ASSIGNMENT

TOPIC: WASHING,DRYING,AND STERILIZATION OF GLASSWARE.DRYING OF SOLVENTS AND CHEMICALS

BASIC CONCEPT IN LABORATORY TECHNIQUES
PGS: 504

SUBMITTED TO,

WASHING OF GLASSWARES:
Introduction: Good laboratory technique demands clean glassware, because the most carefully executed piece of work may give an erroneous result if dirty glassware is used. In all instances, glassware must be physically clean; it must be chemically clean; and in many cases, it must be bacteriologically clean or sterile. All glassware must be absolutely grease-free. The safest criteria of cleanliness is uniform wetting of the surface by distilled water. This is especially important in glassware used for measuring the volume of liquids. Grease and other contaminating materials will prevent the glass from becoming uniformly wetted. This in turn will alter the volume of residue adhering to the walls of the glass container and thus affect the volume of liquid delivered. Furthermore, in pipets and burets, the meniscus will be distorted and the correct adjustments cannot be made. The presence of small amounts of impurities may also alter the meniscus. General Cleaning Methods : Clean glassware is hydrophilic and will have a uniformly wetted surface when distilled water is used as a final rinse. Contaminants such as detergent residues or grease will cause the water to bead and the cleaning procedure should be repeated. Wash glassware as quickly as possible after use. If cleaning is not immediately possible, place the glassware in water to soak. If the glassware is not cleaned immediately, it may become impossible to remove the residue. A non-alkaline detergent should be used. The concentration of detergent should be between 5 and 20% depending on the residue. The water should be hot (~80C). Do not use abrasives or steel wool in the cleaning process. They can scratch the surface of the glassware. During washing, all parts of the glassware should be thoroughly scrubbed with a brush. Brushes with plastic or wooden handles are recommended. Do not use brushes with metal handles as the metal can scratch the glassware. Scratched glassware is more prone to break during experiments. If the glassware is clouded or contains coagulated organic material, it should be cleaned with a chromic acid cleaning solution. The conventional method of washing glassware involves soaking glass in a chromic acid-sulfuric acid bath followed by tap water rinses, distilled water rinses, and finally double-distilled water rinses. Due to the corrosive nature of chromic acid, the use of this procedure has been eliminated except for highly contaminated or soiled glassware. Adequate cleaning of most glassware for tissue culture purposes can be achieved by washing in hot water (70C+) with commercial detergents, rinsing with hot tap water (70C+), and finally rinsing with distilled and double-distilled water. However, highly contaminated glassware should be cleaned in a chromic acidsulfuric acid bath or by some other proven method such as (1) ultrasonic cleaning, (2) washing with sodium pyrophosphate, or (3) boiling in meta-phosphate (Alconox), rinsing then boiling in a dilute hydrochloric acid solution, and then finally re-rinsing. Cleaned glassware should be inspected, dried at 150C in a drying oven, capped with aluminum foil, and stored in a closed cabinet. The following general procedure is recommended for cleaning glassware that contains media and cultures after all data have been collected: 1. Autoclave all glassware with media and cultures still in it. This kills any contaminating microorganisms that may be presents. 2. After the autoclaved media has cooled, but while it is still in a liquid state, pour it into biohazard plastic bags or thick plastic bags, seal, then discard. 3. Wash all glassware in hot soapy water using a suitable bottlebrush to clean the internal parts of the glassware. Any glassware that is stained should be soaked in a concentrated sulfuric acidpotassium dichromate acid bath for 4 hr, then rinsed 10 times before washing it with soapy water. 4. All glassware should be rinsed three times in tap water, three times in deionized water, three times in double-distilled water, dried, and stored in a clean place. 5. Wash all instruments and new glassware in a similar manner.

Removing Grease : Grease is best removed by boiling in a weak solution of sodium carbonate. Acetone or another fat solvent may also be used. Strong alkalis should not be used. Silicone grease can be removed by soaking in decahydronaphthalene for 2 hours. Organic Glassware: This tray should contain glassware which was used solely for organics. First,organic glassware should be attempted to be cleaned with Alconox and warm water followed by rinsing with acetone. If this does not adequately clean the glassware then it should be cycled through the base bath (about 6-8 hours should be sufficient, but can be left overnight) and if necessary the acidbath. When removed from the base bath, the glassware should be rinsed with warm tap water, diluteacid, then distilled water and hung on the rack to dry. Glassware from the acid bath should be rinsed with tap water, and distilled water, and hung on the rack to dry. Before putting glassware in the base bath one should rinse with ethanol, since no water should be transferred to the base bath. Inorganic Glassware: This tray should contain glassware which has been treated with metal. The procedure for washing metal-containing glassware is different from organics and should be strictly followed to remove the trace metal from the surface of the glass. Cyclization through both the acid and base baths is absolutely necessary and should be followed as described in the organic washing procedure. It is most important for this glassware to be scrubbed before being placed in the acid bath, to avoid the build up of metal contaminants in the bath. Fritted Funnels: If you have been using a frit with purely organic compounds or with Celite, you should scrub it and rinse it through with water, alcohol, and acetone immediately after use, and return it to the drawer. Only those frits that cannot be cleaned in this way, particularly those that have insoluble metal contaminants, should be treated in the following manner: (1) Try washing through with dilute HCl. (2) If this fails, try washing through with NoChromix cleaner. (3) If this fails, use the singlet oxygen procedure below. In each case, thorough rinsing with water and alcohol, followed by air-drying, should be performed. Do not soak frits in the base bath, as this will corrode the fritted glass material. Singlet oxygen procedure: - make sure the frit to be cleaned is dry, and that there are no organic solvents around. - add to the frit a little conc. sulfuric acid (1-2 mL) - add a little 30% hydrogen peroxide (about 1 mL), and swirl to mix (caution! generates heat and can spatter!) - add a squirt of Clorox (dilute sodium hypochlorite) solution and swirl caution!! generates a lot of heat!! - pull the mixture through the frit by vacuum. Make sure the receiving flask has no organic material in it such material can ignite in the strong oxidizing conditions of this procedure. - rinse the frit well with water NMR tubes: Again, prompt rinsing with acetone or alcohol should be done immediately. In many cases, this is all that is necessary. If the tube is still dirty, it should be rinsed with dilute HCl, water, and acetone. If this is ineffective, try a pipette or two of the base bath solution, followed immediately by dilute HCl,

water, and acetone. If solid remains, the tube may be scrubbed out with a pipe cleaner. NMR tubes should NOT go in the base bath!! Preparation of Acid Bath: The acid bath contains 10% aqueous HCl, and should be filled to about half full. Preparation of Base Bath: The base bath contains a 1M KOH/ 95% Ethanol solution and should be about half full. Safety: Safety Glasses and a Lab Coat should be worn at all times. If you get solvent in your eyes rinse them liberally with water from the eyewash. PROCEDURE: 1) Put away glassware from the rack. If you do not know where something goes, ASK. Do not put glassware just anywhere. The next person that needs it may never find it. 2) Unload the base bath. This involves taking out each piece of glassware and individually rinsing it with tap water, followed by dilute HCL and D.I. water. Hang it on the rack to dry. 3) Unload the acid bath and transfer its contents to the base bath. Each piece of glassware must be rinsed with water and then ethanol before being tranfered to the base bath. Frits from the acid bath are not transfered to the base bath. Simply put them next to the frit-cleaning station for the frit person to clean. Glassware must be completely sunk in the bath. Flasks floating on the top do not get clean. 4) Load the contents of the bin labeled "metal" into the acid bath. Each piece must first be cleaned with soap and water and ethanol to prevent excesive contamination of the acid bath. Ink must be removed from the glassware before introducing it in the bath. No plastic or rubber items may be placed in the baths. 5) Wash the glassware on the bin labelled "organic" with soap and water, followed by ethanol and a final rinse in D.I. water. This is very important because ethanol dissolves the paint in the rack and leaves paint traces in the glassware. Items that do not come clean this way may be sunk in the base bath 110C (230F). Burets: Remove the stopcock or rubber tip and wash the buret with detergent and water. Rinse with tap water until all the dirt is removed. Then rinse with distilled water and dry. Wash the stopcock or rubber tip separately. Before a glass stopcock is placed in the buret, lubricate the joint with stopcock lubricant. Use only a small amount of lubricant. Burets should always be covered when not in use. Culture Tubes: Culture tubes which have been used previously must be sterilized before cleaning. The best method for sterilizing culture tubes is by autoclaving for 30 minutes at 121C (15 p.s.i. pressure). Media which solidifies on cooling should be poured out while the tubes are hot. After the tubes are emptied, brush with detergent and water, rinse thoroughly with tap water, rinse with distilled water, place in a basket and dry. If tubes are to be filled with a media which is sterilized by autoclaving, do not plug until the media is added. Both media and tubes are thus sterilized with one autoclaving. If the tubes are to be filled with sterile media, plug and sterilize the tubes in the autoclave or dry air sterilizer before adding the media.

Dishes and Culture Bottles: Sterilize and clean as detailed under Culture Tubes. Wrap in heavy paper or place in a petri dish can. Sterilize in the autoclave or dry air sterilizer. Pipets: Place pipets, tips down, in a cylinder or tall jar of water immediately after use. Do not drop them into the jar. This may break or chip the tips and render the pipets useless for accurate measurements. A pad of cotton or glass wool at the bottom of the jar will help to prevent breaking of the tips. Be certain that the water level is high enough to immerse the greater portion or all of each pipet. The pipets may then be drained and placed in a cylinder or jar of dissolved detergent or, if exceptionally dirty, in a jar of chromic acid cleaning solution. After soaking for several hours, or overnight, drain the pipets and run tap water over and through them until all traces of dirt are removed. Soak the pipets in distilled water for at least one hour. Remove from the distilled water, rinse, dry the outside with a cloth, shake the water out and dry. Blood Cell Count Diluting Pipets: After use, rinse thoroughly with cool tap water, distilled water, alcohol, or acetone, and then ether. Dry by suction. Do not blow into the pipets as this will cause moisture to condense on the inside of the pipet. To remove particles of coagulated blood or dirt, a cleaning solution should be used. One type of solution will suffice in one case, whereas a stronger solution may be required in another. It is best to fill the pipet with the cleaning solution and allow to stand overnight. Sodium hypo chlorite (laundry bleach) or a detergent may be used. Hydrogen peroxide is also useful. In difficult cases, use concentrated nitric acid. Some particles may require loosening with a horse hair or piece of fine wire. Take care not to scratch the inside of the pipet. Automatic Pipet Washers: Where a large number of pipets are used daily, it is convenient to use an automatic pipet washer. Some of these, made of metal, can be connected directly by permanent fixtures to the hot and cold water supplies. Others, such as those made with polyethylene, can be attached to the water supplies by rubber hose. Polyethylene baskets and jars may be used for soaking and rinsing pipets in chromic acid cleaning solution. Electrically heated metallic pipet dryers are also available. Serological Tubes: Serological tubes should be chemically clean, but need not be sterile. However, specimens of blood which are to be kept for some time at room temperature should be collected in a sterile container. It may be expedient to sterilize all tubes. To clean and sterilize tubes containing blood, discard the clots in a waste container and place the tubes in a large basket. Put the basket, with others, in a large bucket or boiler. Cover with water, add a fair quantity of soft soap or detergent and boil for 30 minutes. Rinse the tubes, clean with a brush, rinse and dry with the usual precautions. It is imperative when washing serological glassware that all acids, alkali and detergents be completely removed. Acids, alkalis and detergents in small amounts interfere with serologic reactions. Serologic tubes and glassware should be kept separate from all other glassware and used only for serologic procedures. Slides and Cover Glass: It is especially important that microscope slides and cover glass used for the preparation of blood films or bacteriologic smears be perfectly clean and free from scratches. Slides should be washed, placed in glacial acetic acid for 10 minutes, rinsed with distilled water and wiped dry with clean paper towels or cloth. Once the slides have been washed, place them in a wide jar of alcohol. As needed, remove from the jar and wipe dry. If the slides are dry stored, wash them with alcohol before use.

DRYING OF GLASSWARES: Dry test tubes, culture tubes, flasks and other glassware by hanging them on wooden pegs or placing them in baskets with their mouths downward and allowing them to air dry. Alternatively, place them in backsets and dry in an oven. The temperature for drying should not exceed 140C. (Never apply heat directly to empty glassware used for volumetric measurements. Such glassware should be dried at temperatures of not more than 80C to 90C.) Before placing glassware in a basket, cover the bottom of the basket with a clean folded towel or clean piece of cloth. This prevents the mouths of the tubes from becoming dirty. Pegboard drying is not recommended since airborne contaminants in the laboratory will be deposited on the clean glassware. Oven drying is suggested at temperatures ranging from 110 140C. Open-ended glassware such as beakers should be covered with foil and stored in a dust-free cabinet. STERILIZATION OF GLASSWARES: Sterilization is defined as the process where all the living microorganisms, including bacterial spores are killed.Sterilization can be achieved by physical, chemical and physiochemical means. Chemicals used as sterilizing agents are called chemisterilants.

PHYSICAL METHODS OF STERILIZATION: Sunlight: The microbicidal activity of sunlight is mainly due to the presence of ultra violet rays in it. It is responsible for spontaneous sterilization in natural conditions. In tropical countries, the sunlight is more effective in killing germs due to combination of ultraviolet rays and heat. By killing bacteria suspended in water, sunlight provides natural method of disinfection of water bodies such as tanks and lakes. Sunlight is not sporicidal, hence it does not sterilize. Heat: Heat is considered to be most reliable method of sterilization of articles that can withstand heat. Heat acts by oxidative effects as well as denaturation and coagulation of proteins. Those articles that

cannot withstand high temperatures can still be sterilized at lower temperature by prolonging the duration of exposure. Factors affecting sterilization by heat are: o Nature of heat: Moist heat is more effective than dry heat. o Temperature and time: temperature and time are inversely proportional. As temperature increases the time taken decreases. o Number of microorganisms: More the number of microorganisms, higher the temperature or longer the duration required. o Nature of microorganism: Depends on species and strain of microorganism, sensitivity to heat may vary. Spores are highly resistant to heat. o Type of material: Articles that are heavily contaminated require higher temperature or prolonged exposure. Certain heat sensitive articles must be sterilized at lower temperature. o Presence of organic material: Organic materials such as protein, sugars, oils and fats increase the time required. Action of heat: Dry heat acts by protein denaturation, oxidative damage and toxic effects of elevated levels of electrolytes. The moist heat acts by coagulation and denaturation of proteins. Moist heat is superior to dry heat in action. Temperature required to kill microbe by dry heat is more than the moist heat. Thermal death time is the minimum time required to kill a suspension of organisms at a predetermined temperature in a specified environment. DRY HEAT: Red heat: Articles such as bacteriological loops, straight wires, tips of forceps and searing spatulas are sterilized by holding them in Bunsen flame till they become red hot. This is a simple method for effective sterilization of such articles, but is limited to those articles that can be heated to redness in flame. Flaming: This is a method of passing the article over a Bunsen flame, but not heating it to redness. Articles such as scalpels, mouth of test tubes, flasks, glass slides and cover slips are passed through the flame a few times. Even though most vegetative cells are killed, there is no guarantee that spores too would die on such short exposure. This method too is limited to those articles that can be exposed to flame. Cracking of the glassware may occur. Incineration: This is a method of destroying contaminated material by burning them in incinerator. Articles such as soiled dressings; animal carcasses, pathological material and bedding etc should be subjected to incineration. This technique results in the loss of the article, hence is suitable only for those articles that have to be disposed. Burning of polystyrene materials emits dense smoke, and hence they should not be incinerated. Hot air oven: This method was introduced by Louis Pasteur. Articles to be sterilized are exposed to high temperature (160o C) for duration of one hour in an electrically heated oven. Since air is poor conductor of heat, even distribution of heat throughout the chamber is achieved by a fan. The heat is transferred to the article by radiation, conduction and convection. The oven should be fitted with a thermostat control, temperature indicator, meshed shelves and must have adequate insulation. MOIST HEAT: Moist heat acts by coagulation and denaturation of proteins. At temperature below 100oC:

 Pasteurization: This process was originally employed by Louis Pasteur. Currently this procedure is employed in food and dairy industry. There are two methods of pasteurization, the holder method (heated at 63oC for 30 minutes) and flash method (heated at 72oC for 15 seconds) followed by quickly cooling to 13oC. Other pasteurization methods include Ultra-High Temperature (UHT), 140oC for 15 sec and 149oC for 0.5 sec. This method is suitable to destroy most milk borne pathogens like Salmonella, Mycobacteria, Streptococci, Staphylococci and Brucella, however Coxiella may survive pasteurization. Efficacy is tested by phosphatase test and methylene blue test. Vaccine bath: The contaminating bacteria in a vaccine preparation can be inactivated by heating in a water bath at 60oC for one hour. Only vegetative bacteria are killed and spores survive. Serum bath: The contaminating bacteria in a serum preparation can be inactivated by heating in a water bath at 56oC for one hour on several successive days. Proteins in the serum will coagulate at higher temperature. Only vegetative bacteria are killed and spores survive. Inspissation: This is a technique to solidify as well as disinfect egg and serum containing media. The medium containing serum or egg are placed in the slopes of an inspissator and heated at 80-85oC for 30 minutes on three successive days. On the first day, the vegetative bacteria would die and those spores that germinate by next day are then killed the following day. The process depends on germination of spores in between inspissation. If the spores fail to germinate then this technique cannot be considered sterilization. At temperature 100oC: Boiling: Boiling water (100oC) kills most vegetative bacteria and viruses immediately. Certain bacterial toxins such as Staphylococcal enterotoxin are also heat resistant. Some bacterial spores are resistant to boiling and survive; hence this is not a substitute for sterilization. The killing activity can be enhanced by addition of 2% sodium bicarbonate. When absolute sterility is not required, certain metal articles and glasswares can be disinfected by placing them in boiling water for 10-20 minutes. The lid of the boiler must not be opened during the period. Steam at 100oC: Instead of keeping the articles in boiling water, they are subjected to free steam at 100oC. Traditionally Arnolds and Kochs steamers were used. An autoclave (with discharge tap open) can also serve the same purpose. A steamer is a metal cabinet with perforated trays to hold the articles and a conical lid. The bottom of steamer is filled with water and heated. The steam that is generated sterilizes the articles when exposed for a period of 90 minutes. Media such as TCBS, DCA and selenite broth are sterilized by steaming. Sugar and gelatin in medium may get decomposed on autoclaving, hence they are exposed to free steaming for 20 minutes for three successive days. This process is known as tyndallisation (after John Tyndall) or fractional sterilization or intermittent sterilization. The vegetative bacteria are killed in the first exposure and the spores that germinate by next day are killed in subsequent days. The success of process depends on the germination of spores. At temperature above 100oC: Autoclave: Sterilization can be effectively achieved at a temperature above 100oC using an autoclave. Water boils at 100oC at atmospheric pressure, but if pressure is raised, the temperature at which the water boils also increases. In an autoclave the water is boiled in a closed chamber. As the pressure rises, the boiling point of water also raises. At a pressure of 15 lbs inside the autoclave, the temperature is said to be 121oC. Exposure of articles to this temperature for 15 minutes sterilizes them. To destroy the infective agents associated with spongiform encephalopathies (prions), higher temperatures or longer times are used; 135oC or 121oC for at least one hour are recommended. Advantages of steam: It has more penetrative power than dry air, it moistens the spores (moisture is essential for coagulation of proteins), condensation of steam on cooler surface releases latent heat, condensation of steam draws in fresh steam. Different types of autoclave: Simple pressure-cooker type laboratory autoclave, Steam jacketed downward displacement laboratory autoclave and high pressure pre-vacuum autoclave.

RADIATION: Two types of radiation are used, ionizing and non-ionizing. Non-ionizing rays are low energy rays with poor penetrative power while ionizing rays are high-energy rays with good penetrative power. Since radiation does not generate heat, it is termed "cold sterilization". In some parts of Europe, fruits and vegetables are irradiated to increase their shelf life up to 500 percent. Non-ionizing rays: Rays of wavelength longer than the visible light are non-ionizing. Microbicidal wavelength of UV rays lie in the range of 200-280 nm, with 260 nm being most effective. UV rays are generated using a high-pressure mercury vapor lamp. It is at this wavelength that the absorption by the microorganisms is at its maximum, which results in the germicidal effect. UV rays induce formation of thymine-thymine dimers, which ultimately inhibits DNA replication. UV readily induces mutations in cells irradiated with a non-lethal dose. Microorganisms such as bacteria, viruses, yeast, etc. that are exposed to the effective UV radiation are inactivated within seconds. Since UV rays dont kill spores, they are considered to be of use in surface disinfection. UV rays are employed to disinfect hospital wards, operation theatres, virus laboratories, corridors, etc. Disadvantages of using uv rays include low penetrative power, limited life of the uv bulb, some bacteria have DNA repair enzymes that can overcome damage caused by uv rays, organic matter and dust prevents its reach, rays are harmful to skin and eyes. It doesn't penetrate glass, paper or plastic. Ionizing rays: Ionizing rays are of two types, particulate and electromagnetic rays. o Electron beams are particulate in nature while gamma rays are electromagnetic in nature. Highspeed electrons are produced by a linear accelerator from a heated cathode. Electron beams are employed to sterilize articles like syringes, gloves, dressing packs, foods and pharmaceuticals. Sterilization is accomplished in few seconds. Unlike electromagnetic rays, the instruments can be switched off. Disadvantage includes poor penetrative power and requirement of sophisticated equipment. o Electromagnetic rays such as gamma rays emanate from nuclear disintegration of certain radioactive isotopes (Co60, Cs137). They have more penetrative power than electron beam but require longer time of exposure. These high-energy radiations damage the nucleic acid of the microorganism. A dosage of 2.5 megarads kills all bacteria, fungi, viruses and spores. It is used commercially to sterilize disposable petri dishes, plastic syringes, antibiotics, vitamins, hormones, glasswares and fabrics. Disadvantages include; unlike electron beams, they cant be switched off, glasswares tend to become brownish, loss of tensile strength in fabric. Gamma irradiation impairs the flavour of certain foods. Bacillus pumilus E601 is used to evaluate sterilization process. FILTRATION: Filtration does not kill microbes, it separates them out. Membrane filters with pore sizes between 0.20.45 m are commonly used to remove particles from solutions that can't be autoclaved. It is used to remove microbes from heat labile liquids such as serum, antibiotic solutions, sugar solutions, urea solution. Various applications of filtration include removing bacteria from ingredients of culture media, preparing suspensions of viruses and phages free of bacteria, measuring sizes of viruses, separating toxins from culture filtrates, counting bacteria, clarifying fluids and purifying hydatid fluid. Filtration is aided by using either positive or negative pressure using vacuum pumps. The older filters made of earthenware or asbestos are called depth filters. SONIC AND ULTRASONIC VIBRATIONS: Sound waves of frequency >20,000 cycle/second kills bacteria and some viruses on exposing for one hour. Microwaves are not particularly antimicrobial in themselves, rather the killing effect of microwaves are largely due to the heat that they generate. High frequency sound waves disrupt cells. They are used to clean and disinfect instruments as well as to reduce microbial load. This method is not reliable since many viruses and phages are not affected by these waves.

CHEMICAL METHODS OF DISINFECTION: Disinfectants are those chemicals that destroy pathogenic bacteria from inanimate surfaces. Some chemical have very narrow spectrum of activity and some have very wide. Those chemicals that can sterilize are called chemisterilants. Those chemicals that can be safely applied over skin and mucus membranes are called antiseptics. An ideal antiseptic or disinfectant should have following properties: Should have wide spectrum of activity Should be able to destroy microbes within practical period of time Should be active in the presence of organic matter Should make effective contact and be wettable Should be active in any pH Should be stable Should have long shelf life Should be speedy Should have high penetrating power Should be non-toxic, non-allergenic, non-irritative or non-corrosive Should not have bad odour Should not leave non-volatile residue or stain Efficacy should not be lost on reasonable dilution Should not be expensive and must be available easily. PHYSIO-CHEMICAL METHOD: Mode of action: A physio-chemical method adopts both physical and chemical method. Use of steamformaldehyde is a physio-chemical method of sterilization, which takes into account action of steam as well as that of formaldehyde. Saturated steam at a pressure of 263 mm has a temperature of 70oC. The air is removed from the autoclave chamber and saturated steam at sub-atmospheric pressure is flushed in. Formaldehyde is then injected with steam in a series of pulses, each of 5-10 minutes. The articles are held at this holding temperature for one hour. Formaldehyde is then flushed by inflow of steam. Disadvantages: Condensation of formaldehyde occurs and induction of large volume of formaldehyde wets the steam resulting in loss of latent heat. Sterilization control: using paper strips containing 106 spores of G.stearothermophilus. DRYING OF SOLVENTS AND CHEMICALS: Where substances are sufficiently stable, removal of solvents from recrystallized materials presents no problems. The crystals, after filtering at the pump (and perhaps air-drying by suction), are heated in an oven above the boiling point of the solvent (but below their melting point), followed by cooling in a desiccator. Where this treatment is inadvisable, it is still often possible to heat to a lower temperature under reduced pressure, for example in an Abderhalden pistol. This device consists of a small chamber which is heated externally by the vapour of a boiling solvent. Inside this chamber, which can be evacuated by a water pump or some other vacuum pump, is placed a small boat containing the sample to be dried and also a receptacle with a suitable drying agent. Convenient liquids for use as boiling liquids in an Abderhalden pistol, and their temperatures, are given in Table 8. In cases where heating above room temperature cannot be used, drying must be carried out in a vacuum desiccator containing suitable absorbents. For

example, hydrocarbons, such as benzene, cyclohexane and petroleum ether, can be removed by using shredded paraffin wax, and acetic acid and other acids are absorbed by pellets of sodium hydroxide or potassium hydroxide. However, in general, solvent removal is less of a problem than ensuring that the water content of solids and liquids is reduced below an acceptable level. Removal of water: Methods for removing water from solids depend on the thermal stability of the solids or the time available. The safest method is to dry in a vacuum desiccator over concentrated sulphuric acid, phosphorus pentoxide, silica gel, calcium chloride, or some other desiccant. Where substances are stable in air and melt above 100C, drying in an air oven may be adequate. In other cases, use of an Abderhalden pistol may be satisfactory. Often, in drying inorganic salts, the final material that is required is a hydrate. In such cases, the purified substance is left in a dessicator to equilibrate above an aqueous solution having a suitable water-vapour pressure. The choice of desiccants for drying liquids is more restricted because of the need to avoid all substances likely to react with the liquids themselves. In some cases, direct distillation of an organic liquid is a suitable method of drying both solids and liquids, especially if low-boiling azeotropes are formed. Examples include acetone, aniline, benzene, chloroform, carbon tetrachloride, ethylene dichloride, heptane, hexane, methanol, nitrobenzene, petroleum ether, toluene and xylene. Addition of benzene can be used for drying ethanol by distillation. In carrying out distillations intended to yield anhydrous products, the apparatus should be fitted with guardtubes containing calcium chloride or silica gel to prevent entry of moist air into the system. (Many anhydrous organic liquids are appreciably hydroscopic.) Removal of water from gases may be by physical or chemical means, and is commonly by adsorption on to a drying agent in a low temperature trap. The effectiveness of drying agents depends on the vapour pressure of the hydrated compound - the lower the vapour pressure the less the remaining moisture in the gas. Suitability of individual desiccants: Alumina: (Preheated to 175C for about 7h). Mainly as a drying agent in a desiccator or as a column through which liquid is percolated. Aluminium amalgam Mainly used for removing traces of water from alcohols, which are distilled from it after refluxing.

Barium oxide Suitable for drying organic bases. Barium perchlorate Expensive. Used in desiccators. Unsuitable for drying solvents or any organic material where contact is necessary, because of the danger of explosion. Boric anhydride (Prepared by melting boric acid in an air oven at a high temperature, cooling in a desiccator, and powdering.) Mainly used for drying formic acid. Calcium chloride (anhydrous) Cheap. Large capacity for absorption of water, giving the hexahydrate below 30C, but is fairly slow in action and not very efficient. Its main use is for preliminary drying of alkyl and aryl halides, most esters, saturated and aromatic hydrocarbons, and ethers. Unsuitable for drying alcohols and amines (which form addition compounds), fatty acids, amides, aminoacids, ketones, phenols, or some aldehydes and esters. Calcium chloride is suitable for drying the following gases-hydrogen, hydrogen chloride, carbon monoxide, carbon dioxide, sulphur dioxide, nitrogen, methane, oxygen, paraffins, ethers, olefins and alkyl chlorides. Calcium oxide (Preheated to 700-900C before use.) Suitable for alcohols and amines (but does not dry them completely). Need not be removed before distillation, but in that case the head of the distillation column should be packed with glass wool to trap any calcium oxide powder that might be carried over. Unsuitable for acidic compounds or esters. Suitable for drying gaseous amines and ammonia. Calcium sulphate (anhydrous) (Prepared by heating the dihydrate or the hemihydrate in an oven at 235C for 2-3 hours; it can be regenerated.) Available commercially as Drierite. It forms the hemihdrate, CaSO4 H2O, so that its capacity is fairly low (6.6% of its weight of water), and hence is best used on partially dried substances. It is very rapid and efficient (being comparable with phosphorus pentoxide and concentrated sulphuric acid). Suitable for most organic compounds. Solvents boiling below 100C can be dried by direct distillation from calcium sulphate.

Copper(II) sulphate (anhydrous) Suitable for esters and alcohol. Preferable to sodium sulphate in cases where solvents are sparingly soluble in water (for example, benzene or toluene). Magnesium amalgam Mainly used for removing traces of water from alcohols, which are distilled from it after refluxing. Magnesium perchlorate (anhydrous) (Available commercially as Dehydrite. Expensive) Used in desiccators. Unsuitable fro drying solvents or any organic material where contact is necessary, because of the danger of explosion. Magnesium sulphate (anhydrous) (Prepared from the heptahydrate by drying at 300C under reduced pressure.) More rapid and effective than sodium sulphate. It has a large capacity, forming MgSO47 H2O below 48C. Suitable for the preliminary drying of most organic compounds. Phosphorus pentoxide Very rapid and efficient, but difficult to handle and should only be used after the organic material has been partially dried, for example with magnesium sulphate. Suitable for acid anhydrides, alkyl and aryl halides, esters, ethers, hydrocarbons and nitriles, and for use in desiccators. Not suitable with acids, alcohols, amines or ketones, or with organic molecules from which a molecule of water can fairly readily be abstracted by an elimination reaction. Suitable for drying the following gases-hydrogen, oxygen, carbon dioxide, carbon monoxide, sulphur dioxide, nitrogen, methane, ethylene and paraffins. Potassium (metal) Properties and application are similar to those for sodium, and it is a correspondingly hazardous substance. Potassium carbonate (anhydrous) Has a moderate efficiency and capacity, forming the dihydrate. Suitable for an initial drying of alcohols, bases, esters, ketones and nitriles by shaking with them, then filtering off. Also suitable for salting out water-soluble alcohols, amines and ketones. Unsuitable for acids, phenols and other acidic substances.

Potassium hydroxide Solid potassium hydroxide is very rapid and efficient. Its use is limited almost entirely to the initial drying of organic bases. Alternatively, sometimes the base is shaken first with a concentrated solution of potassium hydroxide to remove most of the water present. Unsuitable for acids, aldehydes, amides, esters, ketones, or phenols. Also used for drying gaseous amines and ammonia. Silica gel Granulated silica gel is a commercially available drying agent for use with gases, in desiccators, and (because of its chemical inertness) in physical instruments (pH meters, spectrophotometers, balances). Its drying action depends on physical adsorption, so that silica gel must be used at room temperature or below. By incorporating cobalt chloride into the material it can be made self-indicating, redrying in an oven at 110C being necessary when the colour changes from blue to pink. Sodium (metal) Used as fine wire or as chips, for more completely drying ethers, saturated hydrocarbons and aromatic hydrocarbons which have been partically dried (for example with calcium chloride or magnesium sulphate). Unsuitable for acids, alcohols, aldehydes, amines, esters, organic halides or ketones. Reacts violently if much water is present. Sodium hydroxide Properties and applications are similar to those for potassium hydroxide. Sodium-potassium alloy Used as lumps. Lower metling than sodium, so that its surface is readily renewed by shaking. Properties and applications are similar to those for sodium. Sodium sulphate (anhydrous) Has a large capacity for the absorption of water, forming the decahydrate below 33C, but drying is slow and inefficient, especially for solvents that are sparingly soluble in water. It is suitable for the preliminary drying of most types of organic compounds. Sulphuric acid (concentrated) Widely used in desiccators. Suitable for drying bromine, saturated hydrocarbons, alkyl and aryl halides. Also suitable for drying the following gases - hydrogen, nitrogen, carbon

dioxide, carbon monoxide, chlorine, methane and paraffins. Unsuitable for alcohols, bases, ketones or phenols.