Pharmaceutical Organic Chemistry II Lab Manual PDF

PHARMACEUTICAL ORGANIC CHEMISTRY-II LAB MANUAL PHARMACEUTICAL ORGANIC CHEMISTRY II LAB MANUAL Pharm D 2nd semester Prepared by: Dr Rabia Mehtab By: DR RABIA...

PHARMACEUTICAL ORGANIC CHEMISTRY-II LAB MANUAL PHARMACEUTICAL ORGANIC CHEMISTRY II LAB MANUAL Pharm D 2nd semester Prepared by: Dr Rabia Mehtab By: DR RABIA MEHTAB 1  PHARMACEUTICAL ORGANIC CHEMISTRY-II LAB MANUAL LIST OF EXPERIMENTS Sr. # Date Object Initial LAB SAFETY RULES AND GUIDELINES PART-I (GENERAL LAB TECHNIQUES) 01. Introduction to laboratory Techniques & glassware 02. To perform recrystallization of Benzoic acid 03. To perform recrystallization of Aspirin 04. Compare melting points of recrystallized and pure Benzoic Acid 05. Compare melting points of recrystallized and pure Aspirin 06. To determine purity of recrystallize Benzoic acid using TLC 07. To determine purity of recrystallize Aspirin using TLC PART-II (DERIVATIZATION OF ORGANIC COMPOUNDS) 08. To prepare Calcium Citrate from Citric acid 09. To prepare Calcium Tartrate from tartaric acid 10. To prepare 2,4-dinitrophenyl hydrazine derivative of aldehyde 11. To prepare oximes of aldehydes/ketones 12. To prepare Benzoic acid from Benzamide 13. To prepare Amide-Nitrate derivative (using Urea) By: DR RABIA MEHTAB 2 PHARMACEUTICAL ORGANIC CHEMISTRY-II LAB MANUAL PART-I (INTRODUCTION TO LABORATORY TECHNIQUES) By: DR RABIA MEHTAB 3 PHARMACEUTICAL ORGANIC CHEMISTRY-II LAB MANUAL Chemical Lab Safety Basic Safety rules:  Always read the label on a reagent bottle before using its contents.  Always wear safety goggles when handling chemicals.  Never touch chemicals with your hands.  Never return unused chemicals to their original containers.  To avoid waste, do not take excessive amounts of reagents. You should know how to read the label and MSDS NFPA Diamond Label  National Fire Protection Association diamond indicates flammability, reactivity, health concerns, and other potential hazards associated with the reagent.  Numbers are used to rate potential hazards on a scale from 0 (least hazardous) to 4 (most hazardous) By: DR RABIA MEHTAB 4 PHARMACEUTICAL ORGANIC CHEMISTRY-II LAB MANUAL Chemical Hazard pictogram MSDS A Material Safety Data Sheet (MSDS) is a document that contains information on the potential hazards (health, fire, reactivity and environmental) and how to work safely with the chemical product. It also contains information on the use, storage, handling and emergency procedures all related to the hazards of the material. The MSDS contains much more information about the material than the label. MSDSs are prepared by the supplier or manufacturer of the material. It is intended to tell what the hazards of the product are, how to use the product safely, what to expect if the recommendations are not followed, what to do if accidents occur, how to recognize symptoms of overexposure, and what to do if such incidents occur. What information is on the MSDS? There are nine (9) categories of information that must be present on an MSDS in Canada. These categories are specified in the Controlled Products Regulations and include: 1. Product Information: product identifier (name), manufacturer and suppliers names, addresses, and emergency phone numbers 2. Hazardous Ingredients 3. Physical Data 4. Fire or Explosion Hazard Data 5. Reactivity Data: information on the chemical instability of a product and the substances it may react with By: DR RABIA MEHTAB 5 PHARMACEUTICAL ORGANIC CHEMISTRY-II LAB MANUAL 6. Toxicological Properties: health effects 7. Preventive Measures 8. First Aid Measures 9. Preparation Information: who is responsible for preparation and date of preparation of MSDS Basic Laboratory Techniques 1. Transferring liquids: 1. Using a dropper bottle a. Never lay the dropper on any surface. b. Be sure the dropper never touches your container or its contents. 2. Using a Pasteur Pipet a. Do not pipet directly from the reagent bottle unless instructed to do so. i. Instead, pour some of the liquid into a labeled container and pipet from your container b. Do not touch the container or its contents with the tip of the pipet 2. Mixing Liquids: 1. To dilute concentrated liquids with water, ALWAYS add the concentrated reagent to the water slowly. a. This is especially important to follow when adding a concentrated acid to water. Because of the large amount of heat generated by this combination splattering can occur. 3. Filtering a Mixture: 1. Sometimes it is necessary to separate a solid from a liquid – filtration is necessary. a. Fold a paper circle in half and then in quarters. b. Open the folded paper to form a cone i. One thickness of paper on one side and three thicknesses on the other 2. Put the paper cone in a filter funnel. 1) Place the funnel in an iron ring clamped to a ring stand. By: DR RABIA MEHTAB 6 PHARMACEUTICAL ORGANIC CHEMISTRY-II LAB MANUAL 2) Moisten the filter paper with a small volume of distilled water and gently press the paper against the sides of the funnel. 3) Place a beaker beneath the funnel to collect the filtrate. 4) The tip of the funnel should touch the inside surface of the beaker and extend about one inch below the rim. 3. Decant the liquid from the solid by pouring it down a glass stirring rod into the funnel. 1) Be careful to keep the liquid below the top edge of the cone of filter paper at all times – the liquid must not overflow. 2) Use distilled water from a wash bottle to wash the solid into the filter. 4. When the filtration is complete, wash the solid residue on the filter paper with distilled water to remove traces of solvent – dry the solid. 5. If the filtrate contains a dissolved salt, it may be recovered by evaporation if desired By: DR RABIA MEHTAB 7 PHARMACEUTICAL ORGANIC CHEMISTRY-II LAB MANUAL Experiments involving laboratory techniques Methods of Purification:  Re-crystallization  Sublimation  Chromatography  Extraction  Distillation The methods which we will use in lab are described below; 1. Recrystallization In chemistry, Recrystallization is a procedure for purifying compounds. The most typical situation is that a desired "compound A" is contaminated by a small amount of "impurity B". There are various methods of purification that may be attempted, recrystallization being one of them. A Pure compound is a homogenous sample consisting only of molecules have the same structure. It can be characterized by its physical properties, such as a narrow melting range melting point that matches the accepted value. The method of purification is based on the principle that the solubility of most solids increases with increased temperature. This means that as temperature increases, the amount of solute that By: DR RABIA MEHTAB 8 PHARMACEUTICAL ORGANIC CHEMISTRY-II LAB MANUAL can be dissolved in a solvent increases. During recrystallization, an impure solid compound is dissolved in a hot liquid until the solution is saturated, and then the liquid is allowed to cool. An impure compound is dissolved (the impurities must also be soluble in the solvent), to prepare a highly concentrated solution at a high temperature. The solution is cooled. Decreasing the temperature causes the solubility of the impurities in the solution and the substance being purified to decrease. The pure substance then crystallizes before the impurities- assuming that there was more pure substance than there were impurities. The pure substance will crystallize in a purer form because the impurities won't crystallize yet, therefore leaving the impurities behind in the solution. A filtration process must be used to separate the more pure crystals at this point. The procedure can be repeated. The slower the rate of cooling, the larger the crystals are that form.  The disadvantage of recrystallization is that it takes a long time.  Also, it is very important that the proper solvent is used. This can only be determined by trial and error, based on predictions and observations.  The solution must be soluble at high temperatures and insoluble at low temperatures  The advantage or recrystallization is that, when carried out correctly, it is a very effective way of obtaining a pure sample of some product, or precipitate. General Procedure These are the important steps to the recrystallization process. 1. Dissolve the solute in the solvent: Add boiling solvent to a beaker containing the impure compound. Heat the beaker and keep adding solvent until the solute is completely dissolved. See Step 1 2. Cool the Solution: The solution is cooled in open air first, and then cooled in an ice bath. Slow cooling often leads to purer crystals. Crystals should form on the bottom of the beaker. The process of "seeding" can be used to aid the formation of crystals- this means adding a pure crystal of the compound. The pure crystal forms a surface for the solute to crystallize upon. See Step 2 3. Obtain the crystals from the solute: The purer crystals precipitated from the solute are the desirable part of the mixture, and so they must be removed from the solvent. The process used for isolating the crystals that remain in the beaker still is called By: DR RABIA MEHTAB 9 PHARMACEUTICAL ORGANIC CHEMISTRY-II LAB MANUAL vacuum filtration. Suction is created using an aspirator, and whatever remains in the beaker is poured though a Buchner funnel. If for some reason there are no crystals visible, a gravity filtration can be performed. Activated carbon is added to the solution, the mixture is boiled, and a funnel system is used to transfer the new mixture to a new beaker of boiling solvent. Filter paper is used in the funnel to remove excess carbon. After this mixture cools slowly there should be large crystals present. 4. Dry the resulting crystals: The crystals are dried by leaving them in the aspirator and then by removing them to a glass dish to wait a while longer. The purity of the crystals can be tested by performing a "melting point determination". By: DR RABIA MEHTAB 10 PHARMACEUTICAL ORGANIC CHEMISTRY-II LAB MANUAL 2. Thin layer Chromatography (TLC) Thin-layer chromatography (TLC) is a chromatography technique used to separate non- volatile mixtures. It may be performed on the analytical scale as a means of monitoring the progress of a reaction, or on the preparative scale to purify small amounts of a compound. TLC is an analytical tool widely used because of its simplicity, relative low cost, high sensitivity, and speed of separation. TLC functions on the same principle as all chromatography: a compound will have different affinities for the mobile and stationary phases, and this affects the speed at which it migrates. The goal of TLC is to obtain well defined, well separated spots. Procedure 1- Thin-layer chromatography is performed on a sheet of an inert substrate such as glass, plastic, or aluminium foil, which is coated with a thin layer of adsorbent material, usually silica gel, aluminium oxide (alumina), or cellulose. This layer of adsorbent is known as the stationary phase. 2- After the sample has been applied on the plate, a solvent or solvent mixture (known as the mobile phase) is drawn up the plate via capillary action. Because different analytes ascend the TLC plate at different rates, separation is achieved. 3- The mobile phase has different properties from the stationary phase. For example, with silica gel, a very polar substance, non-polar mobile phases such as heptane are used. The mobile phase may be a mixture, allowing chemists to fine-tune the bulk properties of the mobile phase. 4- After the experiment, the spots are visualized. Often this can be done simply by projecting ultraviolet light onto the sheet; the sheets are often treated with a phosphor, and dark spots appear on the sheet where compounds absorb the light impinging on a certain area. Some common techniques for visualizing the results of a TLC plate include a. UV light b. Iodine Staining: is very useful in detecting carbohydrates since it turns black on contact with Iodine c. KMnO4 stain (organic molecules) By: DR RABIA MEHTAB 11 PHARMACEUTICAL ORGANIC CHEMISTRY-II LAB MANUAL d. Ninhydrin Reagent: often used to detect amino acids and proteins e. Cerric ammonium molybdate (CAM), and p-anisaldehyde 5- To quantify the results, the distance traveled by the substance being considered is divided by the total distance traveled by the mobile phase, this ratio is called the retardation factor (Rf), or sometimes colloquially as retention factor. 6- The retardation factor, RF, is commonly used in paper chromatography and thin layer chromatography for analyzing and comparing different substances. It can be mathematically described by the following ratio Rf value = Distance covered by solute/ Distance covered by solvent 7- An RF value will always be in the range 0 to 1; if the substance moves, it can only move in the direction of the solvent flow, and cannot move faster than the solvent. By: DR RABIA MEHTAB 12 PHARMACEUTICAL ORGANIC CHEMISTRY-II LAB MANUAL TYPES OF CHROMATOGRAPHY By: DR RABIA MEHTAB 13 PHARMACEUTICAL ORGANIC CHEMISTRY-II LAB MANUAL 3. Melting point The melting point is an important physical property of a compound. The melting point can be used to identify a substance and as an indication of its purity. The melting point of solid is defined as the temperature at which the solid exists in equilibrium with its liquid under an external pressure of 1 atmosphere. Factors affecting melting point 1. Size of the molecule: Melting point is higher for higher molecular weight compounds. 2. Force of attraction between the molecules: The force of attraction between the molecules affects the melting point of a compound. Stronger intermolecular interactions result in higher melting points. Ionic compounds usually have high melting points because the electrostatic forces holding the ions (ion-ion interaction) are much stronger. In organic compounds the presence hydrogen bonding, generally leads to higher melting point. 3. Purity: Impurities lower the melting point, hence melting point use as criteria to check the purity of solid compounds. 4. Isomeric form: Trans-isomers are more stable than cis-isomer, therefore they have higher melting point. Types of Melting Point  Sharp melting point: A pure crystalline compound usually possesses a sharp melting point and it melts completely over a narrow range of temperature of not more that 0.5- 1 C. “It can be defined as the temperature at which the solid immediately converts into liquid. This range should not be more than ± 0.5 – 1 C”.  Melting Range: The melting range of a compound is one of the characteristic properties of a pure solid. “The melting range is defined as the span of temperature from the point at which the crystals first begin to liquefy to the point at which the entire sample is liquid”. By: DR RABIA MEHTAB 14 PHARMACEUTICAL ORGANIC CHEMISTRY-II LAB MANUAL Most pure organics melt over a narrow temperature range of 1-2ºC, if heated slowly enough. Impure samples will normally have melting ranges that are greater than 2ºC.  Decomposition Point: The temperature of a substance at which it gets chemically decompose is called decomposition point. This process is normally endothermic as energy is required to break bonds of chemicals undergoing decomposition. Methods of melting point determination: 1. Manual Method Packing a capillary for melting point determination Thin-walled capillary melting point tubes are used to hold melting point samples. This tube needs to be sealed at one end (pre-sealed tubes should be available in the laboratory, or, an open capillary can be sealed by inserting the tip into a Bunsen flame near the base of the flame and turning the tube in your fingers. To pack the tube, the open end is pressed gently into a small amount of the sample of the crystalline material on a watch glass or weighing paper. To transfer the crystals from the open end to the bottom of the tube, tap the bottom gently on the bench top. A densely packed column of crystals about 3 mm high in the tube is all that is required. A packed capillary attached to a normal mercury thermometer is shown below: 2. Instrumental Method Melting point apparatus use to determine melting point of substances with more accuracy and precision in easy and fast track. Simultaneously 3 samples can be placed in a heating block. The melting process can be observed with the help of bright illumination and powerful magnifying glass over heating block. Heating block quickly cools by air pump in apparatus measurement is done. Uses of determining Melting point  Melting point is also used for the identification and characterization of a compound.  If the melting point of two pure samples shows a clear difference in melting points, it indicates that the two compounds must have different structural arrangements. By: DR RABIA MEHTAB 15 PHARMACEUTICAL ORGANIC CHEMISTRY-II LAB MANUAL  If two materials have the same melting point, then they may (not necessarily) have the same structure. 4. Percent Yield calculation To express the efficiency of a reaction, you can calculate the percent yield using this formula: % Yield = (Final weight a / Initial weight b ) x 100. Where, a = weight of product after recrystallization b = weight of product before recrystallization By: DR RABIA MEHTAB 16 PHARMACEUTICAL ORGANIC CHEMISTRY-II LAB MANUAL Introduction to Laboratory Glassware The term laboratory glassware is used to describe a wide range of scientific equipment, that was until the use of plastic became more common, generally made from glass. Glass is a very flexible substance that can be worked into a vast selection of shapes and sizes through molding, cutting and, most commonly, blowing. Working with Laboratory Glassware Glass is extremely durable, resistant to heat and chemicals and can be easily sterilized. Perhaps the greatest benefit, when used in scientific experiments, is its transparent qualities, as this allows experiments to be easily observed. When creating an exothermic reaction, make sure that the experiment takes place in a suitable vessel such as an Erlenmeyer flask. It is also important to avoid warming volumetric equipment on heating plates as this can cause breakage. Type of Glass Materials: Chemistry Lab Glassware can be made of 4 different types of glass materials, each of which possesses different properties and is used for a different application in a laboratory. The most common type of glass material used in any laboratory is ‘Borosilicate 3.3 low expansion Glass’ which is transparent, can withstand thermal shocks, and is resistant to chemical attack. Heavy-walled Boro. 3.3 Glass, lab glassware is generally used for applications to work with high pressure or vacuum. Another, type of glass material is darkened brown or Amber-colored borosilicate 3.3 glass, which can block UV and Infrared radiation. This type of glass is generally used in storage bottle containers for reactive chemicals. Soda-glass is one of the prevailing types of glass, used in the manufacturing of glass containers for beverages, food, and some commodity items. This glass is majorly considered to be used for packaging containers. By: DR RABIA MEHTAB 17 PHARMACEUTICAL ORGANIC CHEMISTRY-II LAB MANUAL Last but not least type of glass material, is Quartz. Also the most expensive of all glass types, it can withstand very high temperatures and is only used in certain specific areas or works in a laboratory. Quartz glass can be used when standard laboratory glassware, made from Borosilicate glass, isn’t suitable. Laboratory Glassware and Chemical Resistance Laboratory Glassware is typically manufactured from borosilicate glass which is resistant to most types of acid. In fact, glass is generally highly chemical resistant but is vulnerable to alkaline substances with a 9.0 or greater pH. Alkalis attack the surface of the glass, breaking down the bonds between the silica and oxygen. The glass surface dissolves, and the overall structure of the equipment is compromised. There are many different kinds of laboratory glassware items: Examples of glassware containers include:  Beakers are simple cylindrical shaped containers used to hold reagents or samples.  Flasks are narrow-necked glass containers, typically conical or spherical, used in a laboratory to hold reagents or samples. Examples flasks include the Erlenmeyer flask, Florence flask.  Reagent bottles are containers with narrow openings generally used to store reagents or samples. Small bottles are called vials.  Jars are cylindrical containers with wide openings that may be sealed. Bell jars are used to contain vacuums.  Test tubes are used by chemists to hold, mix, or heat small quantities of solid or liquid chemicals, especially for qualitative experiments and assays  Desiccators of glass construction are used to dry materials or keep material dry.  Glass evaporating dishes, such as watch glasses, are primarily used as an evaporating surface (though they may be used to cover a beaker.) Examples of glassware used for measurements include:  Graduated cylinders are thin and tall cylindrical containers used for volumetric measurements.  Volumetric flasks are for measuring a specific volume of fluid. By: DR RABIA MEHTAB 18 PHARMACEUTICAL ORGANIC CHEMISTRY-II LAB MANUAL  Burettes are similar to graduated cylinders but have a valve at the end used to disperse precise amounts of liquid reagents often for titrations.  Glass pipettes are used to transfer precise quantities of fluids. Other examples of glassware includes:  Stirring rods are glass rods used to mix chemicals.  Condensers are used to condense vapors by cooling them down and turning them into liquids. A list of Lab Equipment and their basic functions are described in the following chart: By: DR RABIA MEHTAB 19 PHARMACEUTICAL ORGANIC CHEMISTRY-II LAB MANUAL By: DR RABIA MEHTAB 20  PHARMACEUTICAL ORGANIC CHEMISTRY-II LAB MANUAL Equipment Function Equipment Function Equipment Function Heat source in Used to grind Used to the chemistry chemicals into clean lab. Uses powder glassware natural gas. Test tube Mortar and brush/beaker Bunsen Burner Pestle brush Used to hold a Used to Used to crucible above transport hot strongly a flame – used Crucible tongs crucibles and heat in conjunction to remove substances with iron ring their covers Crucible and above a Clay triangle Cover flame Used to hold Used to handle a Used in group of test single test tube. conjunction For transport, with iron tubes. holding test tube ring. Evens in a flame or out the heat Wire Gauze placing test tube of a flame on in hot water bath the bottom of Test Tube Test tube a beaker and holder/clamp supports the Rack beaker on the iron ring. For holding a For clamping For storing pair of burets things to the chemicals on a ring stand. ring stand Clamp Holder Wide mouth Buret Clamp Bottle For stopping for scoring For the flow of glass for grabbing substance Triangular file cutting Forceps substances Pinch Clamp through rubber tubing For controlling Used to filter Used to the flow of solids from transfer substances liquids solid through rubber chemicals Screw Clamp tubing from the Filter Paper Spatula, bottle Spoon, Scoop By: DR RABIA MEHTAB 21  PHARMACEUTICAL ORGANIC CHEMISTRY-II LAB MANUAL For measuring Plastic trays with For cutting temperature. various number of tissue Alcohol have red wells for running bulbs, mercury reactions samples or have a silver cutting colored bulb through layers of tissue in Thermometer Chemplate Scalpel a specimen Equipment Function Equipment Function Equipment Function For growing Used to For cutting bacteria Inoculating loop transfer tissue Dissecting samples Petri Dish cultures bacteria Scissors samples to culture dishes Dissecting Used for Microscope Slide Used for Microscope For mounting Needle probing viewing samples for tissue viewing under a very small samples microscope samples of material. By: DR RABIA MEHTAB 22 PHARMACEUTICAL ORGANIC CHEMISTRY-II LAB MANUAL EXPERIMENT NO 2 OBJECT: To perform recrystallization of Benzoic Acid & calculate its % yield. Requirement: Procedure: ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ __________________________________________________________________________ Calculation: Result: ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ By: DR RABIA MEHTAB 23 PHARMACEUTICAL ORGANIC CHEMISTRY-II LAB MANUAL EXPERIMENT NO 3 OBJECT: To perform recrystallization of Aspirin & calculate its % yield (double solvent). Requirement: Procedure: ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ __________________________________________________________________________ Calculation: Result: ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ By: DR RABIA MEHTAB 24 PHARMACEUTICAL ORGANIC CHEMISTRY-II LAB MANUAL EXPERIMENT NO 4 OBJECT: Compare melting points of recrystallize and pure Benzoic Acid Requirement: Procedure: ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ __________________________________________________________________________ ___________________________________________________________________________ __________________________________________________________________________ ___________________________________________________________________________ __________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ __________________________________________________________________________ Result: ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ By: DR RABIA MEHTAB 25 PHARMACEUTICAL ORGANIC CHEMISTRY-II LAB MANUAL EXPERIMENT NO 5 OBJECT: Compare melting points of recrystallize and pure Aspirin. Requirement: Procedure: ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ __________________________________________________________________________ ___________________________________________________________________________ __________________________________________________________________________ ___________________________________________________________________________ __________________________________________________________________________ ___________________________________________________________________________ Result: ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ By: DR RABIA MEHTAB 26 PHARMACEUTICAL ORGANIC CHEMISTRY-II LAB MANUAL EXPERIMENT NO 6 OBJECT: To determine purity of recrystallize Benzoic Acid using TLC. Theory: ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ Procedure: ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ By: DR RABIA MEHTAB 27 PHARMACEUTICAL ORGANIC CHEMISTRY-II LAB MANUAL ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ Calculation of Rf: Result: ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ By: DR RABIA MEHTAB 28 PHARMACEUTICAL ORGANIC CHEMISTRY-II LAB MANUAL EXPERIMENT NO 7 OBJECT: To determine purity of recrystallize Aspirin using TLC. Theory: ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ Procedure: ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ By: DR RABIA MEHTAB 29 PHARMACEUTICAL ORGANIC CHEMISTRY-II LAB MANUAL ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ Calculation of Rf: Result: ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ By: DR RABIA MEHTAB 30 PHARMACEUTICAL ORGANIC CHEMISTRY-II LAB MANUAL PART-II (DERIVATIZATION OF ORGANIC COMPOUNDS) By: DR RABIA MEHTAB 31 PHARMACEUTICAL ORGANIC CHEMISTRY-II LAB MANUAL THE PREPARATION OF DERIVATIVES OF ORGANIC COMPOUNDS DERIVATIVES/ ANALOGUES: A compound that can be formed from its parent compound by replacing one or more atoms with another atom or group of atoms is called derivative. For example, amides are derivatives of carboxylic aid in which OH is replaced by NH2 group. PURPOSE OF DERIVATIZATION: 1) Preparation of derivative is extensively done in organic chemistry to identify a compound. 2) To measure the correct melting points is easy as compare to measure boiling point. If the initial chemical is impure or gummy liquid, preparing the crystalline derivative is best way to identify it. The preliminary examination and group classification tests indicate the particular class (functional group) to which an unknown organic compound may belong. Further characterisation and identification depends on the selection and preparation of a suitable solid derivative and accurate determination of its melting point (best between 90 – 150). CHOICE OF SUITABLE DERIVATIVE: a. The reaction should be reliable and proceeds to completion and allows a small amount of sample to be used. b. The derivative should be easily and quickly prepared. c. The reaction should be general, allowing a wide range of substrates but enough specific to a single functional group to avoid interference complication. d. The derivatives should be reasonably stable for suitable period of time to facilitate analysis. e. The chemical and physical properties of derivate should be different from the compound, where it derived. f. Solid derivatives should not exceed melting point between 150 – 200 ℃. By: DR RABIA MEHTAB 32 PHARMACEUTICAL ORGANIC CHEMISTRY-II LAB MANUAL The following table lists some of the classes of organic compounds and a selection of derivatives that may be prepared to characterise them. Check with the tables of melting points in Vogel which derivatives are most suitable for the characterisation of your particular compound. CLASS OF COMPOUND DERIVATIVES 1. ALCOHOLS 3,5-dinitrobenzoate 2. PHENOLS benzoate, acetate, bromo-derivative 3. ALDEHYDES AND KETONES semicarbazone, 2,4-dinitrophenyl-hydrazone, oxime 4. ACIDS Anilide, amide, p-toluidide. 5. AMINES benzoyl, acetyl and sulphonamide derivatives We have selected three (3) different class of compounds for derivative preparation. From each class we will prepare two (2) different derivatives for the confirmation of functional groups. Selected compounds and derivatives are as follows; A. DERIVATIVE OF CARBOXYLIC ACID  Calcium Citrate from Citric acid  Calcium Tartrate from Tartaric acid B. DERIVATIVE OF ALDEHYDES AND KETONES  2, 4-dinitrophenyl hydrazone derivative of  Oximes of Aldehyde & Ketone Aldehyde & Ketone C. DERIVATIVES OF AMIDES, IMIDES AND ANILIDES  Benzoic acid from Benzamide  Amide – Nitrate derivative By: DR RABIA MEHTAB 33 PHARMACEUTICAL ORGANIC CHEMISTRY-II LAB MANUAL EXPERIMENT NO 8 OBJECT: To prepare Calcium Citrate from Citric Acid (Carboxylic acid derivative). Theory: ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ Equation: By: DR RABIA MEHTAB 34 PHARMACEUTICAL ORGANIC CHEMISTRY-II LAB MANUAL Procedure: 1. Take a test tube and add some solid citric acid, 10 drops of water and shake it. 2. Add 1ml 10% ammonium hydroxide solution and 5 drops of Calcium Chloride solution. 3. Shake and heat the mixture (not more than 70 ℃). 4. After 2 – 3 minutes white color precipitates has been formed in it. Result: ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ By: DR RABIA MEHTAB 35 PHARMACEUTICAL ORGANIC CHEMISTRY-II LAB MANUAL EXPERIMENT NO 9 OBJECT: To prepare Calcium Tartrate from Tartaric Acid (Carboxylic acid derivative). Theory: ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ Equation: By: DR RABIA MEHTAB 36 PHARMACEUTICAL ORGANIC CHEMISTRY-II LAB MANUAL Procedure: 1. Take a test tube and add some solid Tartaric acid and dissolve it with 1ml of water. 2. Add 10 drops of 10% ammonium hydroxide solution and 5 drops of 0.5M Calcium Chloride solution. 3. Shake well. After 2 – 3 minutes white color precipitates has been formed in it. 4. Filter wash and dry. Result: ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ By: DR RABIA MEHTAB 37 PHARMACEUTICAL ORGANIC CHEMISTRY-II LAB MANUAL EXPERIMENT NO 10 OBJECT: To prepare 2, 4-dinitrophenyl hydrazine derivative of Aldehyde (or ketone). Theory: ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ Equation: By: DR RABIA MEHTAB 38 PHARMACEUTICAL ORGANIC CHEMISTRY-II LAB MANUAL Procedure: 1. In a dry test tube take 0.2 g of 2, 4-dinitrophenyl hydrazine + 10 ml of methanol + 5 drops of conc. HCL. 2. Warm the solution to 60 ℃ and cool it. 3. Add 2 ml of aldehyde solution (0.2 g Glucose in 2 ml Methanol). 4. Again warm in water bath at 70 ℃ for 15 – 20 minutes then cool it at room temperature till precipitation is completed. 5. Filter and recrystallize aq. Ethanol. 6. The melting point of p-amino benzaldehyde derivative is 194 ℃. Result: ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ By: DR RABIA MEHTAB 39 PHARMACEUTICAL ORGANIC CHEMISTRY-II LAB MANUAL EXPERIMENT NO 11 OBJECT: To prepare Oximes of Aldehyde. Theory: ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ Equation: By: DR RABIA MEHTAB 40 PHARMACEUTICAL ORGANIC CHEMISTRY-II LAB MANUAL Procedure: 1. Take an Erlenmeyer flask 250 ml, add 0.5 g of Hydroxylamine hydrochloride and dissolve in 2 ml of water. 2. Add 3 ml of 10% NaOH solution + 0.5 g glucose and shake well. 3. Add 5 ml ethanol and heat for 15 minutes using reflux condenser. 4. Cool the solution in ice and dilute with 10 ml of water. 5. Filter and recrystallize from ethanol. 6. The MP of this glucose derivative is 137 ℃. Result: ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ By: DR RABIA MEHTAB 41 PHARMACEUTICAL ORGANIC CHEMISTRY-II LAB MANUAL EXPERIMENT NO 12 OBJECT: To prepare Benzoic acid from Benzamide. Theory: ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ Equation: By: DR RABIA MEHTAB 42 PHARMACEUTICAL ORGANIC CHEMISTRY-II LAB MANUAL Procedure: 1. Take small amount of Benzamide in a test tube and add 5 ml of 50% H2SO4. 2. Heat well (80℃) and pour the mixture into ice water taken in a beaker. 3. Filter the precipitate of benzoic acid, wash and dry. 4. The MP of Benzamide is 129 ℃ and its derivative benzoic acid is 121℃. Result: ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ By: DR RABIA MEHTAB 43 PHARMACEUTICAL ORGANIC CHEMISTRY-II LAB MANUAL EXPERIMENT NO 13 OBJECT: To prepare Amide-Nitrate derivative. Theory: ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ Equation: By: DR RABIA MEHTAB 44 PHARMACEUTICAL ORGANIC CHEMISTRY-II LAB MANUAL Procedure: 1. Take a test tube and dissolve small amount of urea in it. 2. Now add drop wise conc. HNO3 in a test tube, a white crystalline ppt. is formed. 3. Filter the ppt., wash with small amount of water and dry the ppt. 4. The MP of Urea is 132 C and of its derivative has 163 C. Result: ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ By: DR RABIA MEHTAB 45