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# oxford work shop.pptx

15 de Nov de 2022
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### oxford work shop.pptx

1. Presentation on solution preparation— Industrial Application. Dr. Shivaji.B. M.Sc.,B.Ed.,M.Phil.,Ph.D Associate Professor shivajibio@gmail.com Mobile :9611922895
2.  1.26 gm of oxalic acid crystal is dissolved in 250 ml of the solution. Calculate its molarity  . Calculate the mass of anhydrous sodium carbonate required to prepare 200 ml of decimolar solution.  1.26 gm of oxalic acid crystal is dissolved in 500 ml of the solution. Calculate its normalarity.  Calculate the mass of anhydrous sodium carbonate required to prepare 200 ml of 0.4N solution.  Prepare 10 mM solution 10ml(mol wt;156)  Prepare phosphate buffer of Ph7.2 0.1 M 100 ml.  Ans?
3.  Most chemical reactions, both in nature and in the laboratory, take place in a solution.  Solutions are a mixture of any chemical, and dissolved usually in water. It can be any solute, dissolved in any solvent.  We live in a world of solutions! The air we breath is a huge gaseous solution, the oceans are solutions of about fifty different salts in water, and many of the rocks and minerals of the earth are solid solutions. And we ourselves are largely aqueous solutions, most of it within our cells (whose water content contributes to about half our body weight) and in our blood plasma and the interstitial fluid that bathes our cells (about 5 L in the adult). So in order to understand the world in which we live and the organisms that inhabit it, we need to know something about solutions, and this is where we begin.  Solutions are homogeneous (single-phase) mixtures of two or more components. For convenience, we often refer to the majority component as the solvent; minority components are solutes. But there is really no fundamental distinction between them.
4.  Solution is a binary solution.  Dissolved substance is---solute  Medium in which it is dissolved is----solvent Solution: solute +solvent=solution Composition of a solution can be expressed in two ways Quantity. Is the amount of any solute present in a solvent/solution irrespective of the amount /volume of solvent/solution Concentration: quantity of the solute present in a exact amount of solvent/solution. Ex ; 4g NaOH in 100 ml or 200 ml but amount of NaOH is 4 gm In concentration 4% Amount as the unit of quantity. Per cent is a unit of concentration.
5. Stock solution: stock solution is the one having a concentration many folds higher than that actually required in the experiment. Used frequently and stable at higher concentration for several days and can be used after appropriate dilution just before use. Standard solution: known concentration is referred to as a standard solution.
6.  Concentration is a general term that expresses the quantity of solute contained in a given amount of solution. Various ways of expressing concentration are in use; the choice is usually a matter of convenience in a particular application. You should become familiar with all of them.  How concentrations are expressed  Parts-per concentration  In the consumer and industrial world, the most common method of expressing the concentration is based on the quantity of solute in a fixed quantity of solution. The “quantities” referred to here can be expressed in weight, in volume, or both (i.e., the weight of solute in a given volume of solution.) In order to distinguish among these possibilities, the abbreviations (w/w), (v/v) and (w/v) are used.
7. Making up the solution  Take a watch glass and place it on the balance.  Tare the balance (set it to zero). Carefully weigh out the required mass of substance.  Transfer this amount to a beaker. Add water from a wash bottle to dissolve it. Use some of the water to rinse all the substance off the watch glass. Do this at least twice.  Stir with a glass rod until all the solid is dissolved, then transfer the solution to the volumetric flask. Use more water from the wash bottle to rinse out the beaker and the glass rod. Do this at least twice.  Add water to just below the line on the volumetric flask. Add the final drops with a teat pipette to ensure that the bottom of the meniscus is on the line.  Put the lid on the flask and turn the flask over a couple of times to mix the solution.  Label your solution with your name, the date, and the contents, e.g. 2.0M NaCl. Then tidy up!
8. Criteria for solution preparation  Concentration in terms of Mole:  Normal solution  Molar solution Number of moles: Mass/Molecular mass Mass/Atomic mass Ex : No of moles in 9.2 g sodium and 48 g of oxygen, 11 g of co2, & 20 g sodium hydroxide 9.2/23=0.4 moles 48/16=3.0 moles 11/44=0.25 moles 20/40=0.5 moles
9. Mass= Molecular mass x No of moles Atomic mass x No of moles Ex : calculate mass in gm 1)0.4 mole oxygen molecule 2)2.5 mole co2 32 x 0.4=12.8 gm 44 x 2.5=11.0 gm
10.  Normality: Gram equivalent weight of solute when dissolved in 1000ml of solvent.  Molarity: Number of moles of solute dissolved in one liter of the solution  Normality= No of gm equiv wt of solute/volume of solution(dm3)  Molarity= No of gm molecular wt of solute/volume of solution(dm3)  Equivalent weight: Molecular mass/Basicity of an acid Molecular mass/Acidity of base Molecular mass/Oxidising agent Molecular mass/reducing agent
11. Normality; Mass in dm3/eq mass Molarity: Mass in dm3/molecular mass
12. Equivalent weight of some common acids Acid Mol Wt Basicity Eq Wt HCl 36.5 1 36.5 HNO3 63 1 63 H2SO4 98 2 49 CH3COOH 60 1 60 H3PO4 82 3 32.7 H2C2O4 2H2O 126 2 63
13. Equivalent weights of some common base Formula of base Mol. Wt. Acidity Eq. wt NaOH 40 1 40 KOH 56 1 56 Ca(OH)2 74 2 37 Ba(OH)2 171.4 2 85.7 Equivalent weights of oxidising agent KMNO4 : 5 2KMNO4+3H2SO4 K2SO4+3H2O+5[O] Molecular WT oF KMNO4=158/5=31.6 K2CR2O7=49 it gives 294/6=49 1 molar or 1M H2SO4 contain 98 gm 0.5 M Contain 49 gm 1MH2SO4=2NH2SO4 BUT 1MNaOH=1NNaOH
14. Normality % concentration and specific gravity of some acids and alkalies Acid/Alkali MW Normality Sp Gravity Hydrochloric acid 36.5 11.3 1.18 Sulphuric acid 98 36 1.84 Nitric acid 63 15.7 1.42 O- phosphoric acid 82 44 1.71 Acetic acid 60 17.5 1.06 Potassium hydroxide 56 Sodium hydroxide 40 Ammonium hydroxide 17 15
15. LAB Normaliy 1 N 100 ml 1N 500 ml 1 N 1000ml 2N 100 ml 2N 500ml 2N 1000 ml 5N 100 ml 5N 500 ml 5N 1000 ml HCl 11.3 8.9 44.2 88.4 17.7 88.4 177 44.2 221 442 H2SO4 36.0 2.7 13.8 27.7 5.5 27.7 55.5 13.8 69.4 139 HNO3 16.0 6.25 31.28 62.5 12.5 62.5 125 31.25 156 312.5 CH3COOH 17.4 5.7 28.7 57.4 11.4 57.4 114.9 28.7 143.6 287.2 NH4OH 14.3 6.99 34.9 69.8 13.9 69.9 139.8 34.9 174.8 349 NaOH 40 4gm 20gm 40g 8gm 40g 80gm 20gm 100g 200g
16. BUFFER:  Acid: it is defined as substance which can donate ion concentration i.e proton donor.  Base: it is defined as the substance which can accept ion concentration ie proton acceptor.  It is defined as the negative logarithm of hydrogen ion concentration. i.e pH = - log[H]  Buffer capacity: it is the capacity of buffer to resist change in pH.  it depends on  Actual conc. Of salt and acid present in buffer  Salt: acid conc. Ratio  pH=pKa+log [salt]/[acid]
17. Criteria for buffer preparation :  While choosing a buffer the following factors need to be taken into account. It should  Possess adequate buffering capacity in the required pH range. Generally buffer are most effective over a range of one pH unit on either side of their’ value, eg Tris which has pk’ value of 8.3 has an effective pH range of 7-9 .the pk’ values of some important buffer are given in table.  be chemically inert and not react or bind with biomolecules or other components, particularly, for assaying activities of enzymes which require a metal ion for their functioning  be available in high degree of purity and should not contain impurities which may interfere with estimations  be enzymically and hydrolytically stable  maintain pH that is minimally influenced by the temperature, Ionic composition and concentration or salts effect of the medium  not be toxic  Not absorb light in the visible or ultraviolet regions.
18. Buffer pKa value Buffering range Phosphate 6.86 6.5-7.5 Acetate 4.76 3-6 Citrate 4.74 3-6 Tris 8.1 7-10 Borate 9.24 8.5-10.0
19. Phosphate buffer:  Stock solutions :  Dibasic stock (0.2M):disodium hydrogen phosphate (Na2HPO4)  Monobasic stock (0.2M):Sodium Dihydrogen phosphate (NaH2PO4)
20. WORKING PHOSPHATE BUFFER 0.1M (100 ml) Mix X ml of 0.2M dibasic stock solution with Y ml of monobasic stock and dilute to 100 ml with distilled water pH Xml (dibasic) Yml ( Monobasic) 5.8 4.00 46.00 6.0 6.15 43.75 6.2 9.25 40.75 6.4 13.25 36.75 6.6 18.75 31.25 6.8 24.50 25.50 7.0 30.50 19.50 7.2 36.00 14.00 7.4 40.50 9.50 7.6 43.50 6.50 7.8 45.75 4.25 8.0 47.35 2.65
21. Citrate buffer :Range 3 – 6.2 Stock solution A: 0.1M citric acid monohydrate, dissolve in 21.01 gm citric acid in 1000ml water stock solution B: 0.1M trisodium citrate, dehydrate, dissolve 29.41 gm of sodium citrate in 1000 ml And mix X ml of A and yml of B dilute at 100 ml . pH Solution A( ml) Solution B ( ml) 3.0 46.5 03.5 3.2 43.7 06.3 3.4 40.0 10.0 3.6 37.0 13.0 3.8 35.0 15.0 4.0 33.0 17.0 4.2 31.5 18.5 4.4 28.0 22.0 4.6 25.5 24.5 4.8 23.0 27.0 5.0 20.5 29.5 5.2 18.0 32.0 5.4 16.0 34.0 5.6 13.7 36.3 5.8 11.8 38.2 6.0 9.5 41.5 6.2 7.2 42.8
22. Acetate Buffer : Stock solution A: Acetic acid (0.2M) : Dilute 11.5 mL of glacial acetic acid in 1litre solution. stock solution B:Sodium Acetae( 0.2M): dissolve 16.4 gm of C2H3O2Na or 27.2 gm of C2H3O2Na.3H2O in 1000 ml. (0.1M) WORKING BUFFER: mix X ml of A and y ml of B and dilute to 100 ml with water. pH Solution A( ml) Solution B ( ml) 3.6 46.3 3.7 3.8 44.0 6.0 4.0 41.0 9.0 4.2 36.8 13.2 4.4 30.5 19.5 4.6 25.5 24.5 4.8 20.0 30.0 5.0 14.8 35.2 5.2 10.5 39.5 5.4 8.8 41.2 5.6 4.8 45.2
23.  Percentage solutions and conversion:-  1% w/v = 1 gram in 100ml of solvent  1% w/w= 1gram in 100 grams of solvent  Normally % w/v solution are used
24.  Percentage( % )conversion  To convert %w/v to mg/ml :Multiply by 10 % w/v x 10.0 = mg/ml  Problem: convert 2%w/v to mg/ml  Formula: 2%w/v x 10.0=20mg/ml  Checking : 2% w/v means 2 gram/100ml i.e 2000mg/100ml i.e 20mg/ml  2. To convert mg/ml to % w/v: Divide by 10 = % w/v  3.To convert %w/v to µg/ml: Multiply by 10,000 %w/v 10,000 = µg/ml  4.To convert µg/ml to %w/v: Divide by 10,000 = %w/v
25.  Dilutions  For preparing calibration curves, dilutions have to be prepared from stock solutions.  In dilution the following must be observed:  Minimum quantity of stock solution is to be used  At any step, dilution should not be more than 5 or 10 times( this is to minimize error in dilution ) eg. 1 ml to 5 ml or 1 ml to 10 ml can be done , but not 1 ml to 100ml  The total volume of the solution available should be sufficient for subsequent dilutions  In order to avoid confusion in calculations, it is better to use the following format  Initial volume final volume (concentration/ml)
26. Problem 1: Calibration curve from 10-15µg/ml. The given stock solution is 1mg/ml. (1mg/ml=1000µg/ml) 1000µg/ml 10ml - 100ml (100µg/ml) 1 ml - 10 ml (10µg/ml) 2ml - 10ml (20µg/ml) 3ml - 10ml (30µg/ml) 4ml - 10ml (40µg/ml) 5ml - 10ml (50µg/ml)
27. Contents  INTRODUCTION TO SPECTROSCOPY  TYPES OF SPECTROSCOPY 1.ABSORPTION SPECTROSCOPY 2. MOLECULAR SPECTROSCOPY  COLORIMETRY  PRINCIPLE BEER-LAMBERT’S LAW  INSTRUMENTATION 1.SOURCE OF LIGHT 2.FILTERS 3.MONOCHROMATOR 4.SAMPLE CELL 5.DETECTOR  DEVIATION  DIFFRENCE BETWEEN COLORIMETER AND SPECTROPHOTOMETER  APPLICATION  CONCLUSION
28.  What is SPECTROSCOPY?  Spectroscopy is the measurement and interpretation of electro magnetic radiation (EMR) absorb or emitted when the molecules or ions or atoms of a sample move from one energy state to another energy state. The energy of electromagnetic radiation can be given by the following equation E=hυ
29. TYPES OF SPECTROSCOPY  Can be divided into following types based on  1.Based on atomic or molecular level Atomic spectroscopy Molecular spectroscopy  2. Based on absorption or emission of EMR Absorption spectroscopy Emission spectroscopy  3. Based on electronic or magnetic levels Electronic spectroscopy Magnetic spectroscopy
30.  PRINCIPLE IN COLORIMETRY Colorimetry is concerned with the study of absorption visible radiation whose wavelength ranges from 400nm- 800nm.
31.  What colors are absorbed and how intensely they are absorbed depends on the chemical being used and how the electrons and energy levels within it are arranged.  The color we see is complementary to the color absorbed by the chemical.  Instruments report both A (absorbance) and %T (transmittance). Be sure to use A.  The assumption is that the amount of light absorbed is directly proportional to the concentration of the chemical specie that the light passes through.  Also, the amount of light absorbed is directly proportional to the thickness (or path length) of the solution.  These factors and assumptions can be summarized as Beer's Law and written as the equation, A = abc. In this equation A is absorbance, a is a proportionality factor called the molar absorptive, b is the path length, and c is the molar concentration.
32.  It can be learnt that Transmittance (T)=It / I0 and Absorbance (A) =log 1/T Hence A= log 1 ⁄ I÷I0 A= log I0/It Using equation (iv) & (v) we can get that, A=K ct A= εct (Mathematical equation for Beer-Lambert’s Law)
33. Instrumentation  There are two types of instruments. 1)Colorimeters:- which are usually inexpensive and less accurate. They measures either Absorbance or Transmittance or both and have filters for use with different colored solutions . The range of wavelength used is usually small eg.400-700nm. 2)Spectrometers:- Which are little more expensive than colorimeter. They can be used for a wide wavelength region.eg. 360nm-900nm or sometimes up to 1000nm. The accuracy of instrument is very high since they employ grating monochromators and photo multipliers tube. They are supported by amplifiers, recorders, or plotters for hard copy. The recent ones are either microprocessors or computer based for easy data manipulation.
34. A. Source of Light  It should be provide continues radiation from 400nm -800nm  It should provide adequate intensity  It should be stable and free from fluctuations. Following are the sources of light uses commonly. 1)Tungsten Lamp:-This lamp find it’s place in most of colorimeter & spectrometer. The lamp consist of tungsten filament in a vacuum bulb similar to the one used domestically. But offers sufficient intensity. 2)Carbon arc Lamp:- For a source of very high intensity, carbon arc lamp can be used. It also provides an entire range of visible spectrum.
35. B. Filter and Monochromators  The source of light gives radiation from 400nm to 800nm .  This is Polychromatic in nature . In a colorimeter/ Spectrometer, we require only monochromatic light. Hence a filter or monochromator is used which converts polychromatic light in to monochromatic light. Though the efficiency of each differs considerably. (a)Filters are of two kinds. 1. Absorption Filter 2. Interference Filter (b) Monochromators are two types. 1. Prism type 2. Grating type
36. (a) Filters.  1)Absorption Filter :- These filters are made up of glass, coated with pigments are made up of dyed gelatin. They absorb the unwanted radiation and transmit the rest of the radiation which is required for colorimeter.
37. (b)Monochromators  Monochromators are better and more efficient than filters in convert a polychromatic heterochromatic light into monochromatic light monochromators has the following units: 1)Entrance slit(to get narrow source) 2)Collimator(to render light parallel) 3)Grating or Prism(to disperse radiation) 4) Collimator( to reform the images of entrance slit) 5) Exit slit ( to fall on sample cell)
38. C) Sample cells  Sample cells or cuvettes are used to hold a sample solution.  Their geometry as well as material varies with the instrument and nature of sample handled.  The material of the sample cell should not absorb at the wavelength being observed.  Cells are available which change with the following parameters.  Sample volume - small volume cells and large volume cells  Shape of cell – cylindrical or rectangular  Path length – 1cm upto 10cm cells are available  Material – colored corrected fused glass for visible region polystyrene cells are available for use with aqueous solvents but cannot used with organic solvents. For Uv region this cells must be made up of quartz since ,glass absorbs radiation.
39. D)Detectors  Detectors used in UV/visible spectrophotometers can be called photometric detectors.  The most commonly used detectors are  Barrier Layer cell or photo voltaic cell  Photo tubes or photo emissive cells and  Photo multiplier tubes
40. 1.) Barrier Layer cell or photo voltaic cell
41. 2.)Photo tubes or photo emissive cells photo cathode coated with caesium, potassium, silver oxide which can liberate electron when light falls on it
42. 3.) Photo multiplier tubes
43. Single beam colorimeter
44. Double beam colorimeter
45. DEVIATION FROM BEER’S LAW  Instrumental deviation (improper slit width, band pass,fluctuations)  Physiochemical changes in solutions.  Association(Methylene blue) and dissociation(Cr2O7)
46. APPLICATION  Quality control of purity  Quantitative analysis  Determination of molecular weight of amines  Determination of organic substances and pharmaceuticals  Spectrophotometric titrations.
47. centrifugation  Centrifugation is a process that involves the use of the centrifugal force for the separation of mixtures with a centrifuge, used in industry and in laboratory settings. More-dense components of the mixture migrate away from the axis of the centrifuge, while less- dense components of the mixture migrate towards the axis. Chemists and biologists may increase the effective gravitational force on a test tube so as to more rapidly and completely cause the precipitate ("pellet") to gather on the bottom of the tube. The remaining solution is properly called the "supernatant" or "supernatant liquid“  The rate of centrifugation is specified by the acceleration applied to the sample, typically measured in revolutions per minute (RPM) or g. The particles' settling velocity in centrifugation is a function of their size and shape, centrifugal acceleration, the volume fraction of solids present, the density difference between the particle and the liquid, and the viscosity
48.  When the density of a particle is greater than the density of the solution the particles sediments from a suspension under the influence of applied force.  If a suspension of large particle is allowed to stand the particle will tend to sediment under the influence of gravity.  The rate of sedimentation of a particle depends the force applied.  Particles sediments rapidly when greats force applied.
49. Principle of centrifugation  An object moving in a circle at a steady angular velocity will experience a force, F., directed outwards. This is the basis of centrifugation. Angular velocity in radians, ω, and the radius of rotation, r, in centimeters, collectively determine the magnitude of the force, F. F=ω2r  F might be expressed in terms of earth’s gravitational force if it is divided by 980. The resultant is referred to as relative centrifugal force, RCF. RCF is more frequently referred to as the ‘number times’ g.
50. Types of centrifuges Desktop centrifuge High speed centrifuge Ultracentrifuge
51. Desktop centrifuge These are simple, small and least expensive. They are normally used to collect rapidly sedimenting substances such as red blood cells, yeast cells or bulky precipitates of chemical reactions. Maximum speed is 3000rpm. Separation can be carried out in 10, 20 or 100 cm3  tubes.
52. High speed centrifuge  High speed centrifuges can operate with maximum speed of up to 25,000 rpm.  They are usually equipped with refrigeration equipment for removal of heat generated due to friction between the air and the spinning rotor.  Temperature can be easily be maintained in the range of 0-40,by  means of a thermocouple.  It is used to collect cell debris, large cellular organelles, precipitates of chemical reactions etc.  They are used to isolate lysosome, mitochondria, nuclei etc.
53. Ultracentrifuge  It operates at speeds up to 75,000 rpm providing centrifugal force.  Equipped with refrigeration system.  To prevent the rotor from operating at very high speeds, they are fitted with over speed devices.  It has good resolution power.  Centrifugation for isolation and purification of components is known as preparatory centrifugation.  While the one carried for desire for characterization is known as analytical centrifugation.
54. Types of centrifugation Preparative centrifugation Analytical centrifugation Differential centrifugation Density gradient centrifugation  Rate zonal separation  Isopycnic separation
55. Differential centrifugation  PRINCIPLE:- Separation is achieved primarily based on the size of the particles in differential centrifugation. • This type of separation is commonly used in simple pelleting and in obtaining partially-pure preparation of sub cellular organelles and macromolecules. • For the study of sub cellular organelles, tissue or cells are first disrupted to release their internal contents. • This crude disrupted cell mixture is referred to as a homogenate. • During centrifugation of a cell homogenate, larger particles sediment faster than smaller ones and this provides the basis for obtaining crude organelle fractions by differential centrifugation. • A cell homogenate can be centrifuged at a series of progressively higher g- forces and times to generate pellets of partially-purified organelles.
56. Density gradient centrifugation  Density gradient centrifugation. Density gradient centrifugation is the preferred method to purify sub cellular organelles and macromolecules. Density gradients can be generated by placing layer after layer of gradient media, such as sucrose in a tube with the heaviest layer at the bottom and the lightest at the top in either a discontinuous or continuous mode. The cell fraction to be separated is placed on top of the layer and centrifuged. Density gradient separation can be classified into two categories.  a. Rate-zonal (size) separation.  b. Isopycnic (density) separation.
57. Rate zonal centrifugation  PRINCIPLE:-Rate zonal centrifugation: In rate zonal centrifugation, the sample is applied in a thin zone at the top of the centrifuge tube on a density gradient. Under centrifugal force, the particles will begin sedimenting through the gradient in separate zones according to their size, shape, and density or the sedimentation coefficient(s). The run must be terminated before any of the separated particles reach the bottom of the tube. S is the sedimentation coefficient and is usually expressed in Svedbergs (S) units.
58. Rate zonal separation  Rate-zonal separation takes advantage of particle size and mass instead of particle density for sedimentation.  Figure 2 illustrates a rate-zonal separation process and the criteria for successful rate-zonal separation.  Examples of common applications include separation of cellular organelles such as endosomes or separation of proteins, such as antibodies.  For instance, Antibody classes all have very similar densities, but different masses. Thus, separation based on mass will separate the different classes, whereas separation based on density will not be able to resolve these antibody classes.
59. Density of the sample solution must be less than that of the lowest density portion of the gradient. Density of the sample particle must be greater than that of the highest density portion of the gradient. The path length of the gradient must be sufficient for the separation to occur. Time is important. If you perform too long runs, particles may all pellet at the bottom of the tube Criteria for successful rate-zonal centrifugation
60. Isopycnic separation  In this type of separation, a particle of a particular density will sink during centrifugation until a position is reached where the density of the surrounding solution is exactly the same as the density of the particle. Once this quasi- equilibrium is reached, the length of centrifugation does not have any influence on the migration of the particle.  A common example for this method is separation of nucleic acids in a CsCl gradient. A variety of gradient media can be used for isopycnic separations and their biological applications.
61. Rotor categories  Rotors can be broadly classified into three common categories namely swinging-bucket rotors, fixed-angle rotors, and vertical rotors .Note that each type of rotor has strengths and limitations depending on the type of separation.  swinging-bucket  fixed-angle  vertical
62. Fixed angle rotor These rotors have holes within their body, which allow sliding of the centrifuge tube. Holes are at an angle of 14 to 40 degrees to the vertical of the tube.  They help form the pellet quickly.
63. Vertical rotor They also have holes in their body to slide the centrifuge tubes, BUT, they are not at angle. They lie parallel to the rotor shaft. As the rotor accelerates, after centrifugal force comes into play, the solution reorients itself through an angle of 90 degrees. This reorientation makes it lie perpendicular to the axis of rotation
64. Swinging bucket rotors These rotors swing out to a horizontal position when the rotor accelerates. The solution in the tube reorients to lie perpendicular to the axis of rotation and parallel to the applied centrifugal force. Normally used in density gradient centrifugation.
65. Gradient material It should not affect the biological activity of the sample being separated. It should be easily removable from the purified product. It should not absorb in UV radiation at all It should be non-corrosive to the rotor. Should be cheap and readily available. Examples- sucrose, cesium chloride, silica gels, sorbitol, glycerol etc.
66. Preparation of density gradients o DISCONTINOUS OR STEP DENSITY GRADIENT:- o density decreases abruptly from one layer to another. It is prepared by carefully layering the solutions of continuously decreasing densities over each other with the highest density solution being placed at the bottom of the tube. The sample is then layered at the top and spun under specific conditions. They are useful for the separation of whole cells or sub-cellular organelles from tissue homogenates.
67. o CONTINOUS GRADIENT:- the density decreases linearly from the bottom of the centrifuge tube to the meniscus. It is prepared by allowing the discontinuous gradients to stand for a long time. As they merge, they give rise to a linear gradient. o It takes a longer time if viscous solutions are used.
68. Recovery of the sample  First method, involves puncturing the centrifugation tube at the bottom with the help of a needle and collect the dripping medium in separate tubes in fractions of roughly equal volume.  A peristaltic pump might be employed to suck off the medium from the hole at the bottom.
69. pH meter  A pH meter is an electronic device used for measuring the pH (acidity or alkalinity) of a liquid (though special probes are sometimes used to measure the pH of semi-solid substances). A typical pH meter consists of a special measuring probe (a glass electrode) connected to an electronic meter that measures and displays the pH reading.  The probe  The probe is a key part of a pH meter, it is a rod like structure usually made up of glass. At the bottom of the probe there is a bulb, the bulb is a sensitive part of a probe that contains the sensor. Never touch the bulb by hand and clean it with the help of an absorbent tissue paper with very soft hands, being careful not to rub the tissue against the glass bulb in order to avoid creating static. To measure the pH of a solution, the probe is dipped into the solution. The probe is fitted in an arm known as the probe arm.
70.  Electrode method  Glass electrode ,consisting of a bulb of special glass filled with some standard electrolyte, such as 0.1 NHCl in contact with metallic electrode, is immersed in an unknown solution a potential difference develops between the two solutions. The magnitude of which depends upon the hydrogen ion concentration.  Potential difference is measured as with hydrogen electrode by combining glass electrode with standard half cell like saturated calomel electrode and measuring voltage system.
71.  For very precise work the pH meter should be calibrated before each measurement. For normal use calibration should be performed at the beginning of each day. The reason for this is that the glass electrode does not give a reproducible e.m.f. over longer periods of time. Calibration should be performed with at least two standard buffer solutions that span the range of pH values to be measured. For general purposes buffers at pH 4.01 and pH 10.00 are acceptable.  After each single measurement, the probe is rinsed with distilled water or deionized water to remove any traces of the solution being measured, blotted with a scientific wipe to absorb any remaining water which could dilute the sample and thus alter the reading, and then quickly immersed in another solution.
72.  Storage conditions of the glass probes  When not in use, the glass probe tip must be kept wet at all times to avoid the pH sensing membrane dehydration and the subsequent dysfunction of the electrode.  A glass electrode alone without combined reference electrode is typically stored immersed in an acidic solution of around pH 3.0. In an emergency, acidified tap water can be used, but distilled or deionised water must never be used for longer-term probe storage as the relatively ion less water "sucks" ions out of the probe membrane through diffusion, which degrades it.  Combined electrodes (glass membrane + reference electrode) are better stored immersed in the bridge electrolyte (often KCl 3 M) to avoid the diffusion of the electrolyte (KCl) out of the liquid junction.
73.  Cleaning and troubleshooting of the glass probes  Occasionally (about once a month), the probe may be cleaned using pH-electrode cleaning solution; generally a 0.1 M solution of hydrochloric acid (HCl) is used, having a pH of one.  Alternatively a dilute solution of ammonium fluoride (NH4F) can be used. To avoid unexpected problems, the best practice is however to always refer to the electrode manufacturer recommendations or to a classical textbook of analytical chemistry.
74. Thank you
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