Identification of Active Pharmaceutical Ingredients by Color Test.SANJIV MENONLaboratory of Instrumental Analysis Department of Pharmaceutical Analysis and Medicinal ChemistryFaculty of Pharmacy, Universitas Padjadjaran.

ABSTRACTThere are certain ways to identify functional groups for alkaloid, nitrogen base compounds, sulfonamide and barbiturate and antibiotics in drug molecules using chemical reagents or by color test. There would be specific reaction for functional groups that could be observed by the formation of color and precipitation. The common methods use for alkaloid identifications are using wagner test, concentrated acid and acetone water. Whereas for nitrogen base compounds, it can be tested by using Lieberman test as well as Chen-Kao test therefore for hexamine which belongs to can be nitrogen determine thru red litmus paper. Lastly is identification technique for sulfonamide is such as Koppayi-Zwikker test and by involving vanillin-H2SO4 and for barbiturates, the test is done by using acetone water. This test is done in order to find out which drug belongs to which group and how it is being identified. For the identification alkaloid reagents used are papaverine hydrochloride. Moreover for nitrogen base compounds,ephedrine HCL and hexamine are used as reagents . For sulfonamide, sulfamethoxazole whereas for barbiturate, luminal and barbital being used as the reagents. Concentrated sulphuric acid is used in most of test as catalyst and for speed up the reaction.Keywords : alkaloid, barbiturate, ephedrine, H2SO4, nitrogen base compound, sulfonamide, test.

INTRODUCTIONAlkaloidsAlkaloids are a group of diverse biomolecules.They are all secondary compounds and are derived from amino acids or from the transamination process. From the biological point of view, the alkaloid is any biologically active and heterocyclic chemical compound which contains nitrogen and may have some pharmacological activity and, in many cases, medicinal, or ecological use. They are a special group of chemicals that are active at different cellular levels of organisms. Alkaloid,any of a class of naturally occurring organic nitrogen-containing bases. Alkaloids have diverse and importantphysiological effectson humans and other animals. Well-known alkaloids includemorphine,strychnine,quinine,ephedrine, andnicotine. Alkaloids are derived from L-lysine, L-ornithine, L-tyrosine, L-tryptophan, L-histidine, L-phenylalanine, nicotinic acid, anthranilic acid, or acetate. The terpenoid, steroid, and purine alkaloids are also important. Alkaloids also occur in the animal kingdom. Differently from plants, the source of these molecules in an animal's body can be endogenous or exogenousThe chemical structures of alkaloids are extremely variable. Generally, an alkaloid contains at least onenitrogen atomin anamine-typestructurei.e., one derived from ammonia by replacing hydrogen atoms with hydrogen-carbon groups called hydrocarbons. This or another nitrogen atom can be active as abasein acid-basereactions. The name alkaloid (alkali-like) was originally applied to the substances because, like the inorganic alkalis, they react with acids to form salts. Most alkaloids have one or more of their nitrogen atoms as part of a ring of atoms, frequently called a cyclic system. Alkaloid names generally end in the suffix -ine, a reference to their chemical classification as amines. In their pure form most alkaloids are colourless, nonvolatile, crystalline solids. They also tend to have a bitter taste. A precise definition of the term alkaloid (alkali-like) is somewhat difficult because there is no clear-cut boundary between alkaloids and naturally occurring complex amines. Typical alkaloids are derived from plant sources, they are basic, they contain one or more nitrogen atoms (usually in a heterocyclic ring) and they usually have a marked physiological action on man or other animals. The name proto-alkaloid or amino-alkaloid is sometimes applied to compounds such as hordenine, ephedrine and colchicine which lack one or more of the properties of typical alkaloids. Other alkaloids, not conforming with the general definition, are those synthetic compounds not found in plants but very closely related to the natural alkaloids (e.g. homatropine). In practice, those substances present in plants and giving the standard qualitative tests outlined below are termed alkaloids, and frequently in plant surveys this evidence alone is used to classify a particular plant as alkaloid-containing.Barbiturates are a type of depressant drug that cause relaxation and sleepiness. A barbituate overdose occurs when someone accidentally or intentionally takes more than the normal or recommended amount of this medication. This is life threatening.At relatively low doses, barbiturates may cause you to seem like you are drunk or intoxicated.Barbiturates are addictive, producing physical dependence and a withdrawal syndrome that can be life-threatening. Whereas tolerance to the mood-altering effects of barbiturates develops rapidly with repeated use, tolerance to the lethal effects develops more slowly, and the risk of severe toxicity increases with continued use. They are synthetic drugs derived from barbituric acid , which is a synthetic condensation product of malonic acid and urea. They differ mainly in the substitution pattern at position-5 with some also including an N-methyl at N-1. The most well known derivative, phenobarbitone , has been used medicinally since 1912, mainly in the treatment of epilepsy.Over 2,500 barbiturates have reportedly been synthesized with more than 50 of these presently marketed for clinical use throughout the world. Barbiturates such asphenobarbitalwere long used asanxiolyticsandhypnotics, but today have been largely replaced bybenzodiazepinesfor these purposes because of less potential for lethaloverdoses.[7][8][9]However, barbiturates are still used asanticonvulsants, as para-operative sedatives (ex.sodium thiopental), and analgesics for cluster headaches/ migraines (ex.fioricet).

Sulphonamides is A drug may be classified by the chemical type of the active ingredient or by the way it is used to treat a particular condition. Each drug can be classified into one or more drug classes.Sulphonamides (sulfa drugs) are one of a group of drugs derived from sulphanilamide that prevents the growth of bacteria.Sulphonamides compete with p-aminobenzoic acid (PABA) for the enzyme dihydropteroate synthetase, which is important in the formation of folic acid that is required by the bacteria. Folic acid is required for the synthesis of precursors of DNA and RNA both in bacteria and in mammals. Mammals obtain their folic acid in their diet but bacteria need to synthesize it. Sulphonamides inhibit the growth of bacteria but do not kill them i.e. their action is bacteriostatic.Many sulphonamides are rapidly excreted and very soluble in urine so they are used to treat infections of the urinary tract. Sulfanilamide which was the first compound used has H's at R1 and R4. To date about 15,000 sulfonamide derivatives, analogues, and related compounds have been synthesized. This has lead to the discovery of many useful drugs which are effective for diuretics, antimalerial and leprosy agents, and antithyroid agents. The basic structure of sulfonamide cannot be modified if it is to be an effective competitive "mimic" for p-aminobenzoic acid. Essential structural features are the benzene ring with two substituents para to each other; an amino group in the fourth position; and the singly substituted 1-sulfonamido group. Sulfonamides, as antimetabolites, compete with para-aminobenzoic acid (PABA) for incorporation into folic acid. The action of sulfonamides illustrates the principle of selective toxicity where some difference between mammal cells and bacterial cells is exploited. All cells require folic acid for growth. Folic acid (as a vitamin is in food) diffuses or is transported into human bodyMETHODSAlkaloids A.1 Alkaloid that contains quinolone structureA1.1 Quinine HydrochlorideI. A few milligram of quinine HCL is dissolved in 3 mL of water in a test tube. Add a few drops of H2SO4. Observe the fluorescence under UV lamp 366 nm.II. A few drops of bromine water is added to a saturated solution of quinine HCL, a yellow precipitate is formed, which gradually disappears with formation of rose-red coloration, changing to cherry-red. The colour disappear after a time, but can be reproduced by adding more bromine water, and the reaction is more delicate and prompt if the quinine solution be previously gently warmed.III. Thalleioquin test: a few milligram of quinine HCL is dissolved in 3 mL of water in a test tube. Add 2-3 drops of bromine water. Agitate the mixture. Add 1-2 drops of ammonia water (NH3 liquid). A green color of thalleioquim (ammoniacal solution) indicates positive result.IV. A few milligram of quinine HCL is dissolved in 3 mL of Hg2Cl2 solution. Observe the crystal formed under microscope.V. Erythroquine reaction: a few milligram is dissolved in 3 mL of water in a test tube. Add 2-3 drops of bromine water. Agitate the mixture. Add 1-2 drops of potassium hexacynoferrate and NH4OH.A.2 Alkaloid that contains BenzylisoquinolineA2.1 Papaverine HydrochlorideI. A few milligram of papaverine hydrochloride is dissolved in 3 mL of water. Add 2-3 drops of potassium permanganate solution. Add 2-3 drops of Marquis reagent (formaldehyde in H2SO4 1:9). A green color will be formed and further develops to blue.II. A few milligrams of papaverine hydrochloride is dissolved in 3mL of water. Add 2-3 drops of concentrated H2SO4. Heat the mixture gently. A violet color indicates positive result.III. A few milligrams of papaverine hydrochloride is reacted with 3 mL of acetic anhydride, add cautiously 3 drops of H2SO4 and heat on a water bath for 3-4 minutes, a yellow colour with a green fluorescence is produced.IV. A few milligrams of papaverine hydrochloride is dissolved in 3 mL of Hg2Cl2 solution. Observe the crystal formed under microscope.

Nitrogen Base CompoundB.1 EphedrineI. Liebermann test: A few milligrams of ephedrine Hcl is added with 3 drops of Liebermann reagent (1g of sodium nitrite in 10 mL of H2SO4)II. Chen-Kao test: Dissolved a few milligrams of ephedrine Hcl in 3 mL of water and add 0.1 mL of copper (ii) sulfate. Followed by 2 mL of sodium hydroxide, a violet colour produced. Add 1 mL of ether and shake, a purple colour is produced in the ethereal layer and a blur colour in the aqueous layer.III. Dissolved a few milligrams of ephedrine Hcl in 3 mL of water. Add a few drops of sodium hydroxide and 4 mL of potassium ferricyanide TS, and heat. An odour of benzaldehyde is perceptible.IV. A few milligrams of ephedrine Hcl is dissolved in 3 mL of Hg2Cl2 solution. Observe the crystal formed under microscope.B.2 Hexamine I. In a test tube, mix an equivalent weight of hexamine and salicylic acid. Add 1 mL of H2SO4. Heat the mixture gently.II. In a test tube, mix few milligrams of hexamine with dilute H2SO4 amd a drop of formaldehyde. Place a piece of cotton and a wet red litmus paper on the upper part of the tube. Check the change of litmus color during reaction.SulfonamideA.1 SulfamethoxazoleI. A few milligrams of sulfamethoxazole is dissolved in 3 mL of dilute Hcl. Add 2-3 drops of p-diaminobenzaldehyde solution.II. A few milligrams of sulfamethoxazole is dissolved in 3 mL of NaOH. Add 3 drops of 15% CuSO4 pentahydrate solution.III. Koppayi-Zwikker test: A few milligrams of sulfamethoxazole is dissolved in 2 mL of ethanol. Add 1 drop of Koppayi-Zwikker reagent (1% CO(NO3)2 in ethanol) and 1 drop of pyrolidine.IV. Add vanillin-H2SO4 to a few milligrams of sulfamethoxazole.BarbiturateB.1 Luminal (phenobarbital)I. Parri test: 5mg of luminal is dissolved in 3 mL of methanol. Add 0.1 mL of Parri reagent (contains 1g Co(NO3)2 and 1g of CaCl2 in 10 mL of methanol). Shake the mixture and add 0.1 mL of NaOH solution. A violet-blue color and precipitate are formed.II. A few milligrams of luminal is put onto an object glass. Add 2-3 drops of a mixture of acetone-water. Observe the crystals and microscope.III. Add a few drops of vanillin reagent [1g of vanillin in 20mL of H2SO4] to luminal, dark color change to violet.B.2 BarbitalI. Parri test: 5 mg of barbital is dissolved in 3 mL of methanol. Add 0.1 mL of parri reagent (contains 1g Co(NO3)2 and 1 g of CaCl2 in 10 Ml of methanol). Shake the mixture and add 0.1 mL of NaOH solution. A violet-blue color and precipitate are formed.II. A few milligrams of barbital is put onto an object glass. Add 2-3 drops of a mixture of acetone-water. Observe the crystals under microscope.III. Add a few drops of vanillin reagent [1g of vanillin in 20Ml of H2SO4] to barbital, dark colour changed to violet.AntibioticsA.1 AmoxicillinI. A few milligrams of amoxicillin is put on a metal spatula. Burn the sample. Specific odor is formed.II. A few milligrams of amoxicillin is put on a porcelain spot plate. Add 1-2 drops of concentrate H2SO4. Observe the fluorescence under UV lamp.III. A few milligrams of amoxicillin is put onto an object glass. Add 2-3 drops of a mixture of acetone-water. Observe the crystal under microscope.A.2 ErythromycinI. A few milligrams of erythromycin is put on a porcelain spot plate. Add 1-2 drops of concentrate H2SO4. Observe the fluorescence under UV lamp.II. A few milligrams of erythromycin is put onto an object glass. Add 2-3 drops of a mixture of acetone-water. Observe the crystals under microsope.A.3 ChloramphenicolI. A few milligrams of chloramphenicol is put on a metal spatula. Burn the sample. Specific odor is formed.II. In a test tube, a few milligrams of chloramphenicol is dissolved in 2 mL of freshly prepared NaOH solution. Add 1 mL of pyridine. Heat the mixture gently in a waterbath at 100C for 2 minutes. A positive result is indicated by red color in pyridine layer.III. A few milligrams of chloramphenicol is put onto an object glass. Add 2-3 drops of mixture of acetone-water. Observe the crystals under microscope.A.4 TetracyclineI. Liebermann test: A few milligrams of tetracycline is added with 3 drops pf Liebermann reagent (1g of sodium nitrite in 10mL of H2SO4).II. Marquis test: Add 2-3 drops of Marquis reagent (formaldehyde in H2SO4 1:9) to a few milligrams of tetracycline.Mandelin test: Add 2-3 drops pf mandolin reagent (ammonium vanadate in H2SO4) to a few milligrams of tetracycline. A violet-red-orange color is changed.

RESULTSFunctional GroupReagentsProceduresResult

1.Alkaloid1.1QuinolineStructure

Quinine Hydrochloride

Water+H2SO4

1. A few milligrams of quinine HCl is dissolve in 3mL of water in a test tube.2. Add a few drop of H2SO4.3. Observe the fluorescence under UV lamp 366nm.

Blue colour solution being observed under UV lamp 366nm.

Acetone water1. A few milligram of quinine HCl is dissolved in 3mL of acetone water.2. Observe the crystal formed under the microscope.Crystal formed

1.2Benzylisoquinoline

Papaverin Hydrochloride

Potassium Permanganate+Marquis reagent(formaldehyde in H2SO4 1:9)1. A few milligrams of papverin HCl is dissolve in 3mL of water in a test tube.2. Add 1 drop of formaldehyde and 9 drops of H2SO4 into the test tube.

Negative result:The yellow colour formed

Concentrated H2SO41. A few milligrams of papaverin HCl is dissolve in 3mL of water in a test tube.2. Add 2-3 drops of concentrated H2SO43. Heat the mixture gentally.Negative result:The colour did not change

Acetic anhydride+H2SO4

1. A few milligrams of papverin HCl is reacted with 3mL of acetic anhydride in a test tube.2. Add cautiously 3 drops of H2SO4 3. Heat on a water bath for 3-4 minutes A yellow colour with a green fluorescene is produced.

Acetone water1. A few milligram of papverin HCl is dissolved in 3mL of acetone water.2. Observe the crystal formed under the microscope.A cystal formed

2.Nitrogen base compound2.1 Ephedrine

Lieberman reagent(1g of sodium nitrite in 10 mL of H2SO4)

Liberman Test1. A few milligram of ephedrine HCl is add with 3 drops of Liberman reagent (1g of sodium nitrite in 10 mL of H2SO4)

A yellow colour formed

Water+Copper(II) Sulphate+Sodium Hydroxide

1. Dissolve s few milligrams of ephedrine HCl in 3mL of water 2. Add 2 drops of copper(II) sulphate3. Add 2 mL of Sodium HydroxideA violet colour formed

Water+ sodium hydroxide+ potassium ferricyanide TS1. 1.Dissolve s few milligrams of ephedrine HCl in 3mL of water..2. Add 3 drops of sodium hydroxide.3. Add 4 mL of potassium ferricyanide TS4. Heat the solutionAn odour of almond is produced

Acetone water1. A few milligram of ephedrine HCl is dissolved in 3mL of acetone water.2. Observe the crystal formed under the microscope.

A Crystal formed

2.2 Hexamine( Hexamethylene tetramine)

Salicylic acid1. Mix an equivalent weight of hexamine and salicylic acid in a test tube2. Add 1mL of H2SO43. Heat the solution gentally

Formaldehyde

1. Mix a few milligram of hexamine with dilute H2SO4 in a test tube2. Add a drop of formaldehyde3. Place a piece of cotton and a wet red litmus paper on upper part of test tube

Negative result :The colour of red litmus paper did not change

3Sulfonamide 3.1 Sulfamethoxazole

Dilute HCl+p-diaminobenzaldehyde

1. A few miligrams of sulfamethoxazale is dissolved in 3 mL of dilute HCl2. Add 2-3 drops of p-diaminobenzaldehyde solutionA yellow colour formed

NaOH+ CuSO4 pentahydrate solution

1. A few miligrams of sulfamexazole is dissolve in 3mL of NaOH.2. Add 3 drops of 15% CuSO4 pentahydrate solutionA green-yellowish colour formed

Vanillin-H2SO4

1. Add vanillin-H2SO4 to a few miligrams of sulfomexazoleYellow colour formed

4.Barbiturate

4.1 Luminal (phenobarbital)

Acetone water

1. A few milligram of luminal is put onto an object glass.2. Add 2-3 drops of a mixture of acetone water.3. Observe crystal under microscope

A crystal formed

Vanilin reagent(1g vanillin in 20mL of H2SO4)

1. Add a few drops of vanillin reagent (1g vanillin in 20mL of H2SO4) to luminal

Negative result:A yellow-brownish colour formed

4.2Barbital

Acetone water

1. A few milligram of barbital is put onto an object glass.2. Add 2-3 drops of a mixture of acetone water.3. Observe crystal under microscope

A crystal formed

Vanilin reagent(1g vanillin in 20mL of H2SO4)

1. Add a few drops of vanillin reagent (1g vanillin in 20mL of H2SO4) to luminal

Negative result:Yellow colour formed

5.Antibiotic5.1 Amoxicilin

amoxicilin

1. A few miligrams of amoxicillin is put on a metal spatula.2. Burn the sample

3. Actone water

1. A few milligram of erythromycin is put onto an object glass.2. Add 2-3 drops of a mixture of acetone water.3. Observe crystal under microscope

A crystal formed

5.2 Erythromycin

ConcentratedH2SO41. A few miligrams of erythromycin is put on a porcelain spot plate.2. Add 1-2 drops of concentrated H2SO43. Observe under UV lamo

A green solution being observed

Acetone water1. A few milligram of erythromycin is put onto an object glass.2. Add 2-3 drops of a mixture of acetone water.3. Observe crystal under microscope

A crystal formed

5.3 Chloramphenicol

Chloramphenicol

1. A few miligrams of amoxicillin is put on a metal spatula.2. Burn the sample

Bad smell

NaOH+pyridine

1. In a test tube, a few miligrams of chloramphenicol is dissolved in 2mL of freshly prepared NaOH2. Add 1 mL of pyridine.3. Heat the mixture in a waterbath at 100C for 2 minutes

A red clour formed at pyridine layer

Acetone water

1. A few milligram of erythromycin is put onto an object glass.2. Add 2-3 drops of a mixture of acetone water.3. Observe crystal under microscope

A crystal formed

5.4 Tetracycline Lieberman reagent(1g of sodium nitrite in 10mL of H2SO4) Lieberman test1. A few miligrams of tetracycline is added with 3 drops of Liberman reagent(1g of sodium nitrite in 10mL of H2SO4)

Black-chocolate colour formed

Marquis reagent(Formaldehyde in H2SO41:9) Marquis Test1. Add 2-3 drops of Marquis reagent(formaldehyde in H2SO4 1:9) to a few miligrams of tetracycline.

Chocolate-yellow colour is formed

Mandolin reagent(ammonium vanadate in H2SO4)1. Add 2-3 drops of mandolin reagent (ammonium vanadate in H2SO4) to a few miligrams of tetracycline)

Dark colour near orange colour is formed

DISCUSSION The purpose to carry out the experiment is to identify the alkaloid, nitrogen base compounds, sulphonamide, barbiturate and antibiotic in drug molecule by using chemical reagent.The principle for this experiment is the functional group (such as alkaloid, nitrogen base compound, sulphonamide, barbiturate and antibiotic) provides specific chemical reaction that could be observed by the formation of colour or precipitate in the experiment.The first experiment of Quinine HCl is observation of Quinine Hydrochloride under UV lamp.The reagent used water and sulphuric acid. 3mL of water into the test tube to dissolve the Quinine Hydrochloride.A few drops of the sulphuric acid is added into the test tube.The sulphuric acid will exchange position with the HCl in the Quinine HCL become Quinine H2SO4 which will formed the blue fluorescence under the UV lamp. The blue fluorescence will disappear if adding in a few drop of HCl.The second experiment is to test the alkaloid with sulphuric acid. The reagent used are water and concentrated sulphuric acid. A few milligram of the papaverin HCl is dissolved in 3mL of water.2-3 drops of concentrated H2SO4 is added in the colourless solution. After that the mixture is heated gentally with Bunsen burner.This is done so the concentrated sulphuric acid can react with Benzylisoquonine. This experiment also give the negative result that the colourless solution did not change . this is because of a few reason which the reagent and sample is contaminated with the impurities or the reagent and the sample is expired. The third experiment is testing of the papverin HCl with the acetic anhydride and sulphuric acid. The reagent are acetic anhydride and sulphuric acid. A few miligrams of the papaverin HCl is reacted with 3ml acetic anhydride, Then 2-3 drops of sulphuric acid is added. The sulphuric acid act as the catalyst in the experiment. The mixture is heat gentally in water bath .A yellow colour with green fluorescene was produced. The acetic anhydride is added to dehydrated the hydrogen ion of papavein HCL. The fourth experiment is the crystal test. The reagent used are acetone water. The Hg2Cl2 and the acetone water will cause the forming of the crystal of the sample. The crystal of sample can be seem by microscope.Next up is the experiment of Ephedrine HCl. Ephedrine HCl is one of the Nitrogen base compound. The first experiment of the ephedrine HCl is Lieberman test. The reagent for this test is the Lieberman reagent which contain 1g sodium nitrite in 10mL sulphuric acid. A few miligrams of ephedrine HCl is added with 3 drop of Lieberman reagent.A yellow colour precipitate formed. The second experiment is the Chen-Kao test.The reagents are water, copper(II)sulphate, sodium Hydroxide. A few miligrams of ephedrine HCl was dissolved in 3mL of water. Then the copper(II)sulphate which is blue in colour is added in the test tube, followed by 2mL of sodium hydroxide .There was no reaction between copper sulphate and Hydrochloric acid but in the presence of sodium hydroxide copper hydroxide formed is precipitated which is bluish in color and dissolves in hydrochloric acid due to neutralisation of sodium hydroxide.A violet colour is produced The third test is to test the odour or smell of benzaldehyde. The reagent for this experiment are water, sodium hydroxide and potassium ferricyanide TS. A few miligrams of ephendrine HCl is dissolved in 3mL of water.A few drop of NaOH is added in the text tube. 4 mL of . potassium ferricyanide TS to react with ependrine to form benzaldehyde which produce the almond adour. . The purpose to add the NaOH is to neutralize the HCl of ephendrine The fourth experiment is the crystal test. The reagent is acetone water, supposed the reagent is Hg2Cl2,but because the lab shortage of Hg2Cl2, so we replace the Hg2Cl2. Hg2Cl2 is more effective than acetone water. The Hg2Cl2 and the acetone water will cause the forming of the crystal of the sample. The crystal of sample can be seem by microscope. Hexamine is also one of the nitrogen base compound.The first experiment carry out to test the reaction between hexamine and salicylic acid. The second test is to test the basicity of hexamine with the litmus paper. The reagent for this experiment is dilute sulphuric acid, formaldehyde. Mix a few milligram of hexamine is mixed with dilute H2SO4 and a drop of formaldehyde.A piece of cotton was placed with a wet red litmus paper on the upper part of test tube and then the solution was heated. The reaction of the sample produces NH3 gas which is base that will turns the re litmus paper to blue colour. .The first experiment pf sulfamethoxazole is to test the reaction of sulfamethoxazole during acid form. The reagent that used in this experiment is dilute HCl and p-diaminobenzaldehyde. A few miligrams of sulfamethoxazole was dissolved in 3 mL of dilute HCl.2-3 drops p-diaminobenzaldehyde is adeed in the test tube of The HCl will react with the NH2 in sulfamethoxazole to form NH3+ which will later react with p-diaminobenzaldehyde to produce yellow precipitate solution. The second experiment is to test the reaction of sulfamethoxazole in base condition.The reagent that used in this experiment is NaOH, 15% CuSO4 pentahydrate. A few miligrams of sulfamethoxazole is dissolved in 3mL of NaOH. 3 drops of NaOH, 15% CuSO4 pentahydrate is added in the test tube. The NaOH added to sulfamethaxazole is to react with the NH which is acid in sulfamethoxazole to form N- ions which will react with 15% CuSO4 pentahydrate to form the green-yellow precipitate solution. The third experiment is to test the reaction of vanillin with the sulfamethoxazole. The reagent used in this experiment is vanillin-H2SO4. The vanillinH2SO4 was added to a few milligram of sulfamethoxazole. The sulphuric acid will react with NH2 to form NH3+ that will react with vanillin to form yellow colour solution.

Luminal (phenobarbital) is one chemical that form from barbiturate. The first experiment that form barbiturate is is the crystal test. The reagent is acetone water The crystal of sample can be seem by microscope. The second experiment is vanillin test. The reagent used in this experiment is the vanillin reagent which contain 1g vanillin in 20mL of sulphuric acid.A few drops of vanillin reagent is added to luminal. The sulphuric acid will react with NH2 to form NH3+ that will react with vanillin The first experiment which is been used for is the crystal test as the parri test was not carried out. The reagent is acetone water, supposed the reagent is Hg2Cl2,but because the lab shortage of Hg2Cl2, so we replace the Hg2Cl2. Hg2Cl2 is more effective than acetone water. The Hg2Cl2 and the acetone water will cause the forming of the crystal of the sample. The crystal of sample can be seem by microscope. The third experiment is vanillin test. The reagent used in this experiment is the vanillin reagent which contain 1g vanillin in 20mL of sulphuric acid. A few drops of vanillin reagent is added to barbital. The H2SO4 will react with NH2 to form NH3+ that will react with vanillin.

For antibiotics there are four sample was used to test. Firstly is Amoxicilin which is one of the antibiotic. For the first experiment a few milligram of the amoxicillin is put on a metal spatula. Then, the sample was burn. A specific odour is produced. The second experiment is the testing of the amoxicillin under UV Lamp. The reagent that used in this experiment is concentrated sulphuric acid . A few miligrams of amoxicillin is put on a porcelain spot plate.1-2 drops of concentrated sulphuric acid is added to the porcelain spot plate. The blue fluorescene is formed under the UV lamp. This is because the concentrated H2SO4 replace the NH in the amoxicillin structure. The third experiment is the crystal test. The reagent is acetone water, supposed the reagent is Hg2Cl2,but because the lab shortage of Hg2Cl2, so we replace the Hg2Cl2. Hg2Cl2 is more effective than acetone water. The Hg2Cl2 and the acetone water will cause the forming of the crystal of the sample. The crystal of sample can be seem by microscope. For Erythromycin The first experiment is to test it under UV Lamp.The reagent that used in this experiment is concentrated sulphuric acid. A few milligrams Erythromycin of is put on a porcelain spot plate.1-2 drops of concentrated H2SO4 is added to the porcelain spot plate. The green fluorescene is formed under the UV lamp. This is because the concentrated H2SO4 replace the NH in the amoxicillin structure. The second experiment is the crystal test. The reagent is acetone water, supposed the reagent is Hg2Cl2,but because the lab shortage of Hg2Cl2, so we replace the Hg2Cl2. Hg2Cl2 is more effective than acetone water. The Hg2Cl2 and the acetone water will cause the forming of the crystal of the sample. The crystal of sample can be seem by microscope. Chloramphenicol is one of the antibiotic. The first experiment is to test the odour of the amoxicillin. A few milligram Chloramphenicol of the is put on a metal spatula. Then, burn the sample. A specific odour which is a bad spell is produced. The second experiment is to test the reaction of chloramphenicol in pyridine layer. The reagent that used in this experiment is NaOH and the pyridine. A few miligrams of chloramphenicol is dissolved in the 2mL of freshly prepared NaOH. 1mL of the pyridine is added in the test tube. The mixture is heated in the waterbath at 100C for 2 minutes. The red colour formed in pyridine layer is obtained. The NaOH added to sulfamethaxazole is to react with the NH which is acid in sulfamethoxazole to form N- ions which will react with pyridine layer to formed red colour. The third experiment is the crystal test. The reagent is acetone water, supposed the reagent is Hg2Cl2,but because the lab shortage of Hg2Cl2, so we replace the Hg2Cl2. Hg2Cl2 is more effective than acetone water. The Hg2Cl2 and the acetone water will cause the forming of the crystal of the sample. The crystal of sample can be seem by microscope. Tetracycline is also one of the antibiotic.The first experiment is Lieberman test.The reagent is the Lieberman reagent which contain 1g sodium nitrite in 10mL H2SO4. A few miligrams of tetracycline is add with 3 drop of Lieberman reagent. The dark-brown colour precipitate formed. The second test is Marquis test. The reagent of this experiment have marquis reagent which contain formaldehyde in H2SO4 with ratio of 1:9.The experiment start with 2-3 drops of marquis reagent is added to a few milligrams of Tetracycline. A brown-yellow precipitate is formed. The third test in this experiment is mandolin test. The reagent that used in this experiment is mandolin reagent which contain ammonium vanadate in sulphuric acid to a few miligrams of tetracycline.A dark colour near orange colour form because the ammonium vanadate react with th NH3+ ion that formed from the reaction of H2SO4 and NH2. CONCLUSIONBy using chemical reagents the identification of alkaloid nitrogen base compound sulphonamide barbiturate and antibiotics can be done

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