acids may be defined as

a group of organic molecules that consist of a basic amino group (−NH2), an acidic carboxyl group (−COOH), and an organic R group (or side chain) that is unique to each amino acid. The term is usually understood to mean α-Amino acids with the general formula:

Proteins and amino acids are of primary importance to the continuing functioning of life on

Earth. Proteins catalyze the vast majority of chemical reactions that occur in the cell. They

provide many of the structural elements of a cell, and they help to bind cells together into tissues.

Some proteins act as contractile elements to make movement possible. Others are responsible for

the transport of vital materials from the outside of the cell (“extracellular”) to its inside

(“intracellular”). Proteins, in the form of antibodies, protect animals from disease and, in the

form of interferon, mount an intracellular attack against viruses that have eluded destruction by

the antibodies and other immune system defenses. Many hormones are proteins. Last but

certainly not least, proteins control the activity of genes (“gene expression”).

Hence amino acids as building blocks of proteins find great application in nutrition. They help in

repairing muscle tissues, food flavoring et al. However free amino acids that are consumed in

diet are also of immense use since they synthesize a variety of standard and non standard

biomolecules and contribute to the urea cycle.

Also, in recent years the free amino acid content in naturally occurring substances has been

exploited to produce biocompatible materials. For example, changing the amino acid structure of

keratin can help develop the compound used for biodegradable plastic.

Thus, in account of the above it becomes very important to analyze the content of amino acids in

various food stuffs.


Chemistry Project Vinay A Iyer

Amino acids or more specifically Proteinogenic amino acids are 22 (including proline where the

amino group is secondary due to cyclization and hence an imino acid) amino acids that are

found in proteins and that are coded for in the standard genetic code. Proteinogenic literally

means protein building. Proteinogenic amino acids are assembled into a polypeptide (the

subunit of a protein) through a process known as translation

The precise amino acid content, and the sequence of those amino acids, of a specific protein, is

determined by the sequence of the bases in the gene that encodes that protein. The chemical

properties of the amino acids of proteins determine the biological activity of the protein.

The amino acids are usually distinguished by the R group on the

α Carbon and all the standard amino acids are known to exist stereochemically in the L form, though D-alanine has been discovered in some bacterial cell walls and gycine is not chiral. Amino acids also differ by the polarity of the


Nonstandard amino acids refer to those amino acids that have been chemically modified after they have been incorporated into a protein (termed a “post-translational modification”) and those amino acids that occur in living organisms but are not found in proteins.

Another important feature of free amino acids is the existence of both a basic and an acidic group at the α-carbon. Compounds such as amino acids that can act as either an acid or a base are called amphoteric. The basic amino group typically has a pKa between 9 and 10, while the acidic α-carboxyl group has a pKa that is usually close to 2. The pKa of a group is the pH value at which the concentration of the protonated group equals that of the unprotonated group. Thus, at physiological pH (about 7–7.4), the free amino acids exist largely as dipolar ions or zwitter ions. There is a pH called the isoelectric point at which the molecule has a net zero charge.

Free amino acids combine with each other through polypeptide bonds to form proteins. This is the principal use of amino acids.


Amino acid Abbreviation Symbol Structure

Alanine ala A CH3-CH(NH2)-COOH

Arginine arg R HN=C(NH2)-NH-(CH2)3-CH(NH2)-COOH

Asparagine asn N H2N-CO-CH2-CH(NH2)-COOH

Aspartic Acid asp D HOOC-CH2-CH(NH2)-COOH

Cysteine cys C HS-CH2-CH(NH2)-COOH

Glutamic Acid glu E HOOC-(CH2)2-CH(NH2)-COOH

Glutamine gln Q H2N-CO-(CH2)2-CH(NH2)-COOH

Glycine gly G NH2-CH2-COOH

Histidine his H NH-CH=N-CH=C-CH2-CH(NH2)-COOH Isoleucine ile I CH3-CH2-CH(CH3)-CH(NH2)-COOH

Leucine leu L (CH3)2-CH-CH2-CH(NH2)-COOH

Lysine lys K H2N-(CH2)4-CH(NH2)-COOH

Methionine met M CH3-S-(CH2)2-CH(NH2)-COOH

Phenylalanine phe F Ph-CH2-CH(NH2)-COOH

Proline pro P NH-(CH2)3-CH-COOH

Serine ser S HO-CH2-CH(NH2)-COOH

Threonine thr T CH3-CH(OH)-CH(NH2)-COOH

Tryptophan trp W Ph-NH-CH=C-CH2-CH(NH2)-COOH Tyrosine tyr Y HO-Ph-CH2-CH(NH2)-COOH

Valine val V (CH3)2-CH-CH(NH2)-COOH

Apart from these 20 classical standard proteinogenic amino acids two others namely;

Selenocysteine (Sec / U) with CH2SeH as R group and Pyrrolysine (Pyl / O) with C4H8-NH-CO-3-CH3-3,4-dihydro-2H-pyrrol-2-yl as the R group are recommended by the IUPAC and

IUBMB as recent additions to the group.

The 3D structure of Asparagine, one of the proteinogenic amino acids


Chemistry Project Vinay A Iyer

Essential amino acids:


Has been used in the treatment of arthritis and anemia; is essential for the growth and repair of tissues; is needed for the production blood cells; protects the body from radiation damage; lowers blood pressure.


Is needed for hemoglobin formation; stabilizes and regulates blood sugar and energy levels; is valuable to athletes because it aids in the healing and repair of muscle tissue, skin and bones.


Works with Isoleucine and Valine to promote the healing of muscle tissue, skin, and bones lowers blood sugar levels; aids in increasing growth hormone production.


Participates in hydrogen bonding and as a general base in catalysis; treatment of cancer; beneficial for those with herpes simplex infections.


A powerful anti-oxidant and a good source of sulfur, which prevents disorders of the hair, skin, and nails; detoxifies harmful agents such as lead and other heavy metals; helps diminish muscle weakness.


Used by the brain to produce norepinephrine, a chemical that transmits signals between nerve cells in the brain; promotes alertness and vitality; elevates mood; decreases pain; aids memory and learning.


Helps maintain proper protein balance in the body; is important for the formation of collagen, elastin and tooth enamel; assists metabolism and assimilation.


Chemistry Project Vinay A Iyer

A natural relaxant, helps alleviate insomnia by inducing normal sleep; reduces anxiety and depression and stabilizes mood; enhances the release of growth hormones; helps control hyperactivity in children.


Needed for muscle metabolism and coordination, tissue repair and for the maintenance of proper nitrogen balance in the body. Sickle cell disease results when Valine substitutes Glutamic acid reducing folding.

Non-Essential amino acids:


Plays a major role in the transfer of nitrogen from peripheral tissue to the liver; aids in the metabolism of glucose; guards against the buildup of toxic substances; strengthens the immune system.


Retards the growth of tumors; aids in liver detoxification by neutralizing ammonia; used in treating sterility in men ; facilitates an increase in muscle mass and a reduction of body fat; assists the growth.


Used in baking; the nervous system requires Asparagine; plays an important role in the synthesis of ammonia.


Increases stamina; rejuvenates cellular activity, cell formation and metabolism; protects the liver by aiding the expulsion of ammonia.


Functions as a powerful anti-oxidant in detoxifying harmful toxins; protects the body from radiation damage; protects the liver and brain from damage due to alcohol.


Is an excitatory neurotransmitter; important in the metabolism of sugars and fats; aids in the transportation of potassium; acts as fuel for the brain; helps correct personality disorders.


Chemistry Project Vinay A Iyer


The most abundant amino acid found in muscles; helps build and maintain muscle tissue; increases mental activity; assists in maintaining the proper acid/alkaline balance in the body;


Retards muscle degeneration; improves glycogen storage, thus freeing up glucose for energy needs; promotes a healthy central nervous system, and immune system; useful for repairing damaged tissue.


Improves skin texture by aiding the production of collagen; helps in the healing of cartilage and the strengthening of joints, tendons, and heart muscle


Needed for the proper metabolism of fats and fatty acids, the growth of muscle, and the

maintenance of a healthy immune system; is a component of the protective myelin sheaths that cover nerve fibers


Is important for overall metabolism; regulates mood and stimulates metabolism and the nervous system; helps reduce body fat; aids in the production of melanin


Used to prepare selenoproteins and as a source of selenium.


Catalyses methyltransferases; incorporated during translation


Use of dietary amino acids in humans. The classification of essential and non essential amino acids as made above is strictly for humans.


Chemistry Project Vinay A Iyer

Based on the polarity


Amino acids like alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine and tryptophan are non polar. The R groups of these amino acids have either aliphatic or aromatic groups. This makes them hydrophobic (“water fearing”). In aqueous solutions, globular proteins will fold into a three-dimensional shape to bury these hydrophobic side chains in the protein interior.

On the other hand amino acids such as glycine, serine, cysteine, threonine, tyrosine, asparagine, and glutamine are polar and uncharged . The side chains in this group possess a spectrum of functional groups. However, most have at least one atom (nitrogen, oxygen, or sulfur) with electron pairs available for hydrogen bonding to water and other molecules. They are hydrophilic. The carbonyl group can function as a hydrogen bond acceptor, and the amino group (NH2) can function as a hydrogen bond donor.

Based on acidic nature:

Both aspartic acid and glutamic acid have a carboxylic acid on their side chain that gives them acidic (proton-donating) properties. In an aqueous solution at physiological pH, all three functional groups on these amino acids will ionize, thus giving an overall charge of −1.

Arginine, histidine, and lysine are called basic amino acids. Each side chain is basic (i.e., can accept a proton). Lysine and arginine both exist with an overall charge of +1 at physiological pH. The guanidino group in arginine’s side chain is the most basic of all R groups (a fact reflected in its pKa value of 12.5). Both acidic and basic amino acids are generally hydrophilic.

Hydrophilic Character:

Polar amino acids are hydrophilic and neutral ones are hydrophobic.

The nature of constituent amino acids decides nature of proteins. For example, soluble proteins have surfaces rich with polar amino acids like serine and threonine, while integral membrane proteins tend to have outer rings of hydrophobic amino acids that anchor them into the lipid bilayer. In the case part-way between these two extremes, peripheral membrane proteins have a patch of hydrophobic amino acids on their surface that locks onto the membrane.

Non polar amino acids form globular proteins with hydrophobic part inwards

Hydrophilic amino acids


Chemistry Project Vinay A Iyer

Amino acids via their various chemical functionalities (carboxyls, amino, and R groups) can undergo numerous chemical reactions. However, two reactions (peptide bond and cysteine oxidation) are of particular importance because of their effect on protein structure.

Peptide bond formation:

Amino acids can be linked by a condensation reaction in which an −OH is lost from the carboxyl group of one amino acid along with a hydrogen from the amino group of a second, forming a molecule of water and leaving the two amino acids linked via an amide—called, in this case, a peptide bond.

In cells, this reaction does not occur directly; instead the amino acid is first activated by attachment to a transfer RNA molecule through an ester bond.

Small polymers of amino acids (fewer than 50) are termed oligopeptides, while larger ones (more than 50) are referred to as polypeptides. Hence, a protein molecule is a polypeptide chain composed of many amino acid residues, with each residue joined to the next by a peptide bond. The lengths for different proteins range from a few dozen to thousands of amino acids, and each protein contains different relative proportions of the 20 standard amino acids, each linked via peptide bond formation.

Other reactions:

Amino acids can undergo other reactions like nucleophilic addition, imine formation, esterification and decarboxylation reactions due to the amino and carboxylic acid groups as also other reactions due to side chains.


Chemistry Project Vinay A Iyer

Amino acids are useful components in a variety of metabolisms. Even though, some roles can be highlighted as a function of an amino acid, it is important to be aware that they are part of complex pathways and biological systems. The function and use of an amino acid is dependent on other amino acids, mineral elements, carbohydrate and fatty acids and has indirect effects that are manifested in myriad metabolisms. They are one of the chief constituents of cell protoplasm

No biosynthesis can happen without the help of amino acids. 20 of them are required for effective human biosynthesis. While essential amino acids are to be supplemented in diet so as to aid in effective human biosynthesis. There are also semi-essential amino acids that are needed by lactating mothers and growing children, like Arginine and Histidine.

Apart from protein synthesis amino acids are also important in forming parts of coenzymes or as precursors for biosynthesis. Note that the human body, minus water, is 75% amino acids.

Non standard amino acids have their own uses. For example, they determine the localization of the protein, e.g., the addition of long hydrophobic groups can cause a protein to bind to a phospholipid membrane.

Humans consume many foods that contain free amino acids. Foods from animal sources are typically rich in essential amino acids. These include chicken, fish, eggs, dairy products, beef, and pork. Plant sources include dried beans (black, kidney, red, and white beans), peas, soy, nuts, and seeds. Although plant sources generally lack one or more of the essential amino acids, when combined with whole grains such as rice, or by eating nuts or seeds with legumes all the amino acids can be obtained. A large quantity of dissolved free amino acids has been reported in ocean water as well. But it is to be remembered that the major source of amino acids remain breaking down of dietary protein.

Glycine and alanine have a sweet taste, valine and leucine have a bitter taste, and aspartic acid and glutamate have sour tastes. Though called a bitter amino acid, valine has a slightly sweet taste as well. The sweetness of glycine and alanine is lighter than that of sugar. Combination of amino acids with their respective tastes is a key determinant for the taste of food.

Amino acids are also present in hormones, enzymes and other bio-fluids.


Chemistry Project Vinay A Iyer

Colorimetry can be defined as the procedure for quantitative chemical analysis, based on comparison of the color developed in a solution of the test material with that in a standard solution; the two solutions being observed simultaneously in a colorimeter and quantitated on the basis of the absorption of light. Most of the species in water do not have any color, meaning that the species do not absorb light in the visible region. To measure the absorbance of the colorless molecules, a reaction must be found which will produce a color that can be measured. Thus we react the species with a reagent to produce a new

compound which has one or more chromophores. Following this we may analyze its color with respect to a standard or blank.

Laws used in colorimetric analysis are:

Beer’s Law: It states that for a parallel beam of monochromatic radiation passing through

homogeneous solutions of equal path length the absorbance is proportional to the concentration. Alternatively, the rate of decrease of intensity is exponential as the concentration of absorbing substance increases arithmetically.

Lambert’s Law: It states that for a parallel beam of monochromatic radiation passing through

homogeneous solutions of equal concentration the absorbance is proportional to the path length. Alternatively, the rate of decrease of intensity with thickness is proportional to the intensity of light.

Beer-Lambert’s Law: It states that there is a logarithmic dependence between the transmission

(or transmissivity), T, of light through a substance and the product of the absorption coefficient of the substance, α, and the distance the light travels through the material (i.e. the path length), ℓ. Mathematically;

where A’ is transmission, N is concentration and I0 and I are the intensity of the incident light and the transmitted light, respectively

A colorimeter- instrument used for colorimetric analysis.

Depiction of the


Chemistry Project Vinay A Iyer

The essential parts of a colorimeter are:

 a light source (often an ordinary low-voltage filament lamp)  an adjustable aperture

 a set of colored filters

 a cuvette to hold the working solution

 a detector (usually a photoresistor) to measure the transmitted light  a meter to display the output from the detector


Changeable optics filters are used in the colorimeter to select the wavelength of light which the solute absorbs the most, in order to maximize accuracy. The usual wavelength range is from 400 to 700 nanometres (nm).

The choice of the correct wavelength for testing is important. It is interesting to note that the wavelength that gives the most sensitivity (lower detection limit) for a test factor is the complementary color of the test sample.

A spectrophotometer is a similar type of instrument that can measure intensity as a function of the color, or more specifically, the wavelength of light.

(1) Wavelength selection, (2) Printer button, (3) Concentration factor adjustment, (4) UV mode selector (Deuterium lamp), (5) Readout, (6) Sample compartment, (7) Zero control (100% T), (8) Sensitivity switch.


Chemistry Project Vinay A Iyer

Over the years, the study of amino acids has gained a variety of applications in science and technology. For example, Ninhydrin is most commonly used to detect fingerprints, as amino acids left over from peptides and proteins sloughed off in fingerprints react with Ninhydrin. The method employed in this process is similar in principal to the one in this project.

Though the content of free amino acids in various food stuffs is not necessarily an indicator of the level of proteins; and hence the amount of amino acids that can be obtained on hydrolysis during digestion, it certainly gives a fair idea of the ‘protein- richness’ of a particular material. Hence in the background of the efforts being made by international nutrition experts in isolating the ‘perfect diet’ for each individual, this becomes a small step in that direction.

Amino acids became popular as dietary supplements by the end of the twentieth century for various uses, including fitness training, weight loss, and certain chronic diseases. Claims exist in holistic medicine that indicate amino acid supplements taken in the proper dosage can aid also in fighting depression, allergies, heart disease, gastrointestinal problems, high cholesterol, muscle weakness, blood sugar problems, arthritis, insomnia, bipolar illness, epilepsy, chronic fatigue syndrome, autism, attention-deficit

hyperactivity disorder (ADHD), and mental exhaustion. Thus, in the next decade the importance of amino acids will increase manifold and it will be detrimental for us to increase our knowledge about them. Also this project has given us a fair view of the processes involved in chemical qualitative and quantitative analysis and has increased our interest in the same.

Forensic investigations involving fingerprints are largely dependent on amino acid analysis.


Chemistry Project Vinay A Iyer






Chemistry Project Vinay A Iyer


To estimate the percentage of free amino acids in various food samples.


Food samples, Ninhydrin, Glycerin, Sodium Hydroxide solution, Citric Acid, Stannous Chloride crystals, Ethanol, Distilled Water, Digital Colorimeter, Chemical balance, Test Tubes, Beakers, Measuring Cylinder, hot water trough & Propanol.


Free Amino acids undergo special unique reaction with Ninhydrin (Triketohydrindene hydrate). Among the products is a purple colour imino derivative, which provides as a useful colour test.




Ninhydrin Reagent : 1 gm Ninhydrin dissolved in 50 ml Glycerin

Citrate Buffer : 2 gm citric acid crystals dissolved in 200ml of 0.1 N NaOH

made up to 500 ml.

Stannous Chloride- Citrate Buffer : 80 mg stannous chloride dissolved in 50 ml citrate buffer Ninhydrin-SnCl2 Citrate Buffer : Ninhydrin solution and stannous chloride-Citrate Buffer mixed

in equal volumes.

Propanol-Water : Propanol and Distilled Water mixed in equal volumes.


• Prepare 5% homogenate (5 g in 100 ml) of sample in ethanol.

• Centrifuge till supernatant is clear.

• Blank: Add 1 ml of Ninhydrin-SnCl2 Citrate Buffer and 3 ml of Propanol Water to 1 ml of Ethanol.

• Sample: Add 1 ml of Ninhydrin-SnCl2 Citrate Buffer to 1 ml of supernatant and heat for 10 mints. Cool the solution and add 3 ml of Propanol water.

• Record Optical Density of sample against that of Blank.

• Record your observations.


• Wavelength of Light (Colorimeter) = 540mm

• 0.3 OD of Sample = 0.4 mg of Amino Acids

• Percentage of Amino Acids: (M x 100/5 g) M: g (Amino Acids)


• Ninhydrin is Carcinogenic and a strong Oxidizing agent. Hence proper caution should be Taken and excessive contact is unadvisable.

• Heating of supernatant and Ninhydrin-SbCL2 citrate Buffer mixture should not be Direct: a water bath should be used.

• All reagents should be prepared freshly if possible; for best results Ninhydrin and Stannous Chloride-Citrate Buffer should be refrigerated and mixed only at time of analysis.


Chemistry Project Vinay A Iyer


No Sample

OD(540 nm)

Quantity of free amino acids(mg)

Percentage of free amino acids 1. Rice 0.06 0.08 1.60% 2. Wheat 0.04 0.053 1.06% 3. Corn 0.19 0.253 5.06% 4. Ragi 0.03 0.04 0.80% 5. Maida 0.16 0.213 4.26% 6. Pea 0.14 0.187 3.74% 7. Urad Dal 0.40 0.533 10.66%

8. Black eye pea 0.20 0.267 5.34%

9. Rajma 0.31 0.413 8.26%

10. Green gram 0.09 0.12 2.40%


Chemistry Project Vinay A Iyer


Chemistry Project Vinay A Iyer

 Among the 10 food samples analyzed, white lentils were found to possess the highest amount of free amino acid content followed by rajma. This is expected due to the common knowledge that lentils are a very good source of proteins.

 As a general trend, the free amino acid content in cereals is shown to be abysmally low when seen in the light of lentils and peas. This indicates that for a well rounded diet the latter is an absolute necessity, more so on the Indian platter which is largely composed of cereals.

 Among cereals, though it is known that ragi is richer in proteins when compared to rice or wheat, the experiment seems to suggest otherwise. This may be due to the fact that the amount of free amino acids is not directly related to the amount of proteins. This statement is true even for wheat and maida.

 Though green pea and black eye pea are expected to have a similar amount of free amino acids, the latter has a higher content of free amino acids. This may be attributed to the fact that the latter is usually marketed in processed form which may reduce the free amino acid content.


Chemistry Project Vinay A Iyer

 Encyclopedia Britannica

 Wikipedia- The free encyclopedia

 The Biology Project- University of Arizona  West Virginia University - Online archives  About.com: Chemistry

 IUBMB – Online articles  PeptideGuide.com

 Links for Chemists- www virtual library  Austin Nutritional Research

 Organic Chemistry By Robert Thornton Morrison & Robert Neilson Boyd  IIT JEE Organic Chemistry By Dr Jagdamba Singh


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