Extraction of Amino acids from Human Hair “Waste” and Used as a Natural Fertilizer Sharanabasappa B. Patil1, Shreya K2 and Kruti S2

1Department of Chemistry, Ramaiah Institute of Technology, Bangalore-560054, India 2Department of Chemical Engineering, Ramaiah Institute of Technology, Bangalore-560054, India

Abstract Hair is a protein filament that grows from follicles found in the dermis. Hair is one of the defining characteristics of mammals. The human body, apart from areas of glabrous skin, is covered in follicles which produce thick terminal and fine vellus hair. Most common interest in hair is focused on hair growth, hair types, and hair care, but hair is also an important biomaterial primarily composed of protein, notably alpha-keratin. Attitudes towards different forms of hair, such as hairstyles and hair removal, vary widely across different cultures and historical periods, but it is often used to indicate a person's personal beliefs or social position, such as their age, sex, or religion. Human hair is a material considered useless in most societies and therefore is found in the municipal waste streams in almost all cities and towns of the world.

Key Words: Human Hair, Amino acids and Fertilizer

INTRODUCTION: 1.1Discription: The word “hair” usually refers to two distinct structures: 1. The part beneath the skin, called the hair follicle, or,when pulled from the skin, the bulb. This organ is locatedin the dermis and maintains stem cells, which not only re-grow the hair after it falls out, but also are recruited to re-grow skin after a wound.2. The shaft, which is the hard filamentous part thatextends above the skin surface. A cross section of the hairshaft may be divided roughly into three zones.The hair’s structure can be divided into three distinctparts:3. Medulla: innermost layer of the hair shaftcomposed of an amorphous, soft, oily substance.4. Cuticle: thin protective outer layer that containsthe nourishing portion essential to hair growth. It ishighly keratinized, composed of cells shaped likescales that are layered one over the other, measuringabout 60 micrometers long and about 6 micrometerswide.5 Cortex: main component of the hair, containinglong keratin chains that add elasticity, suppleness andresistance to the hair. The cells of the cortex arejoined together by an intercellular cement rich inlipids and proteins. Each cell is composed of bundlesthat lie in the direction of the hair length: these aremacro-fibrils which are made up of micro-fibrils,which in turn contain proto-fibrils.The shape of the follicle determines the shape of thecortex, and the shape of the fiber is related to how straightor curly the hair is. People with straight hair have roundhair fibers. Oval and other shaped fibers are generallymore wavy or curly. The cuticle is the outer covering. Itscomplex structure slides as the hair swells and is coveredwith a single molecular layer of lipid that makes the hairrepel water. The diameter of human hair varies from 0.017to 0.18mm (0.00067 to 0.00709 in). There are two millionsmall, tubular glands and sweat glands that produce wateryfluids that cool the body by evaporation. The glands at theopening of the hair produce a fatty secretion that lubricatesthe hair.

Hair growth begins inside the hair follicle. The only "living" portion of the hair is found in the follicle. The hair that is visible is the hair shaft, which exhibits no biochemical activity and is considered "dead". The base of a hair's root (the "bulb") contains the cells that produce the hair shaft. Other structures of the hair follicle include the oil producing sebaceous gland which lubricates the hair and the arrector pili muscles, which are responsible for causing hairs to stand up. In humans with little body hair, the effect results in goose bumps.

Cross Section of Hair Strand

Root of the hair

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1.2 Natural Color All natural hair colors are the result of two types of hair pigments. Both of these pigments are melanin types, produced inside the hair follicle and packed into granules found in the fibers. Blonde hair is the result of having little pigmentation in the hair strand. Gray hair occurs when melanin production decreases or stops, while poliosis is hair (and often the skin to which the hair is attached), typically in spots, that never possessed melanin at all in the first place, or ceased for natural genetic reasons, generally, in the first years of life. 1.3 Texture Hair exists in a variety of textures. Three main aspects of hair texture are the curl pattern, volume, and consistency. The derivations of hair texture are not fully understood. All mammalian hair is composed of keratin, so the make-up of hair follicles is not the source of varying hair patterns. There are a range of theories pertaining to the curl patterns of hair. Scientists have come to believe that the shape of the hair shaft has an effect on the curliness of the individual's hair. A very round shaft allows for fewer disulfide bonds to be present in the hair strand. This means the bonds present are directly in line with one another, resulting in straight hair. The flatter the hair shaft becomes, the curlier hair gets, because the shape allows more cystines to become compacted together resulting in a bent shape that, with every additional disulfide bond, becomes curlier in form. As the hair follicle shape determines curl pattern, the hair follicle size determines thickness. While the circumference of the hair follicle expands, so does the thickness of the hair follicle. An individual's hair volume, as a result, can be thin, normal, or thick. The consistency of hair can almost always be grouped into three categories: fine, medium, and coarse. This trait is determined by the hair follicle volume and the condition of the strand. Fine hair has the smallest circumference, coarse hair has the largest circumference, and medium hair is anywhere between the other two. Coarse hair has a more open cuticle than thin or medium hair causing it to be the most porous. 2.1Hair Composition Human hair is an appendage which grows from follicles, tube like sacs in the scalp or skin containing the hair root. The hair that we cut, relax, color and style is a non-living fiber comprised of keratinized protein. Within the hair follicle cells are produced. These cells mature in an upward moving process through the follicle. This maturing process is known as keratinization. During keratinization cells absorb keratin, a fibrous protein. As the cells continue to move upward they lose their nucleus and die off, producing the non-living keratinized cells (appendage) that emerge from the scalp. Hair is comprised of many contributing factors. Proteins, raw elements, amino acids and bonds work together in forming hair fiber. The dominant contributor in the composition of hair is protein, accounting for 91 percent of hair fiber. Amino acids, the building blocks of protein,

are made up of C,O,H,N and S elements, (Carbon, Oxygen, Hydrogen, Nitrogen and Sulfur). 2.2 The percentage of COHNS elements in hair is as follows:

Element Percentagein Normal hair

Carbon 51% Oxygen 21% Nitrogen 17% Hydrogen 6%

Sulfur 5% These elements form bonds called side bonds which link together the long chain of amino acids known as the polypeptide chain. This chain forms a helix by creating spiral movement that intertwines. 2.3 The following are the amino acids and the percentage found in hair fiber:

Amino acid Percentage in Normal hair

Cysteine 17.50% Serine 11.70%

Glutamic Acid 11.10% Threonine 6.90% Glycine 6.50% Leucine 6.10% Valine 5.90%

Arginine 5.60% Aspartic Acid 5.00%

Alanine 4.80% Proline 3.60%

Isoleucine 2.70% Tyrosine 1.90%

Phenylalanine 1.40% Histidine 0.80%

Methionine 0.50% Millions of polypeptide chains reside in the cortex layer. Side bonds such as hydrogen bonds, salt bonds and disulfide bonds link together these polypeptide chains. Hair fibers are held in place by the side bonds which attribute to the elasticity and strength of hair. A hydrogen bond can easily be broken by water or heat, and is a physical side bond. Collectively, hydrogen bonds account for one-third of hair’s strength. Salt bonds are also physical side bonds. Strong acidic or alkaline solutions break salt bonds because they are affected by changes in pH. Like hydrogen bonds, salt bonds also account for approximately one-third of hair’s strength. Disulfide bonds differ from hydrogen and salt bonds because they are not physical side bonds. Disulfide bonds are chemical side bonds. Disulfide bonds link together two sulfur atoms attached to cysteine amino acids within the polypeptide chains. Chemical hair relaxers and permanent waves chemically alter the hair’s disulfide bond. Disulfide bonds cannot be broken by water or heat1.

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3. Uses of Human Hair The unique properties of human hair such as its unique chemical composition, slow degradation rate, high tensile strength, thermal insulation, elastic recovery, scaly surface, and unique interactions with water and oils, along with its socio-cultural roles, have led to many diverse uses. These uses also depend on the variety of hair available, varying in terms of five parameters: length, color, straightness or curliness, hair damage, and contamination. This variation depends on the culture, ethnicity, hair styles, and the hair care practices in the region. For example, in areas with malnutrition or in areas where hair treatments such as permanent waving, dyeing, and chemical shampoos are common, hair is more damaged. Chemical contamination in hair is observed due to use of toxic dyes and chemicals in hair care, or due to the presence of toxic chemicals in the atmosphere or food chain of the area. For example, in many areas of the world where electronic waste recycling is carried out, hair is found contaminated with brominated flame retardants2. This section describes uses for different kinds of human hair according to the field of application. 4. Applications of Human Hair 4.1 Fashion, Theatre, and Cosmetics Industry 4.1.1. Wigs, Hair Extensions, Eyelashes, Moustaches, Beards, and Other Beauty Accessories. This is one of the most ancient and currently the largest of the human hair-based industries, with a constantly increasing scale due to global expansion of the fashion industry. The oldest known wigs are from1400 B.C. Egypt, some of which are still intact today after3400 years3. In the eastern countries, such products primarily catered to the theatre world, but in the west they evolved significantly with the fashion industry. This application predominantly uses good quality, long hair of almost all colors. In addition, hair in which all strands have surface scales in the same direction (similar to hair on the human body), known as Remy hair, is preferred because it tangles much less during working. Non-Remy hair is often used after chemically removing the outer scale containing cuticle layer. Pure Remy hair products are expensive, while those with non-Remy hair or human hair mixed with other fibers are cheaper. The users include hair care researchers, product manufacturers, hair stylists, and trainees in both labs and salons. These tests use hair of different colors, range of curliness, and different levels of damage. 4.1.2. For Making Cosmetic Brushes. Scales on hair can hold cosmetic powder particles and apply it uniformly on skin or a surface. Therefore, human hair is used in making

cosmetic brushes4. Almost all kinds of straight hair can be used for brushes. 4.2 Agriculture 4.2.1.As Fertilizer. Human hair is one of the highest nitrogen containing (∼16%) organic material in nature because it is predominantly made up of (nitrogen-containing) proteins. For comparison, cattle dung contains only ∼0.2-0.3% nitrogen. In addition, human hair also contains sulfur, carbon, and 20 other elements essential for plants5. In the atmosphere, hair decomposes very slowly, but moisture and keratinolytic fungi present in soil, animal manure, and sewage sludge can degrade hair within a few months6. In traditional Chinese agriculture, human hair was mixed with cattle dung to prepare compost that was applied to the fields in the winter season. In some communities in India, hair has been used directly as fertilizer for many fruit and vegetable crops and in making organic manures. Recent experiments on horticulture plants show that direct application of human hair to soil provides the necessary plant nutrients for over two to three cropping seasons. A company named Smart Grow has popularized the fertilizer use of human hair in the USA by selling it in the form of hair mats for potted plants. Small entrepreneurs in the USA are also promoting hair as fertilizer by packaging it in various user-friendly forms such as in tea bags. By mixing human hair with cattle dung and feeding worms on the mixture, it is also possible to make good quality vermin compost within a period of about 2 months. Non composted hair, however, has advantages than composted hair because composting can lead to some loss of nitrogen. While the biological decomposition pathways take a few months, human hair can also be decomposed within a few hours by chemically hydrolyzing it at high temperatures in acid or base solutions. The hydrolyzed solution, which mainly consists of amino acids with some fatty acids and nucleotides, can be used as a liquid fertilizer after neutralization. Experiments using this hydrolyzed as foliar spray show enhancement of the chlorophyll content as well as biomass in spinach and wheat plants. Application of the solution to soil also shows improvements in the color and size of Amarant husdubius and hot pepper plants. Experiments on the hot pepper plants also show increased diversity of soil-intrinsic bacteria, which significantly reduces the spread of a wilt disease in these plants caused by the bacterium Ralstoniasolanacearum. Long term impacts of this use, however, need to be assessed. Any kind of hair without toxic contamination can be used for fertilizers. Finely shredded hair, however, is better for faster decomposition.

4.2.2.Pestcontrol. Human hair is also known to address problems arising from many animals as well as insect pests, although by different mechanisms. Among large animals, it has been used to repel rabbits in Mauritius, rodents, and wild boar in India, and deer in the USA. Typically, the hair is spread along the boundary of the fields/farms or near rat holes in the field. Rabbits, rodents, and wild boar finds their food by sniffing, and hair supposedly causes them discomfort during sniffing by coming into their nostrils. In the case of deer, repulsionis

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supposedly caused by human smell emanating from the hair because hanging hair in nylon bags also works, and the technique does not seem to work well in the areas where deer are unafraid of humans. Among insects, human hair is used for deterring rhinoceros beetles in India. Small balls of human hair are placed at the nodes of the affected plant such as coconut tree. Beetles get tangled in the hair becoming unable to move. By using human hair mats, farmers in Florida(USA) were able to save ∼$45,000 on pesticides on about 1million plants in the year 2007 in addition to the labor savings and benefits as fertilizer.

4.3 Composite Materials 4.3.1. Reinforcement of Construction Materials. Due to high tensile strength and high friction coefficient, human hair has been used for reinforcing clay-based constructions. In rural areas in Uttar Pradesh and Madhya Pradesh, India, Bangladesh, Syria, and in European countries, human hair/clay mixture (along with other binders) is used in plastering house walls, lining ovens, making wheels, and so forth. The addition of hair significantly reduces cracking and prolongs the life of these structures. Research shows that human hair reinforcement enhances the structural strength as well as the thermal insulation capacity of the claystructures7-8. Although such clay-based constructions are now decreasing in rural areas, they are gaining importance in sustainable architecture. Human hair reinforcement also reduces cracks in cement mortar caused by plastic shrinkage by as much as 92%and increases the compressive strength of fly ash/cement concrete by over three times. Any kind of hair can be used in these applications. 4.3.2. Molded Furniture and Objects. A UK-based entrepreneur, Ronald Thompson, has developed a method for making composite materials which includes first weaving human hair into a web or mat and then adding a structural additive like resin or flexible polymer (preferably a recyclable or biodegradable material). The composite has good strength and can be used for making molded structures such as furniture and mannequins. A similar composite with unwoven hair has also been used for making biodegradable eye glasses. 4.3.3. Composites for Superconducting Systems. Superconducting power equipmentoften use fiber-glass-based composites for cryogenic insulations. Michael et al have that composite laminate of human hair (and several other natural fibers) with epoxy resin has dielectric breakdown properties suitable for insulation in cryogenic systems. Compared to currently used glass-fiber composites, these composites can significantly lower the production costs of cryogenic equipment. 4.4 Pollution Control and Remediation 4.4.1. Oil-Water Separation and Oil Spill Remediation. Human hair surface has a high affinity for oils—much higher than its affinity for water. This property is very useful in oil-water separation. After the pioneering work of Phillip A. McCrery from Alabama, USA, booms and mats of human hair have been used to clean up coastal oil

spills in the Philippines and the USA. In this method, oil can be recovered by wringing out the hair, which then can be reused up to 100 times—advantages not present in other oil spill remediation methods. With this method, up to 98% of the spilled oil can be recovered. The oily hair can then be used to grow oyster mushrooms, which decompose the oil. The hair then left can be composted. Human hair can also separate emulsified oil in water, which is very expensive to clean by other methods. 4.4.2. Removing Phenols, Aldehydes, Dyes, and Heavy Metal Pollutants from Water. Human hair absorbs several chemicals from aqueous solutions. Experiments show that human hair can absorb organic pollutants such as formaldehyde and phenol, and heavy metals such as mercury (Hg),copper (Cu), cadmium (Cd), and silver (Ag) from aqueous solution. The capacity for metal absorption can be enhanced by pre-treating the hair with an alkali. Powdered human hair has good absorptive capacity for Ni (II) and Cr(VI) ions at acidic pH, and partially burned human hair9 shows selective absorption of mercury (Hg2+) and silver(Ag+) ions over cobalt (Co2+), copper (Cu2+), and iron (Fe3+).Thus, human hair can be a low-cost absorbent for purification of polluted waters. The recycling/disposal of contaminated hair, however, can become a problem. Partial recovery of absorbed metals from the hair has been explored, but further research is needed to develop recovery/recycling methods for such chemicals from the contaminated hair. 4.5 Pharmaceuticals and Biomedical Applications 4.5.1. Pharmaceuticals. Human hair proteins typically contain 20 essential amino acids, which can be extracted by complete hydrolysis of hair. Some of the amino acids obtained in good yield from human hair are L-cysteine, Leucine,L-isoleucine, and L-valine. L-cysteine and its chemical derivatives are used in many cosmetics and pharmaceutical formulations. For example, L-cysteine is used for permanent wave lotions and wound healing formulations, while one of its derivatives, N-acetyl L-cysteine (NAC),is used to treat conditions such as chest congestions and acetaminophen poisoning. Hair from certain demographic regions is better for extracting certain amino acids. For example, black hair from Asia has more cysteine than blonde hair. In addition, hair that is chemically not altered by any styling treatment is better, because some of these treatments can change the chemical structure of the hair. For example, increasing trend of permanent waving in some parts in China has made cysteine extraction from this hair difficult. 4.5.2.HydrolyzedHair Keratin. Mixture of amino acids and polypeptides obtained by the hydrolysis of keratin protein from human hair, known as hydrolyzed human hair keratin protein (HHKP), is used in hair care products by manycompanies10-11. It is reported to repair hair damage caused by various hair styling treatments supposedly because its constituents are similar to the native hair protein. 4.5.3. Ethno medicinal Uses. Several cultures have been using human hair for preparing traditional medicines. Carbonized human hair has been used in Traditional

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Chinese Medicine for treating hemorrhage, burns, wounds, and scars. It is also used in veterinary medicine to stop bleeding and to promote urination. In rural communities in Chhattisgarh, India, hair ash is applied to open wounds for immediate pain reliefs well as long term recovery. In addition, these communities use formulations made from powdered hair, hair ash, and hair decoction for the treatment of mouth ulcers, ringworm, and blisters due to burns. Another ethno medicinal studying India has revealed therapies using human hair for treatments of anemia, asthma, urinary calculus, piles, rat bite poisoning, foot sprains, sexual problems, and childbirth pain. The quality and purity of hair is of essence in these preparations. 4.5.4. Suturing Material in Surgery. Human hair has sufficiently high strength for use as suture in most surgeries. It is relatively easy to tie knots with and is noninfectious (because of its slow decomposition rate and high compatibility with the human body). Its use as suture was known in European the Middle Ages. Studies have now established the potential of human hair sutures in cataract and conjunctiva wound repair surgeries, general surgeries on humans and animals, and in microsurgery. It can be easily sterilized by autoclaving. Long, undamaged hair of medium thickness (not too coarse or too thin) is best for suturing. 4.5.5. Keratin-Based Engineering Biomaterials.In 2002, Nakamura etal. developed the method to extract proteins rapidly and efficiently from human hair, opening possibilities of reengineering human hair proteins into new materials. Based on this method, many materials with novel properties have been developed such as hair protein based thin films, hairprotein gellan chitosan hybrid fibers, and protein scaffolds and hydro gels for tissue engineering. These materials have several potential applications in bioengineering and medical science such as for wound dressing and soft tissue regeneration with the advantage ofgood biocompatibility with the human body. Investigations on effects of these materials in surgical applications are under progress. 4.5.6. Human Hair Follicle Cell Cultures and Tissue Regeneration. Biomedical studies show that certain cells from human hair follicles such as outer root sheath cells also are useful in wound treatments, antilogous grafting of chronic wounds, and treatment of alopecia. The follicles are collected from fallen hair or hair plucked from volunteers.Therefore, this application is not likely to utilize much of the hair waste. 4.5.7. Flexible Microelectrodes. Human hair by itself is not a good conductor of electricity, but Xu et al. from China have developed a human hair microelectrode by coating its surface with an ultrathin layer of gold. Currently used carbon fiber microelectrodes have good electrical conductivity, chemical stability, and low cost but are brittle and have weaksignal strength and limited biocompatibility. The humanhair microelectrode is flexible and its signal strength canbe tailored by changing the hair length. The gold coating ischemically stable and the electrode is likely to be compatiblewith biological systems. In addition, these electrodes can alsobe useful as

microsensors for small biomolecules. Furtherexperiments are needed to test their efficacy in variousbiological systems. 4.6 Food Industry Many amino acids obtained from human hair such as L-cysteine are also used in the food industry as leavening agent for pizza dough and doughnuts, for artificial meat flavor, in nutritional supplements, and so forth. The use of human hair derived amino acids in the food industry, however, is a big source of concern in many countries (videinfra). 4.7 Scientific Instrumentation

Human hair expands in length on absorbing moisture, and this expansion is reversible. This property was used by Horace-Benedict de Saussure in1783 to make a hair hygrometer for humidity measurements. Since then, the instrument has undergone improvements and modifications in design12-13. Although more sophisticated electronic instruments are now available for more precise measurements, hair hygrometers are still cheap and simple for reasonably good estimations of humidity and are still used in many metrological stations in the world. This application requires long hair (∼12 inches or longer). 4.8 Textiles, Fiber stuffing, and Other Artifacts 4.8.1. Stuffing Toys, Mattresses, and Other Household Items. Due to its elastic and cushiony nature and good thermal insulation properties, human hair has been used to stuff household items such as hair-pin cushions and toys in the USA14-15. And toys, furniture, mattresses, quilts, jackets, and so forth, in India. In pin cushions, natural oil of the hair prevents the pins and needles from rusting. For toys and mattress stuffing, human hair is usually mixed with cotton or other fibers. 4.8.2. Fabrics. High thermal insulation, elasticity, and good tensile strength also make human hair useful for making various kinds of fabrics. In Arunachal Pradesh, India, people have traditionally been making fabrics by mixing human hair with yak hair, nettle fiber, and cotton. In China, human hair, yak hair, and cotton are used to make interlining cloth for coats and jackets. In these applications, mostly cotton yarn is used as warp and the hair is used as weft.Human hair blended with animal fibers is used for making blankets in Panipat, India. A company al. Kishore’s (Chennai, India) has also started making purely human hair fabric and clothing. Making felt from human hair is more difficult than from animal hair, but it is made by many for use as doormats, thermal padding of furniture, artwork, and so forth. A Serbian artist produced 1200 sq. m. of felt from human hair in 2009. 4.8.3. Oil Filters. Tightly woven human hair cloths were used in the 1920s as filters for heavy oils in refineries and distilleries because these processes involved high pressure that many natural fibers could not withstand16. Haircloths, by contrast, were tough and almost untreatable. Later, synthetic fibers with higher tensile strength and smaller diameters became available. Filters of these fibers could

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filter even smaller particles and therefore replaced the human hair filters. 4.8.4. Ropes. Due to good tensile strength, human hair has-been used to make ropes in many cultures, for example, to lift heavy beams and bells in the construction of Japanese temples and for household purposes by Native Americans17.These ropes are still valued for horse riding. 4.9 Artwork Two art traditions evolved in the world around human hair as the key material. The first—hair embroidery flourished in China between the7th and 13th centuries, when women made images of Buddha with their own hair. This art revived again in the late 20thcenturies and now has surpassed its past in color and variety. Earlier, natural shades of hair were used, but now dyed hair is also used. The tradition has even developed a rich school of portrait embroidery. The second tradition hair work evolved in the 19thcentury Scandinavian countries and then spread to England and America18-19. Hair work included intricate jewelry, flowers, buttons, brooches, and so forth, made of human hair with gold and resins. It also included the craft of hair embroidery. Dissolved hair and finely chopped hair mixed with oil were used as paint. The 1853 Crystal Palace Exposition in New York included a full line of hair work jewellery, buttons, flowers, and even a tea-set made completely of hair. Socially, hair work pieces were mainly associated with special affection such as between spouses and in memories of a dead person (with hair work piece made from the deceased person’s hair). Increasing symbolism of hair work with death and mourning led to fading of this tradition by the early 20th century; however, recently, there is increasing interest in reviving this art and a society has been established to connect and promote hair work artists in theworld20. 4.10 Miscellaneous Uses Human hair is placed with other fibers as nesting material to increase breeding of birds in places where bird populations are declining. Long fibers can entangle and cause injuries to the birds; therefore, short hair (3 inches or less) is recommended for the use. A stringed musical instrument named “git-git” is also made using human hair as strings21.

5. Materials Used • Human hair • NaOH • HCl • Ethanol • Acetone • Ninhydrin • H2SO4

6. Extraction of Amino acids Collecting the initial weighed hair samples and dissolved in 0.05 N (20 ml) HCl in a beaker. Mix properly and manually stirred for 15 minutes. After 15 minutes 0.1N NaOH solution added drop wise until the solid separates and residue is washed with 20 ml distilled water. After washing the residue is dissolved in 20 ml of ethanol and heated for 10 min then filtered.

After it is dried, test the residue for the presence of amino acids using following methods. 7. Amino Acid Tests 7.1 Ninhydrin Test Ninhydrin (triketohydrindene hydrate) is a chemical used to detect ammoniaor primary and secondary amines. Amino acids also react with ninhydrin at pH=4. The reduction product obtained from ninhydrin then reacts with NH3 and excess ninhydrin to yield a blue colored substance. This reaction provides an extremely sensitive test for amino acids. Apply this test to any of the amino acids you choose.

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Procedure: - To 0.1 gram of amino acid dissolved in water, add 200mg of ninhydrin dissolved in 10ml acetone. - Notice color changes. - Measure the absorbance of the solution using calorimeter at 550nm. Observation: Direct reaction of ninhydrin solution with amino acid gave dark blue color. Inference :Amino acids are detected. 7.2 Xanthoproteic Test Some amino acids contain aromatic groups that are derivatives of benzene. These aromatic groups can undergo reactions that are characteristics of benzene and benzene derivatives. One such reaction is the nitration of a benzene ring with nitric acid. The amino acids that have activated benzene ring can readily undergo nitration. This nitration reaction, in the presence of activated benzene ring, forms yellow product. Apply this test to tyrosine, tryptophan, phenylalanine and glutamic acid.

Procedure: - To 2 mL amino acid solution in a boiling test tube, add equal volume of concentrated HNO3. - Heat over a flame for 2 min and observe the color.

- Now cool thoroughly under the tap and cautiously run in sufficient 40% NaOH to make the solution strongly alkaline. - Observe the color of the nitro derivative of aromatic nucleus. Observation: Yellow color is seen. Inference : Amino acid is detected. 7.3 Millon’s Test Millon’s test is specific to phenol containing structures (tyrosine is the only common phenolic amino acid). Millon’s reagent is concentrated HNO3, in which mercury is dissolved. As a result of the reaction ared precipitate or a red solution is considered as positive test. A yellow precipitate of HgO is NOT a positive reaction but usually indicates that the solution is too alkaline. Apply this test to tyrosine, phenylalanine, glycine and β-naphtol.

Procedure:

- To 2 mL amino acid solution in a test tube, add 1-2 drops of Millon’s reagent. - Warm the tube in a boiling water bath for 10 min. - A brick red color is a positive reaction.

- Note that this is a test for phenols, and the ninhydrin test should also be positive if it is to be concluded that the substance is a phenolic amino acid. 7.4 Hopkin’s Cole Test The indole group of tryptophan reacts with glyoxylic acid (glacial acetic acid, which has been exposed to light, always contains glyoxylic acid CHOCOOH as an impurity) in the presence of concentrated H2SO4 to give a purple color. Apply this test to glycine, tryptophan and tyrosine.

Procedure: - To a few mL of glacial acetic acid containing glyoxylic acid, add 1-2 drops of the amino acid solution. - Pour 1-2 mL H2SO4 down the side of the sloping test tube to form a layer underneath the acetic acid. - The development of a purple color at the interface proves a positive reaction. Observation :Purple color wasn’t detected Inference :Negative test 7.5 Lead-Sulfide Test When cystine is boiled with 40% NaOH, some of sulfur in its structure is coverted to sodium sulfide (Na2S). The Na2S can be detected by using sodium plumbate solution which causes the precipitation of PbS from an alkaline solution. In order to apply this test, first the sodium plumbate solution should be prepared. Apply this test to cysteine and cystine.

Procedure: - Sodium Plumbate Solution Preparation: - Add 5 mL dilute NaOH to 2 mL dilute lead acetate. - A white precipitate of lead hydroxide forms. - Boil until the precipitate dissolves with the formation of sodium plumbate. - Boil 2 mL amino acid solution with a few drops of 40% NaOH for 2 min. - Cool and add a few drops of the sodium plumbate solution. - A brown color or precipitate is a positive test for sulfides.

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