Buffers and Buffer Capacity

Buffers and Buffer Capacity

Buffers are compounds that resist changes in pH upon the addition of limited amounts of acids or bases. Buffer systems are usually composed of a weak acid or base and its conjugate salt. The components act in such a way that addition of an acid or base results in the formulation of a salt causing only a small change in pH.

The pH of a buffer system is given by the Henderson-Hasselbach equation:

 (for a weak acid and its salt)

 (for a weak base and its salt)

where [salt], [acid] and [base] are the molar concentrations of salt, acid and base.

Buffer capacity is a measure of the efficiency of a buffer in resisting changes in pH. Conventionally, the buffer capacity () is expressed as the amount of strong acid or base, in gram-equivalents, that must be added to 1 liter of the solution to change its pH by one unit.

Calculate the buffer capacity as:

 = gram equivalent of strong acid/base to change pH of 1 liter of buffer solution
 = the pH change caused by the addition of strong acid/base

In practice, smaller pH changes are measured and the buffer capacity is quantitatively expressed as the ratio of acid or base added to the change in pH produced (e.g., mEq./pH for x volume). The buffer capacity depends essentially on 2 factors:

1. Ratio of the salt to the acid or base. The buffer capacity is optimal when the ratio is 1:1; that is, when pH = pKa
2. Total buffer concentration. For example, it will take more acid or base to deplete a 0.5 M buffer than a 0.05 M buffer.

The relationship between buffer capacity and buffer concentrations is given by the Van Slyke equation:

where C = the total buffer concentration (i.e. the sum of the molar concentrations of acid and salt).

Just as we must often compromise the optimal pH for a product, so must we compromise on the optimal buffer capacity of our solution. On the one hand, buffer capacity must be large enough to maintain the product pH for a reasonably long shelf-life. Changes in product pH may result from interaction of solution components with one another or with the product package (glass, plastic, rubber closures, etc.). On the other hand, the buffer capacity of ophthalmic and parenteral products must be low enough to allow rapid readjustment of the product to physiologic pH upon administration. The pH, chemical nature, and volume of the solution to be administered must all be considered. Buffer capacities ranging from 0.01 - 0.1 are usually adequate for most pharmaceutical solutions.

Stereochemistry

Stereochemistry

 

Stereochemistry is an important principle in biology, when we consider drugs and receptors. Pharmaceutical drugs often have a family of different isomers, of which only one of these members will successfully carry out the desired role in the body. Other isomers may have no effect or may promote adverse side effects, the most well known example being thalidomide. A basic knowledge of stereochemistry is therefore essential to truly understand some key aspects in pharmacology and biochemistry. 

 

An isomer is a compound with the same molecular formula, but a different structural formula. The same atoms present but they are arranged differently in space. 

 

Stereoisomers are structures which cannot be superimposed onto each other. The structures are arranged differently despite having the same elements in the compound. There are two different types of stereoisomerism. The first is Geometric isomerism, and the second is Optical isomerism.

 

Geometric isomerism

 

Geometric isomers are associated with structures which contain double bonds. Groups attached to atoms which form single covalent bonds to other atoms are free to rotate. With double carbon bonds the substituents cannot rotate, they are confined to a single side of the compound. This is due to the double bond having restricted rotation. The figure below shows how this can lead to isomerism.

 

Figure 1

When it comes to naming geometric isomers, we use phrases to describe the location of a substituent with reference to the double bond. The old phrases used were:

 

Cis                =together

Trans             =opposite   

 

The other notation which is more commonly used is E and Z:

 

E = Entgegen which means opposite. So the substituents of a double bond are opposite each other- Trans (think of E standing for enemies in a fight which are on opposite sides. So the groups of highest priority are on opposite sides

Z = Zusammen which is together- Cis. So the highest priority groups are on the same side of double bond.

 

Which is highest priority?

 

If there are two substituents attached to the carbon double bond, how do we know which one is the highest priority when naming? We choose the substituent with the highest priority. The highest priority is the atom with the highest atomic number. Using Figure 2 as an example, the following points provide a step-by-step guide on how to name it.

 

1)    We start by splitting the compound into two, so that each carbon is considered separately. We'll call them carbon A and carbon B.

2)    Start by looking at carbon A; notice how it is bonded to a hydrogen atom and a methyl group.

3)    Which one has the higher atomic number - hydrogen or carbon? Using the periodic table we know that carbon has a greater atomic number hence we can say that the highest priority for carbon A is the methyl group. (Carbon = 6 and Hydrogen = 1).

4)    Consider carbon B; notice how it is bonded to a bromine atom and a methyl group. Which has higher atomic number bromine or carbon? Using the periodic table we know that bromine is higher so it has greater priority. (Bromine = 35 and carbon = 6).

5)    Therefore the priority substituents for carbon A is the methyl group and the priority for carbon B is the bromine atom.

6)    One priority group is on the bottom face, whilst the other priority is on the top face, so they are opposite each other.

7)    So this compound is trans 2 bromo but-2-ene, or we can say it is (E) 2 bromo but-2-ene, because E is opposite which is the same as trans.

 

Figure 3 shows an example of how to name isomers using both the cis-trans and E-Z notation.

Figure 3

Optical isomerism

 

Before we go into optical isomers you must be familiar with the term chirality.

 

An atom of carbon is described as being chiral if it is bonded to 4 different groups. Look at Figure 4. The carbon (labelled α) is chiral since it has 4 bonds attached to 4 different substituents. One bond is to a methyl group (CH3), another to an amino group (NH2), one to a carboxyl group (COOH) and finally one to hydrogen.

 

Chiral carbon

 

Another rule that must be followed for carbon to be chiral is that the carbon is sp3 hybridised. This means the carbon contains 4 sigma bonds. If we consider the carbon atom in the COOH group in Figure 4, we notice it has one sigma bond to another carbon, one to the hydroxyl group and a double bond to oxygen. Therefore this carbon contains 3 sigma bonds and one pi bond, hence it is sp2 hybridised so it is not chiral. The important conclusion for all carbon atoms is that if they contain a double bond, they are not chiral.

 

In order for a compound to be optically active it must have a chiral carbon. Compounds that do not have a chiral centre are optically inactive. The chiral carbon is sometimes referred to as the stereogenic centre. When dealing with chiral carbons we usually reference the carbon as having S or R configuration. We base this characteristic depending on the priorities of each different group attached to the chiral carbon. 

 

The best known examples of chiral molecules in biochemistry include:

 

1.    Oranges and lemons: Limonene is a chiral molecule which gives oranges and lemons their distinctive smell. Oranges contain the left-handed molecule and lemons contain the right handed. Just like a left shoe will only fit on your left foot these molecules will only fit on the left or right handed smell receptors.

2.    Thalidomide: Thalidomide was a drug used in the mid-20th century to treat morning sickness but was later found to be teratogenic. Only one enantiomer is thought to cause this effect but the two interconvert in the body so it is not possible to administer only one.

3.    Amino acids: Amino acids are the building blocks of protein. Usually when describing the handedness of amino acids L- and D- notation is used instead of R- and S- (see References for an article describing this in more detail). L-amino acids are the sort found in proteins but D-amino acids are also found in nature, particularly on the surface of bacteria, where they protect against some attacking enzymes.  

 

R or S conformation

 

Look at Figure 5.    

1)    Ensure the carbon is sp3 hybridised, in other words check that the carbon has four sigma bonds (No double bonds).

2)    Check that this carbon is attached to four different groups to ensure it is chiral.

3)    Identify the group which has the highest atomic number- highest priority. In this scenario we notice that the chlorine is the highest priority so we label it 1 (chlorine =17)

 

Figure 6

 

4)    The second highest priority group is slightly tricky, since we have two bonds going to two carbon atoms, so we look at both carbons and see which atoms are bonded to these carbon atoms. For the methyl carbon it is bonded to three hydrogen atoms only, whereas the propyl carbon is bonded to two hydrogen atoms and a carbon atom. Since this carbon is attached to another carbon which is higher priority compared to the carbon attached to the hydrogen we can conclude that the propyl group is second highest priority. We label it 2.

5)    The third highest priority would be the methyl group, labelled 3.

6)    The fourth highest priority is the hydrogen atom, labelled 4.

7)    So now we have prioritised each group we can move onto the final step, which is drawing a curved arrow from the first priority group (1) to the second priority group (2) shown in Figure 5.

8) If the arrow is going clockwise then the chiral carbon is R configuration

    If the arrow is going anticlockwise then it is S configuration

 

Hint: If you find it difficult to remember S and R directions, use this trick. When you turn the steering wheel of a car clockwise the car moves to the right R.

 

In this example the arrow is going anticlockwise hence we know that this carbon is S configuration.

 

Enantiomers

Enantiomers: are compounds with one stereogenic centre which are exactly mirror images of each other. Enantiomers are non-superimposable images. Notice how one enantiomer is R configuration whilst the other is S configuration.

 

 

Diastereoisomers

If a compound has more than one stereogenic centre (chiral carbon) then it is described as being a diastereoisomer. Diastereoisomers have different configurations around one or more chiral carbons but are not mirror images of each other. If only one stereogenic centre has a different conformation between isomers then they are called epimers. The most important difference between diastereoisomers and enantiomers is that the former have differing physical properties but the latter do not.

 

Mesoforms

Mesoforms are achiral (not chiral) compounds which contain two stereogenic centres. Each stereogenic centre has an opposite configuration e.g. one carbon atom is S whilst the other carbon atom is R configuration. The two opposite configurations cancel each other out, so the molecule is not optically active.

 

Naming molecules with two chiral centres

What happens if we have a compound with more than one chiral carbon? Usually in this case we have to start at one chiral carbon and determine whether it is R or S configuration. We then repeat the same process for the second chiral centre.

 

Compound conformation in space

In the previous section we mentioned briefly how single carbon bonds are able to freely rotate, however we will find that not every conformation is approved in free space. Conformation of a compound may be described as:

 

Staggered when the bonds of adjacent carbons are at a maximum distance away opposite from each other.

Eclipsed when the bonds are in line with each other.

 

When the bonds are in line with each other (eclipsed conformation) the electrons in the bonds repel each other. This means that rotation around the carbon bond is not completely free because the staggered conformation is more energetically favourable. The dihedral angle is a measure between the hydrogen atoms of adjacent carbons, which has a value of 0° in eclipsed conformation and 60° in staggered conformation. Figure 8 shows the example ethane. Newman projections (an alternative way of representing this) are shown next to the ball and stick diagrams as it is likely that your would need to know how to read or draw one in an exam.

 

Figure 8

 

Using this graph (Figure 9) we can explain why compounds such as hexane etc. exist as staggered shapes. The staggered conformations have a lower energy value than the eclipsed conformations. The eclipsed forms of a compound correspond to the peaks of the graphs, with the highest potential energy. The troughs of the graph are reflective of the staggered conformation of the compound with the lowest potential energy.

 

This results in the activation barrier of the formation of this compound being low. Since reactions proceed via the lowest energy pathway most compounds are formed via the staggered barrier. The eclipsed structure requires more input energy to form, since there is repulsion between the bonds leading to additional energy- high activation energy.

However the trend of most compounds existing as staggered configurations is not strictly true, some compounds favour eclipsed conformations. An example is ethane 1, 2 diol. Eclipsed formation is favoured due to hydrogen bonding between the hydrogen and oxygen bonded to adjacent carbon atoms. The force of hydrogen bonding overpowers the steric repulsion.

 

Gauche and Anti

Ethane is a useful example to start with because all the groups are hydrogen, so the molecule can only rotate between the staggered and eclipsed conformations. But what happens when we have different groups around the carbon? Taking the example of butane, the larger methyl groups are going to repel each other more than the smaller hydrogens. The conformations of butane in order of increasing stability (shown in Figure 10) are;

 

·         Fully Eclipsed: where the two methyl groups are eclipsed. This is the most energetically unfavourable conformation.

·         Eclipsed: where the methyl groups each eclipse a hydrogen.

·         Gauche: where the methyl groups are staggered at a 60 degree angle to each other.

·         Anti: where the methyl groups are staggered at 180 degrees to each other. This is the most energetically favourable conformation.

 

This changes the shape of the graph in Figure 9. The fully eclipsed peak of the graph would be higher than the two eclipsed peaks as fully eclipsed is the state with the highest potential energy. The anti conformation trough would be lower than the two gauche troughs because anti conformation is the most energetically favourable state.

 

Stereochemistry

• chemistry in three dimensions
• includes both structure and reactivity effects

Enantiomers

• mirror-image stereoisomers
• like left and right hands 
  (see page 172 in your text)
• observed when a carbon atom has four different groups attached to it 
  CHXYZ or CX1X2X3X4

Enantiomer Examples






Chirality

• property of having "handedness"
  (different from its mirror image)
• a molecule with any element of symmetry (e.g., a mirror plane) must be achiral

Stereogenic Centers

• chiral centers or stereocenters
• a molecule with a stereogenic center (e.g., CX1X2X3X4) will be chiral
• a stereogenic center cannot be:
  sp- or sp2-hybridized (must be sp3)
  an atom with 2 identical substituents (e.g., any -CH2- group)

Identifying Chiral Molecules

• achiral
  
• chiral
  

Properties of Enantiomers

• enantiomers have identical physical and chemical properties, 
  EXCEPT they
• interact with another chiral molecule differently 
  (like trying on left- or right-handed gloves - left and right hands react differently)
• rotate the plane of plane-polarized light by equal amounts but in opposite directions

Optical Activity

• chiral compounds rotate the plane of plane-polarized light
• rotation measured in degrees
  clockwise (dextrorotatory or +) or
  counterclockwise (levorotatory or -)
• polarimeter - instrument for measuring optical activity

Specific Rotation

• standard amount of optical rotation by 1 g/mL of compound 
  in a standard 1 decimeter (10 cm) cell
• [a] = a / l C
• where [a] is specific rotation
  a = observed rotation in degrees 
  l = path length in dm
  C = concentration in g/mL

Absolute Configuration

• nomenclature method for designating the specific arrangement of groups about a stereogenic center
• differentiates between enantiomers
• uses the same sequence rules for establishing priority of groups as was used for E and Z

R and S Designations

• assign priorities 1-4 (or a-d) to the four different groups on the stereogenic center
• align the lowest priority group (4 or d) behind the stereogenic carbon
• if the direction of a-b-c is clockwise, it is R
• if a-b-c is counterclockwise, it is S

Right- and Left-Hand Views

• textbook analogy - steering wheel
• alternative analogy - your hands 
  assign priorities to your fingers in order of height
  a = middle finger, b = pointer finger, c = thumb, d = wrist 
  R - this works for your right hand 
  S - this works for your left hand 

Drawing 3-D Structures

• practice with models
• dotted-line & wedge
• Fischer projections
  

Fischer Projections

• a method for depicting stereochemistry at a series of chiral centers
• arrange the chiral center so that:
• horizontal groups are forward
• vertical groups are oriented backward

• Note that there are numerous ways to show a given chiral center
• 12 different Fischer projections represent (R)
• 12 different Fischer projections represent (S)

Multiple Stereogenic Centers

• compounds with more than 2 stereocenters have more than 2 stereoisomers 
  e.g., 2-bromo-3-chlorobutane 
  (2R,3R) and (2S,3S) are enantiomers 
  (2R,3S) and (2S,3R) are enantiomers
• in general, n stereocenters give 2^n stereoisomers

Diastereomers

• stereoisomers that are not enantiomers 
  e.g., (2R,3R) and (2R,3S)
  (not mirror images, but not the same either)
• diastereomers may have different chemical and physical properties

Meso Compounds

• compounds with stereogenic centers but which are not chiral 
  e.g., (2R,3S)-2,3-dibromobutane 
  (same as its mirror image)
  

Identifying Meso Compounds

• mirror plane of symmetry
• one stereocenter is the mirror image of the other
• cis-1,2-disubstituted cycloalkanes are meso if the two substituents are identical
  

Cyclohexane Derivatives

• chair interconversions affect conformation, but not configuration
• trans-1,2-dichlorocyclohexane is (R,R) or (S,S)
• cis-1,2-dichlorocyclohexane is (R,S)
• one chair has the R stereocenter with axial Cl and S with equatorial
• the other chair has R equatorial and S axial
• the two chair forms are enantiomers but not isolatable

Racemic Mixtures

• an equal mix of both enantiomers (also called a racemate)
• a common form in the laboratory (but not in nature)
• optical resolution - separating enantiomers from a mix (typically difficult)

Optical Purity / Enantiomeric Excess

• unequal mixtures of enantiomers may occur
• optical purity - compare actual rotation with what a pure enantiomer would give (in %)
• enantiomeric excess - % excess of one pure enantiomer over the other
• % optical purity = % enantiomeric excess
• example - consider a mix of 75% (R) + 25% (S)
• optical rotation would be 50% (50% inactive racemic + 50% R)
• enantiomeric excess is also 50% (75% - 25%)

Optical Resolution

• for acids or bases - formation of diastereomeric salts from a naturally ocurring acid or base
• enzymatic resolution - preferential binding or reaction of just one enantiomer

Isomerism - Summary

• isomers - same molecular formula (same collection of atoms used)
• constitutional isomers -differ in the connections between atoms
  different carbon skeletons
  different functional groups
  different locations of a functional group

 

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Dry Heat Sterilization

Sterilization (or sterilisation) is a term referring to any process that eliminates (removes) or kills all forms of microbial life, including transmissible agents (such as fungi, bacteria, viruses, spore forms, etc.) present on a surface, contained in a fluid, in medication, or in a compound such as biological culture media.^[1][2]Sterilization can be achieved by applying heat, chemicals, irradiation, high pressure, and filtration or combinations thereof.

The term has evolved to include the disabling or destruction of infectious proteins such as prions related to Transmissible Spongiform Encephalopathies (TSE).^[3]

Heat[edit source]

See also: Dry heat sterilization and Moist heat sterilization

Steam sterilization[edit source]

Front-loading autoclaves

A widely used method for heat sterilization is the autoclave, sometimes called a converter. Autoclaves commonly use steam heated to 121–134 °C (250–273 °F). To achieve sterility, a holding time of at least 15 minutes at 121 °C (250 °F) at 100 kPa (15 psi), or 3 minutes at 134 °C (273 °F) at 100 kPa (15 psi) is required. Additional sterilizing time is usually required for liquids and instruments packed in layers of cloth, as they may take longer to reach the required temperature (unnecessary in machines that grind the contents prior to sterilization). Following sterilization, liquids in a pressurized autoclave must be cooled slowly to avoid boiling over when the pressure is released. Modern converters operate around this problem by gradually depressing the sterilization chamber and allowing liquids to evaporate under a negative pressure, while cooling the contents.

Proper autoclave treatment will inactivate all fungi, bacteria, viruses and also bacterial spores, which can be quite resistant. It will not necessarily eliminate allprions.

For prion elimination, various recommendations state 121–132 °C (250–270 °F) for 60 minutes or 134 °C (273 °F) for at least 18 minutes. The prion that causes the disease scrapie (strain 263K) is inactivated relatively quickly by such sterilization procedures; however, other strains of scrapie, as well as strains of CJD andBSE are more resistant. Using mice as test animals, one experiment showed that heating BSE positive brain tissue at 134–138 °C (273–280 °F) for 18 minutes resulted in only a 2.5 log decrease in prion infectivity. (The initial BSE concentration in the tissue was relatively low). For a significant margin of safety, cleaning should reduce infectivity by 4 logs, and the sterilization method should reduce it a further 5 logs.

To ensure the autoclaving process was able to cause sterilization, most autoclaves have meters and charts that record or display pertinent information such as temperature and pressure as a function of time. Indicator tape is often placed on packages of products prior to autoclaving. A chemical in the tape will change color when the appropriate conditions have been met. Some types of packaging have built-in indicators on them.

Biological indicators ("bioindicators") can also be used to independently confirm autoclave performance. Simple bioindicator devices are commercially available based on microbial spores. Most contain spores of the heat resistant microbe Geobacillus stearothermophilus (formerly Bacillus stearothermophilus), among the toughest organisms for an autoclave to destroy. Typically these devices have a self-contained liquid growth medium and a growth indicator. After autoclaving an internal glass ampule is shattered, releasing the spores into the growth medium. The vial is then incubated (typically at 56 °C (133 °F)) for 24 hours. If the autoclave destroyed the spores, the medium will retain its original color. If autoclaving was unsuccessful the B. sterothermophilus will metabolize during incubation, causing a color change during the incubation.

For effective sterilization, steam needs to penetrate the autoclave load uniformly, so an autoclave must not be overcrowded, and the lids of bottles and containers must be left ajar. Alternatively steam penetration can be achieved by shredding the waste in some Autoclave models that also render the end product unrecognizable. During the initial heating of the chamber, residual air must be removed. Indicators should be placed in the most difficult places for the steam to reach to ensure that steam actually penetrates there.

For autoclaving, as for all disinfection or sterilization methods, cleaning is critical. Extraneous biological matter or grime may shield organisms from the property intended to kill them, whether it physical or chemical. Cleaning can also remove a large number of organisms. Proper cleaning can be achieved by physical scrubbing. This should be done with detergent and warm water to get the best results. Cleaning instruments or utensils with organic matter, cool water must be used because warm or hot water may cause organic debris to coagulate. Treatment with ultrasound or pulsed air can also be used to remove debris.

Heat sterilization of foods

See also: Food safety

Although imperfect, cooking and canning are the most common applications of heat sterilization. Boiling water kills the vegetative stage of all common microbes. Roasting meat until it is well done typically completely sterilizes the surface. Since the surface is also the part of food most likely to be contaminated by microbes, roasting usually prevents food poisoning. Note that the common methods of cooking food do not sterilize food - they simply reduce the number of disease-causing micro-organisms to a level that is not dangerous for people with normal digestive and immune systems.

Pressure cooking is analogous to autoclaving and when performed correctly renders food sterile. However, some foods are notoriously difficult to sterilize with home canning equipment, so expert recommendations should be followed for home processing to avoid food poisoning.

Other heat sterilization methods[edit source]

Other heat methods include flaming, incineration, boiling, tindalization, and using dry heat.

Flaming is done to loops and straight-wires in microbiology labs. Leaving the loop in the flame of a Bunsen burner or alcohol lamp until it glows red ensures that any infectious agent gets inactivated. This is commonly used for small metal or glass objects, but not for large objects (see Incineration below). However, during the initial heating infectious material may be "sprayed" from the wire surface before it is killed, contaminating nearby surfaces and objects. Therefore, special heaters have been developed that surround the inoculating loop with a heated cage, ensuring that such sprayed material does not further contaminate the area. Another problem is that gas flames may leave residues on the object, e.g. carbon, if the object is not heated enough.

A variation on flaming is to dip the object in 70% ethanol (or a higher concentration) and merely touch the object briefly to the Bunsen burner flame, but not hold it in the gas flame. The ethanol will ignite and burn off in a few seconds. 70% ethanol kills many, but not all, bacteria and viruses, and has the advantage that it leaves less residue than a gas flame. This method works well for the glass "hockey stick"-shaped bacteria spreaders.

Incineration will also burn any organism to ash. It is used to sanitize medical and other biohazardous waste before it is discarded with non-hazardous waste.

Boiling in water for fifteen minutes will kill most vegetative bacteria and inactivate viruses, but boiling is ineffective against prions and many bacterial and fungalspores; therefore boiling is unsuitable for sterilization. However, since boiling does kill most vegetative microbes and viruses, it is useful for reducing viable levels if no better method is available. Boiling is a simple process, and is an option available to most people, requiring only water, enough heat, and a container that can withstand the heat; however, boiling can be hazardous and cumbersome.

Tindalization^[6] /Tyndallization^[7] named after John Tyndall is a lengthy process designed to reduce the level of activity of sporulating bacteria that are left by a simple boiling water method. The process involves boiling for a period (typically 20 minutes) at atmospheric pressure, cooling, incubating for a day, boiling, cooling, incubating for a day, boiling, cooling, incubating for a day, and finally boiling again. The three incubation periods are to allow heat-resistant spores surviving the previous boiling period to germinate to form the heat-sensitive vegetative (growing) stage, which can be killed by the next boiling step. This is effective because many spores are stimulated to grow by the heat shock. The procedure only works for media that can support bacterial growth - it will not sterilize plain water. Tindalization/tyndallization is ineffective against prions.

Dry heat sterilizer

Dry heat can be used to sterilize items, but as the heat takes much longer to be transferred to the organism, both the time and the temperature must usually be increased, unless forced ventilation of the hot air is used. The standard setting for a hot air oven is at least two hours at 160 °C (320 °F). A rapid method heats air to 190 °C (374 °F) for 6 minutes for unwrapped objects and 12 minutes for wrapped objects.^[8][9] Dry heat has the advantage that it can be used on powders and other heat-stable items that are adversely affected by steam (for instance, it does not cause rusting of steel objects).

Prions can be inactivated by immersion in sodium hydroxide (NaOH 0.09N) for two hours plus one hour autoclaving (121 °C or 250 °F). Several investigators have shown complete (>7.4 logs) inactivation with this combined treatment. However, sodium hydroxide may corrode surgical instruments, especially at the elevated temperatures of the autoclave.

Glass bead sterilizer, once a common sterilization method employed in dental offices as well as biologic laboratories,^[10] is not approved by the U.S. Food and Drug Administration (FDA) and Centers for Disease Control and Prevention (CDC) to be used as inter-patients sterilizer since 1997.^[11] Still it is popular in European as well as Israeli dental practice although there are no current evidence-based guidelines for using this sterilizer.^[10]

Gold Number

Gold Number is a term used in colloidal chemistry. It is defined as the minimum amount of lyophilic colloid in milligrams which prevents the flocculation of 10mlgold sol by the addition of 1 ml of 10%NaCl solution.

Coagulation of gold sol is indicated by colour change from red to blue when particle size just increases. More is the gold number less is the protective power of the lyophilic colloid since it means that the amount required is more.It was first used by Zsigmondy.The amount is taken in terms of weight in milligrams.

The gold number of some colloids is given below.

Protective Colloids	Gold Number
Gelatin	0.005-.01
Haemoglobin	0.03-0.07
Egg Albumin	0.15-0.25
Potato Starch	25
Gum arabic	0.15-0.25
Caseinate	0.01
Sodium Oleate	1-5
Dextrin	6-20

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Number of pharmaceutical companies are there all over the world which require number of managing experts. MBA in pharmaceutical management have very big opportunities all over the world.

They are getting attracting payment packages and placement being offered in college years only. They are the main ladder for corporate world. So, anyhow they are strongly grow up their future prospectus and have a very respective life than any other fields.