assignment on bac

7/28/2019 Assignment on Bac

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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 bephysically 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 wettingof 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 becominguniformly wetted. This in turn will alter the volume of residue adhering to the walls of the glasscontainer and thus affect the volume of liquid delivered. Furthermore, in pipets and burets, themeniscus will be distorted and the correct adjustments cannot be made. The presence of small amountsof 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 beadand 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 notcleaned 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 (~80°C). Do not use abrasives or steel wool in the cleaning process. They canscratch 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 becleaned with a chromic acid cleaning solution. The conventional method of washing glassware involvessoaking 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 thisprocedure 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 (70°C+) withcommercial detergents, rinsing with hot tap water (70°C+), and finally rinsing with distilled anddouble-distilled water. However, highly contaminated glassware should be cleaned in a chromic acid-sulfuric acid bath or by some other proven method such as (1) ultrasonic cleaning, (2) washing withsodium pyrophosphate, or (3) boiling in meta-phosphate (Alconox), rinsing then boiling in a dilutehydrochloric acid solution, and then finally re-rinsing. Cleaned glassware should be inspected, dried at150°C 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 andcultures after all data have been collected:

1. Autoclave all glassware with media and cultures still in it. This kills any contaminatingmicroorganisms that may be presents.

2. After the autoclaved media has cooled, but while it is still in a liquid state, pour it into biohazardplastic 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 acid-potassium 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 6-8hours should be sufficient, but can be left overnight) and if necessary the acidbath. When removedfrom the base bath, the glassware should be rinsed with warm tap water, diluteacid, then distilled waterand 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 withethanol, 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 removethe trace metal from the surface of the glass. Cyclization through both the acid and base baths isabsolutely necessary and should be followed as described in the organic washing procedure. It is mostimportant 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 metalcontaminants, 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. Donot 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 andcan spatter!)- add a squirt of Clorox (dilute sodium hypochlorite) solution and swirl – caution!! generates a lotof heat!!- pull the mixture through the frit by vacuum. Make sure the receiving flask has no organicmaterial 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 tubesshould 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 putglassware 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 berinsed with water and then ethanol before being tranfered to the base bath. Frits from the acid bath arenot transfered to the base bath. Simply put them next to the frit-cleaning station for the frit person toclean. 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 withsoap and water and ethanol to prevent excesive contamination of the acid bath. Ink must be removedfrom 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 finalrinse in D.I. water. This is very important because ethanol dissolves the paint in the rack and leavespaint traces in the glassware. Items that do not come clean this way may be sunk in the base bath 110°C(230°F).

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 rubbertip 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 121°C (15 p.s.i. pressure). Media whichsolidifies 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 basketand dry. If tubes are to be filled with a media which is sterilized by autoclaving, do not plug until themedia is added. Both media and tubes are thus sterilized with one autoclaving. If the tubes are to befilled with sterile media, plug and sterilize the tubes in the autoclave or dry air sterilizer before addingthe 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 certainthat 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 runtap water over and through them until all traces of dirt are removed. Soak the pipets in distilled waterfor 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 fillthe 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, useconcentrated 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. Itmay be expedient to sterilize all tubes. To clean and sterilize tubes containing blood, discard the clots ina 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 thetubes, clean with a brush, rinse and dry with the usual precautions. It is imperative when washingserological glassware that all acids, alkali and detergents be completely removed. Acids, alkalis anddetergents in small amounts interfere with serologic reactions. Serologic tubes and glassware should bekept 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, placedin glacial acetic acid for 10 minutes, rinsed with distilled water and wiped dry with clean paper towels orcloth. Once the slides have been washed, place them in a wide jar of alcohol. As needed, remove fromthe 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, placethem in backsets and dry in an oven. The temperature for drying should not exceed 140°C. (Never apply heat directly to empty glassware used for volumetric measurements. Such glassware should be dried attemperatures of not more than 80°C to 90°C.) 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 110-140°C. Open-ended glass ware such as beakers should be covered with foil and stored in a dust-freecabinet.

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 usedas 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 isresponsible for spontaneous sterilization in natural conditions. In tropical countries, the sunlight is moreeffective 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. Sunlightis 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 theduration 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 thetime taken decreases.o Number of microorganisms: More the number of microorganisms, higher the temperature or longer theduration 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 prolongedexposure. 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 timerequired.

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 todry 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 predeterminedtemperature in a specified environment.DRY HEAT:Red heat:

Articles such as bacteriological loops, straight wires, tips of forceps and searing spatulas aresterilized by holding them in Bunsen flame till they become red hot. This is a simple method for effectivesterilization 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 afew times. Even though most vegetative cells are killed, there is no guarantee that spores too would dieon 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 suchas soiled dressings; animal carcasses, pathological material and bedding etc should be subjected toincineration. This technique results in the loss of the article, hence is suitable only for those articles thathave to be disposed. Burning of polystyrene materials emits dense smoke, and hence they should not beincinerated.Hot air oven:

This method was introduced by Louis Pasteur. Articles to be sterilized are exposed to hightemperature (160o C) for duration of one hour in an electrically heated oven. Since air is poor conductorof heat, even distribution of heat throughout the chamber is achieved by a fan. The heat is transferred tothe 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 isemployed 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 secand 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 highertemperature. Only vegetative bacteria are killed and spores survive. Inspissation: This is a technique to solidify as well as disinfect egg and serum containing media. Themedium containing serum or egg are placed in the slopes of an inspissator and heated at 80-85oC for 30minutes on three successive days. On the first day, the vegetative bacteria would die and those sporesthat 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 bacterialtoxins such as Staphylococcal enterotoxin are also heat resistant. Some bacterial spores are resistant toboiling 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 andglasswares can be disinfected by placing them in boiling water for 10-20 minutes. The lid of the boilermust not be opened during the period. Steam at 100oC: Instead of keeping the articles in boiling water, they are subjected to free steam at100oC. Traditionally Arnold’s and Koch’s steamers were used. An autoclave (with discharge tap open) canalso 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 sterilizesthe articles when exposed for a period of 90 minutes. Media such as TCBS, DCA and selenite broth aresterilized by steaming. Sugar and gelatin in medium may get decomposed on autoclaving, hence they areexposed 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 arekilled in the first exposure and the spores that germinate by next day are killed in subsequent days. Thesuccess 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 saidto be 121oC. Exposure of articles to this temperature for 15 minutes sterilizes them. To destroy theinfective agents associated with spongiform encephalopathies (prions), higher temperatures or longertimes 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 isessential 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 withpoor penetrative power while ionizing rays are high-energy rays with good penetrative power. Sinceradiation 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 aregenerated using a high-pressure mercury vapor lamp. It is at this wavelength that the absorption by themicroorganisms 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 incells irradiated with a non-lethal dose. Microorganisms such as bacteria, viruses, yeast, etc. that areexposed to the effective UV radiation are inactivated within seconds. Since UV rays don’t 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 lowpenetrative power, limited life of the uv bulb, some bacteria have DNA repair enzymes that can overcomedamage 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. Highspeedelectrons are produced by a linear accelerator from a heated cathode. Electron beams are employed tosterilize articles like syringes, gloves, dressing packs, foods and pharmaceuticals. Sterilization isaccomplished 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 radioactiveisotopes (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 2.5megarads kills all bacteria, fungi, viruses and spores. It is used commercially to sterilize disposable petridishes, plastic syringes, antibiotics, vitamins, hormones, glasswares and fabrics. Disadvantages include;

unlike electron beams, they can’t be switched off, glasswares tend to become brownish, loss of tensilestrength in fabric. Gamma irradiation impairs the flavour of certain foods. Bacillus pumilus E601 is used toevaluate sterilization process.FILTRATION:Filtration does not kill microbes, it separates them out. Membrane filters with pore sizes between 0.2-0.45 μm are commonly used to remove particles from solutions that can't be autoclaved. It is used toremove microbes from heat labile liquids such as serum, antibiotic solutions, sugar solutions, ureasolution. 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 toxinsfrom 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 orasbestos 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 notreliable 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 cansterilize are called chemisterilants. Those chemicals that can be safely applied over skin and mucusmembranes 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 isflushed in. Formaldehyde is then injected with steam in a series of pulses, each of 5-10 minutes. Thearticles 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. Thisdevice 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 100°C, 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 guard-

tubes 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 175°C for about 7h). Mainly as a drying agent in a desiccator or as a column through whichliquid 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 30°C, 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 700-900°C 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 235°C for 2-3 hours; it

can be regenerated.) Available commercially as Drierite. It forms the hemihdrate, CaSO4· ½H2O, 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 100°C 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 300°C under reduced pressure.) More rapid

and effective than sodium sulphate. It has a large capacity, forming MgSO4·7 H2O below

48°C. 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 110°C 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 33°C, but

drying is slow and inefficient, especially for solvents that are sparingly soluble in water. It issuitable 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.

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