GROUP 8 FINAL, ORGANIC CHEMISTRY TWO.pptx

Describes chirsrity and drug test of enatiomers

1. GROUP 8 ORGANIC CHEMISTRY 2, 2.1 MEMEBERS HAMALA INNOCENT LUCKY. VU-BPC-2307-0058-DAY KUTEGANA SEMUTEWO. VU-BPC-2307-0245-DAY NASSIWA RITAH. VU-BPC-2307-0057-DAY

2. Question Discuss the stereo chemistry and test or analytical methods for both qualitative and quantitative analysis of these different organic compounds in the pharmaceutical analytical laboratory. • Benzoic acid • Aspirin • Acetanilide • Sugars or carbohydrates (use examples of different sugars or carbohydrates)

3. BENZOIC ACID Benzoic acid is an aromatic carboxylic acid characterized by a carboxyl group attached to a benzene ring.

4. PHYSICAL PROPERTIES 1.Benzoic acid has a colorless appearance in its solid state, which is of a crystalline nature. 2.The crystal structure is monoclinic. 3.The presence of the aromatic ring gives this compound a faintly pleasant odour. 4.At a temperature of 130 o C, the density of this compound reduces to 1.075 grams per cubic centimeter. 5.boiling point of 250 °C and melting point 122.3 °C

5. STEREOCHEMISTRY OF BENENE 1.planarity. Benzene ring is planar contributing to overall molecular planarity. The carboxyl group is also coplanar with the benzene ring due to the resonance,leading to conjugation between the pie electrons of the ring and the carbonyl part of the carboxylic group.

6. 2.STEREOGENIC CENTERS. It has no stereogenic centers(chiral centers) because the two carbons directly involved are either symmetrically substituted or share multiple identical groups thus lacking chirality. 3.CONFIGURATION. Because benzoic acid lacks chiral centers, it does not exhibit stereoisomerism. Consquently,there is only one structural form of benzoic acid, meaning no R/S notation is applicable.

7. 4.NO STERIC ISOMERS. Even though benzoic acid contains functional groups, the absence of chiral centers results in no geometrical stereo isomers (cis and trans)or conformational isomers. 5.DERIVATIVES AND ISOMERISM. Derivatives such as ortho,para and meta substituted benzoic acids can exhibit stereoisomerism if they have substituents that allow for differential spatial arrangements.

8. QUALITATIVE ANALYSIS OF BENOIC ACID 1. SODIUM BICARBONATE TEST benzoic acid reacts with sodium bicarbonate forming sodium benzoate and carbon dioxide gas and water. 2.ESTER TEST. Benzoic acid reacts with alcohol in presence of concentrated sulphuric acid forming an ester identified by a sweat fruity smell. Such a process is called esterification.

9. 3.NEUTRAL FERRIC CHLORIDE TEST benzoic acid reacts with ferric chloride forming a buff colored complex with the Fe3 ions. The acid is first reacted with ammonium hydroxide to form ammonium acetate which is later reacted with the ferric chloride.

10. QUANTITATIVE ANALYSIS OF BENOIC ACID High-performance liquid chromatography (HPLC) is a technique for the separation of components of mixtures by differential migration through a column containing a micro particulate solid stationary phase. Solutes are transported through the column by a pressurized flow of liquid mobile phase, and are detected as they are eluted. The mobile phase is either a single solvent or a blend of two or more having the appropriate eluting power for the sample components. It ranges from a nonpolar liquid to aqueous buffers mixed with an organic solvent. The solvent delivery system comprises a means of degassing, filtering and blending up to four solvents which are then delivered to the top of the column under pressure by a constant flow pump.

11. Liquid samples or solutions are introduced into the flowing mobile phase at the top of the column through a constant or variable volume loop and valve injector that is loaded with a syringe. Columns are straight lengths of stainless steel tubing tightly packed with a micro particulate stationary phase. The column packings are chemically modified silicas, unmodified silica or polymeric resins or gels. Solutes are detected in the mobile phase as they are eluted from the end of the column. The detector generates an electrical signal that can be amplified and presented in the form of a chromatogram of solute concentration as a function of time. A dedicated microcomputer is an integral part of a modern high-performance liquid chromatograph. Software packages facilitate the control and monitoring of instrumental parameters, and the display and processing of data.

12. ASPIRIN/ACETYLSALICYLIC ACID ASA

13. STEREO CHEMISTRY ASA does not exhibit stereochemistry in the conventional sense because it lacks a chiral center. However, its stereochemistry can be discussed in terms of its structural configuration and the orientation of its functional groups. Structure of Aspirin consists of a benzene ring (aromatic ring) attached to a carboxylic acid group (-COOH) and an ester group (-COOCH3_33​) derived from acetic acid.

14. Lack of chirality. Aspirin lacks a chiral center (a carbon atom with four different substituents), so it is not an optically active compound and does not exist in enantiomeric forms. This is because its molecular structure is symmetric in terms of the positioning of the functional groups on the benzene ring . Planar structure. The benzene ring in Aspirin is planar due to the delocalized π-electron system, which stabilizes the ring and keeps it flat. The carboxylic acid and ester groups are attached to this aromatic ring and are also in the same plane, contributing to a relatively rigid, flat structure.

15. Conformation of Functional Groups . Aspirin is not chiral, the orientation of its ester and carboxylic acid functional groups relative to the benzene ring can impact its reactivity and interactions with biological molecules. The ester group (-COOCH3_33​) is typically oriented in such a way to minimize steric hindrance, which means the spatial arrangement of atoms can influence how Aspirin interacts with its biological target, cyclooxygenase (COX) enzymes. Molecular Interactions and Enzyme Binding . Aspirin functions by acetylating a serine residue in the active site of the COX enzymes, which is crucial for its anti-inflammatory and analgesic effects. The orientation and accessibility of its acetyl group are vital for this interaction. The flat, planar nature of the benzene ring allows for π-π interactions with aromatic residues within the COX enzyme binding site, stabilizing the enzyme-substrate complex and enabling the transfer of the acetyl group to the serine residue.

16. Stereochemistry in Relation to Aspirin's Derivatives While Aspirin itself is achiral, derivatives of Aspirin can be designed to have chiral centers. For example: Chiral Aspirin Derivatives : By substituting groups on the aromatic ring or modifying the ester moiety, researchers can create chiral derivatives that may have different pharmacokinetic properties or selectivities for COX-1 and COX-2 enzymes.

17. Synthesis of aspirin Aspirin is synthesized by producing an esterification reaction of salicylic acid with acetic anhydride

18. Physical properties of aspirin It's crystalline, white, odor less and aromatic in nature It has an acidic pH ( 3.5) It has a melting point of 136°c It is soluble in organic solvents such as ethanol,methanol and ether It predominantly exists in monoclinic crystal system

19. Chemical reactions undergo by the different functional groups in aspirin Aromatic ring This undergoes electrophilic aromatic substitution reaction For example nitration to introduce a nitro group to the benzene ring

20. Continuation Carboxylic group Neutralization The carboxylic group can react with bases to form salts It can also undergo esterification whereby it reacts with alcohols in presence of an acid catalyst to form esters

21. Continuation Ester group undergoes hydrolysis and transesterification Transesterification Reaction with Alcohols: Aspirin can react with alcohols in the presence of an acid catalyst to form different esters. Hydrolysis When aspirin is treated with a strong acid (e.g., hydrochloric acid) in water, the ester bond is hydrolyzed to yield salicylic acid and acetic acid.

22. QUALITATIVE TESTS OF ASA In a pharmaceutical laboratory, qualitative tests are used to confirm the identity and assess the purity of ASA. 1 . Ferric Chloride Test Detects the presence of phenolic groups, which can indicate the presence of salicylic acid, a degradation product of Aspirin. Procedure : Dissolve a small amount of Aspirin in ethanol or water. Add a few drops of ferric chloride solution (FeCl3_33​ ). Observation : A purple or violet color indicates the presence of salicylic acid. Pure Aspirin should not produce this color unless it has degraded to salicylic acid.

23. 2. Melting Point Determination Checks the purity of Aspirin based on its melting point range. Procedure : Place a small sample of Aspirin in a melting point capillary tube. Gradually heat the tube in a melting point apparatus and observe the temperature range over which the sample melts. Observation : Pure Aspirin melts at around 135-136°C. A melting point lower than this range or a broad melting range suggests impurities or degradation.

24. 3. Thin Layer Chromatography (TLC) Identifies Aspirin by comparing its Rf value with a standard. Procedure : Prepare a TLC plate and spot a small amount of Aspirin and a standard Aspirin solution on the plate. Develop the plate in a suitable solvent system (e.g., ethyl acetate and hexane). Visualize the spots under UV light or by staining with iodine vapor. Observation : Compare the Rf value (ratio of the distance traveled by the compound to the distance traveled by the solvent front) of the sample with that of the standard. Identical Rf values indicate the presence of Aspirin.

25. 4. Infrared (IR) Spectroscopy Purpose : To confirm the presence of characteristic functional groups in Aspirin. Procedure : Obtain the IR spectrum of the Aspirin sample using an IR spectrometer. Observation : Look for specific absorption bands corresponding to the ester functional group (around 1750 cm⁻¹) and the carboxylic acid group (around 1680-1725 cm⁻¹). The presence of these peaks confirms the presence of Aspirin . 5. Reaction with Sodium Hydroxide Confirms the presence of the ester group in Aspirin. Procedure : Dissolve a small amount of Aspirin in water and add a few drops of sodium hydroxide ( NaOH ) solution. Heat the mixture gently for a few minutes. Add a few drops of phenolphthalein indicator. Observation : The solution will turn pink if hydrolysis has occurred, indicating the presence of the ester group in Aspirin.

26. 6 . Reaction with Acetic Anhydride and Sulfuric Acid (Acetylation Test) Purpose : To confirm the presence of salicylic acid impurity by re-acetylation. Procedure :Mix a small amount of Aspirin with acetic anhydride and a few drops of concentrated sulfuric acid. Heat the mixture gently. Allow it to cool and then add water. Perform the ferric chloride test on the resulting solution. Observation : If the original sample had free salicylic acid, the ferric chloride test will now show a reduced or absent purple color, indicating successful acetylation of salicylic acid.

27. Quantitative tests for Aspirin Quantitative tests for ASA in a pharmaceutical laboratory are designed to determine the exact amount of Aspirin in a sample, ensuring its potency and compliance with pharmacopeial standards . 1. UV-Visible Spectrophotometry Purpose : To quantify the concentration of Aspirin in a sample based on its absorbance. Procedure : Sample Preparation : Dissolve a known weight of Aspirin in ethanol or methanol. Hydrolyze the Aspirin to salicylic acid using a base, such as sodium hydroxide ( NaOH ). Reaction with Ferric Chloride : After neutralization, react the hydrolyzed sample with ferric chloride to form a violet-colored complex. Measurement : Measure the absorbance of the violet complex at 525 nm using a UV-visible spectrophotometer. Calculation : Use a calibration curve of known concentrations of salicylic acid to determine the concentration of Aspirin in the sample. Observation : The intensity of the color correlates with the concentration of salicylic acid, allowing calculation of the original Aspirin content.

28. 2. High-Performance Liquid Chromatography (HPLC) Purpose : To accurately quantify the amount of Aspirin and any impurities in a sample. Procedure : Sample Preparation : Dissolve a known quantity of Aspirin in a suitable solvent, like acetonitrile or methanol. Chromatographic Conditions : Use an HPLC system with a C18 column and an appropriate mobile phase (often a mixture of water and acetonitrile or methanol with a small amount of acid like phosphoric acid). Detection : Aspirin is detected at around 230 nm using a UV detector. Quantification : Compare the peak area of the sample with a standard calibration curve prepared with known concentrations of Aspirin. Observation : The area under the peak corresponding to Aspirin is directly proportional to its concentration in the sample.

29. 3. Potentiometric Titration Purpose : To determine the amount of Aspirin by titration with a strong base. Procedure : Sample Preparation : Dissolve a known weight of Aspirin in a mixture of ethanol and water. Titration : Titrate with a standardized sodium hydroxide ( NaOH ) solution. Detection of Endpoint : Use a pH meter to detect the endpoint, which occurs when all the acetylsalicylic acid has been hydrolyzed to salicylic acid and the carboxylic acid groups have been neutralized. Calculation : Calculate the amount of Aspirin based on the volume of NaOH used to reach the endpoint. Observation : The titration curve will show a sharp change in pH at the endpoint, corresponding to the complete neutralization of the Aspirin.

30. 4. Gas Chromatography-Mass Spectrometry (GC-MS) Purpose : To quantify Aspirin and its degradation products with high sensitivity and specificity. Procedure : Sample Preparation : Convert Aspirin to a more volatile derivative, if necessary, by silylation or methylation. Chromatographic Conditions : Inject the sample into a gas chromatograph equipped with a capillary column and connected to a mass spectrometer. Detection : Aspirin and its metabolites are identified based on their retention times and mass spectra. Quantification : Compare the peak areas of the sample with a standard calibration curve for Aspirin. Observation : The mass spectrum provides a highly specific identification and quantification of Aspirin based on its molecular ions and fragmentation pattern.

31. 5. Back Titration . Determines the amount of Aspirin in samples with poor solubility/ other interfering substances. Procedure : Sample Preparation : Dissolve a known amount of Aspirin in an excess of standardized sodium hydroxide solution to hydrolyze the ester bond, forming salicylic acid and sodium acetate. Back Titration : Titrate the remaining NaOH with a standardized acid solution, such as hydrochloric acid ( HCl ). Calculation : The difference between the amount of NaOH added and the amount remaining after hydrolysis gives the amount of NaOH that reacted with the Aspirin, allowing for the calculation of the Aspirin content. Observation : The volume of acid required to neutralize the remaining base is used to determine the concentration of Aspirin in the sample.

32. Application of stereo chemistry of aspirin inpractice 1 . Pharmacological Activity. Chira l Centers : Aspirin does not have a chiral center, but its metabolite, salicylic acid, can be conjugated to form chiral derivatives. The stereochemistry of these metabolites can affect their pharmacological activity and interactions with biological targets. Enzyme Interaction : The stereochemistry of aspirin affects its interaction with enzymes like cyclooxygenase (COX). Aspirin irreversibly inhibits COX enzymes by acetylating a serine residue, which is stereoselective . This acetylation is responsible for its anti-inflammatory, analgesic, and antipyretic properties. 2. Stereoselective Metabolism Metabolic Pathways : The metabolism of aspirin involves stereoselective processes that can influence the drug's efficacy and safety. For example, enzymes in the liver may preferentially metabolize one stereoisomer over another, affecting the duration and intensity of the drug's action. 3. Drug Design and Development Stereochemical Modifications : Understanding the stereochemistry of aspirin and its derivatives allows for the design of more selective and potent analogs. Modifying the stereochemistry can lead to drugs with improved therapeutic profiles, such as reduced side effects or enhanced efficacy .

33. Prodrugs : Aspirin's stereochemistry can be exploited to design prodrugs that are activated only under specific conditions, such as in the presence of a particular enzyme or at a particular pH, allowing for targeted drug delivery. 4. Chiral Interactions with Proteins Binding Specificity : Proteins, including enzymes and receptors, often have chiral binding sites. Aspirin’s interaction with these proteins is dependent on its stereochemistry, influencing its effectiveness as an anti-inflammatory or anticoagulant. 5. Stereochemistry in Pharmacokinetics Absorption, Distribution, Metabolism, and Excretion (ADME) : The stereochemistry of aspirin can influence its pharmacokinetics. For example, the rate of absorption and the route of elimination can be affected by the stereochemical properties of the molecule. 6. Enantiomeric Purity and Regulation Regulatory Requirements : In pharmaceutical development, the enantiomeric purity of drugs is a key regulatory concern. Stereochemistry can affect the safety and efficacy of the drug, necessitating rigorous control during manufacturing

34. Applications of qualitative and quantitative tests of aspirin in medicine and pharmacy 1 . Quality Control in Pharmaceutical Manufacturing Qualitative Tests : These tests help confirm the identity and purity of aspirin in raw materials, intermediates, and finished products. Quantitative Tests : These tests determine the concentration or amount of aspirin in a sample to ensure that it meets the specified dosage. 2 . Pharmaceutical Research and Development Qualitative Analysis : Identifies the chemical structure and confirms the presence of aspirin in new formulations or combination therapies. Methods like nuclear magnetic resonance (NMR) and mass spectrometry (MS) are often used for structural elucidation. Quantitative Analysis : Assesses the stability, bioavailability, and release profile of aspirin in different formulations, such as tablets, capsules, or suspensions. HPLC and dissolution testing are commonly employed to measure the amount of aspirin released over time under various conditions.

35. 3. Clinical Diagnostics Qualitative Tests : Used in emergency settings to detect aspirin overdose. For example, the ferric chloride test is a rapid qualitative test where a purple color indicates the presence of salicylates (the breakdown products of aspirin) in urine. Quantitative Tests : Determine the concentration of aspirin or its metabolites (salicylates) in biological fluids (like blood or urine) to assess compliance, therapeutic levels, or toxicity in patients. Techniques include: Gas Chromatography-Mass Spectrometry (GC-MS) : Provides precise quantification of salicylate levels in serum, crucial for managing overdose cases. Enzyme Immunoassays (EIA) : Used in clinical laboratories to measure aspirin or salicylate concentrations in blood samples, offering a quick assessment of drug levels. 4. Monitoring and Compliance Qualitative Tests : Used to confirm the presence of aspirin in patient samples to ensure adherence to prescribed medication regimens. Simple spot tests or chromatographic techniques can provide quick results. Quantitative Tests : Employed to monitor therapeutic drug levels in patients, especially in those requiring long-term aspirin therapy for cardiovascular protection or anti-inflammatory purposes. Regular monitoring helps adjust dosing to maintain efficacy while minimizing adverse effects.

36. 5. Regulatory Compliance and Standardization Qualitative and Quantitative Tests : Both types of tests are critical for regulatory submissions to health authorities (. They ensure that aspirin products meet established standards for identity, strength, quality, purity, and potency. This is essential for gaining approval to market the drug and for post-marketing surveillance to monitor product quality over time. 6. Detection of Counterfeit Drugs Qualitative Tests : Quick screening methods like TLC or IR spectroscopy are used to detect counterfeit aspirin products in the market. These tests can identify incorrect or missing active ingredients. Quantitative Tests : Assure the amount of aspirin in the product matches the labeled claim, helping to identify substandard or falsified products.

37. ACETANILIDE / N-PHENYLACETAMIDE An organic chemical compound classified as an amide ;>CONH in terms of functional group. The carbonyl group is bonded directly to a nitrogen atom. It also contains an aromatic ring, with six carbon atoms and alternating double-single-double-single bonding pattern around the ring.

38. Molecular Formula; C8H9NO Structure ;

39. Synthesis of acetanilide Method 1. -Aniline when reacted with acetic anhydride/glacial acetic acid in the presence of zinc dust, Acetanilide is formed -Reflux a mixture of aniline, acetic anhydride ,glacial acetic acid and zinc dust under anhydrous condition , -and then poured the mixture into ice cold water to get acetic anhydride precipitate

40. - The crude precipitate of acetic anhydride is recrystallized to get pure crystals of acetanilide. - oxidation of aniline during the chemical reaction is prevented by use of zinc. Below is the chemical reaction .

41. Method 2. - Acetanilide can be prepared by acetylating aniline with acetic anhydride in the presence of concentrated hydrochloric acid. -Aniline is dissolved in hydrochloric acid, add acetic anhydride, then stir well. -Sodium acetate mixture is poured into water and then Acetanilide is formed .

42. STEREO CHEMISTRY - Acetanilide has unsaturated amide group which is achiral. -Acetanilide exhibit conformational isomerism due to the presence of the benzene ring and the amide group . conform ational Isomer ; Z-Conformer: The amide group and the benzene ring are in a staggered conformation, with the benzene ring and the methyl group on the same side of the amide group

43. E -Conformer : The amide group and benzene ring are in an eclipsed conformation, with the benzene ring and the methyl group on opposite sides of the amide group . Cis-Trans Isomerism : Due to the presence of the amide group acetanilide can exhibit cis- trans isomerism. However, this isomerism is not significant in this compound

44. Chirality : Acetanilide does not have a chiral center and so does not exhibit optical activity

45. QUALITATIVE ANALYSIS Appearance : White to grey crystalline powder. The melting points: can be used for characterization and identification of acetanilide. That is 126-128°C Solubility ; soluble in chloroform and ethanol ,Slightly soluble in water .

46. Quantitative Analysis; Volumetric Analysis Titration: Reacting acetanilide with a standardized solution such as sodium hydroxide , -Detect the endpoint using a indicator -Calculate the concentration or amount of acetanilide based on the volume of titrant used

47. APPLICATIONS OF ACETANILIDE -used as an antipyretic agent ,fever reducing agent. - used as an inhibitor of hydrogen peroxide decomposition - used in the synthesis of penicillin and in other pharmaceuticals . -used to stabilize cellulose ester varnishes .

48. SUGARS AND CARBOHYDRATES Stereoisomerism : Sugars are prime examples of chiral molecules. Examples: Glucose and fructose are monosaccharides that differ in their stereochemistry .

49. Starch and cellulose are polysaccharides with complex stereo isomeric structures .

50. QUALITATIVE ANALYSIS OF CARBOHYDRATE There are different test is use in qualitative analysis of carbohydrate like; Molisch test . Benedict’s test. Silver mirror test (Tollen’s reagent ). Molisch test Molisch test is used to distinguish between carbohydrates and non- carbohydrates. It is the preliminary test used to detect the presence of carbohydrates in a sample.

51. Principle Molisch’s test, which is based on the dehydration reaction, identifies carbohydrate content. By adding concentrated H2SO4, the carbohydrates in the sample are dehydrated and converted to aldehyde. Furfural (made by the dehydration of pentoses or pentosans) or hydroxymethylfurfural (generated by the dehydration of pentoses or pentosans) are the aldehydes which are created (produced by the dehydration of hexoses or hexosans). The Molisch reagent’s contain α- naphthol which interacts with the aldehyde to produce a purple color condensation product

52. Reagents Molisch reagent, Sample, Concentrated H2SO4 Procedure Add 1 drop of Molisch’s reagent (10 percent - naphthol in ethanol) to 2 mL of a sample solution present in a test tube. Pour 1-2 mL of concentrated H2 SO4 down the edge of the test tube, forming a layer at the bottom. Compare your results to a control (which contain water instead of test sample) by observing the purple colour complex at the interface between two layers

53. Benedict’s test Benedict test is use for identifies reducing sugars, which have free ketone or aldehyde functional group. Principle By interacting with an alkaline reagent, such as Benedict’s solution, the reducing sugar will be converted to enediols. Depending on the sugar content, the reducing sugar produces a green to brick red precipitate. The color shift is caused by the reduction of copper (II) to copper (I) in the solution, which results in the formation of a red-colored precipitate. The resulting precipitate is insoluble in water. The alkalinity in the redox reaction solution is provided or maintained by the sodium carbonate. Sodium citrate bind to copper- II ions, preventing copper I reduction . Reagent Benedict reagent, Test sample. Procedure Make a 1 mL solution by a test sample. In the same test tube, pour 2 mL of Benedict’s solution. Then boil it in a water bath, for 3-5 minutes. Keep an eye on the test tube for the formation of brick red precipitate

54. Silver mirror test (Tollen’s test) This test is given positive by reducing sugars. Detect presence of aldehyde containing carbohydrates also differentiate them from ketone containing carbohydrates. Principle Tollen’s reagent ( ammoniacal solution of silver nitrate) reacts with reducing sugars or aldehydes to create free silver metal. This reagent is an alkaline solution of silver nitrate ( AgNO3) dissolved in l iquid ammonia ( NH3) that forms a complex. When AgNO3 combines with NaOH , a brown precipitates of silver oxide ( Ag2O ) is produced. Aqueous ammonia dissolves silver oxide, forming the combination [Ag(NH3)2]NO3

55. . Tollen’s reagent contains this complex, which is a strong oxidizing agent that converts the aldehyde group in certain carbohydrates to a carboxylic acid. The reagent’s silver ions are reduced to free metallic silver, which produces a silver mirror on the test tube’s bottom and sides .

56. Reagent Tollen’s reagent, Test sample. Procedure Take two clean, dry test tubes and fill one with 1 ml of the test sample and the other with 1 ml of distilled water as a blank. To both test tubes, add 2 mL Tollen’s reagent. Put both the test tube in hot water bath. If a dark grey precipitate e or silver mirror is formed on the bottom and sided of test tube, it indicates the positive result for silver mirror test. Also it confirms the presence of reducing sugar or aldose or α- hydroxy ketoses in the given test sample. Quantitative analysis of carbohydrate There are number of quantitative tests are available. The method mostly depend upon the type of sugar whether it is reducing sugar or not. This method use for the measurement of total carbohydrate present in the given test sample. Here, some of quantitative method is mentioned below.

57. 1 . Anthrone test Anthrone test is used to determine and quantify carbohydrates in a variety of samples such as milk, blood serum, and its variations. Principle The concentrated acid in the Anthrone reagent first hydrolyzes carbohydrate into component monosaccharide if it is present in the form of free carbohydrate as poly- or monosaccharide or bound as in a glycoprotein or glycolipid. The concentrated acid also catalyses the dehydration of monosaccharides, resulting in furfural (from pentoses) or hydroxyl furfural (from hexoses). Anthranol , the enol tautomer of anthrone, is the active form of the reagent, which combines with the carbohydrate furfural derivative to produce a green colour in dilute solutions and a blue colour in concentrated solutions, which can be detected colorimetrically . The blue - green solution shows absorption maximum at 620 nm.

58. Reagents Anthrone reagent, sugar stock solution, sample. Procedure First take varied volume of sugar solution (in μl ) from the given stock solution into a succession of test tubes and make up the volume to 1 ml with distilled water. Then take suitable volumes of unknown sample and make up final volume to 1.0ml distilled water. In each tube, add 5 ml of anthrone reagent and vortex thoroughly to mix. Cool test tubes. Cover the tubes with caps on top and incubate for 17 minutes at 90°C or 10 minutes in a boiling water bath. Cool the tubes to room temperature before comparing the optical densities of the solutions to a blank at 620 nm. Make an absorbance vs. sugar concentration standard curve to determine quantity of sugar in the unknown sample .

59. 2.DNSA (Dinitrosalicylic acid) method DNSA is an aromatic compound that reacts with react with reducing sugars to estimate the amount of carbohydrates. Principle 3, 5- Dinitrosalicylic acid (DNSA) that forms 3-amino-5-nitrosalicylic acid (ANSA) when it combines with reducing sugars [with a free carbonyl group (C=O)]. The aldehyde functional group (in glucose) and the ketone functional group (in fructose) are both oxidised in this process. DNSA is reduced to ANSA, which under alkaline condition is transformed to a reddish brown colored complex. The absorbance is measured at 540 nm.

60. Reagents DNSA reagent, standard sugar solution, 40% potassium sodium tartrate, test sample. Procedure Dilution of sugar standard (in μl) with various concentration made by transferring respective quantity of sugar from the standard sugar solution and by adding distilled water adjusting it to a total volume of 200 µl. In each of test tubes, add 0.5 ml of DNSA reagent. Mix thoroughly. Allow 15 minutes in a boiling water bath. Then, mix with 0.5 ml of a 40% potassium sodium tartrate ( Rochell’s Salt) solution. Using a spectrophotometer set to 540nm records the absorbance and find sugar concentration of test sample from standard curve absorbance v/s sugar concentration in µg/200 µl

61. Q uestions Describe the stereochemistry, qualitative and quantitave tests of Aspirin and their application in the medical sector. Discuss the stereochemistry of Acetanilide.