Here are the steps to prepare the calibration curve: 1. Stock solution concentration: 100 μg/ml 2. Dilute 1 ml stock solution to 10 ml. This gives 10 μg/ml. Initial volume: 1 ml Final volume: 10 ml Concentration: 10 μg/ml 3. Dilute 2 ml of the above solution to 10 ml. This gives 12 μg/ml Initial volume: 2 ml Final volume: 10 ml Concentration: 12 μg/ml 4. Dilute 3 ml of the above solution to 10 ml. This gives 13.5 μg/ml Initial volume: 3 ml Final volume: 10 ml Concentration: 13.5 μg/

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.

32.  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.

33.  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)

34. 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.

35. 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.

36. 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

37. (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.

38. (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)

40. 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.

41. 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

42. 1.) Barrier Layer cell or photo voltaic cell

43. 2.)Photo tubes or photo emissive cells
photo cathode coated with caesium, potassium, silver oxide which can liberate
electron when light falls on it

44. 3.) Photo multiplier tubes

45. Single beam colorimeter

46. Double beam colorimeter

47. DEVIATION FROM BEER’S LAW
 Instrumental deviation (improper slit width, band
pass,fluctuations)
 Physiochemical changes in solutions.
 Association(Methylene blue) and dissociation(Cr2O7)

48. APPLICATION
 Quality control of purity
 Quantitative analysis
 Determination of molecular weight of amines
 Determination of organic substances and
pharmaceuticals
 Spectrophotometric titrations.

49. 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

50.  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.

51. 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.

53. Types of centrifuges
Desktop centrifuge
High speed centrifuge
Ultracentrifuge

54. 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.

55. 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.

56. 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.

57. Types of centrifugation
Preparative centrifugation
Analytical centrifugation
Differential centrifugation
Density gradient centrifugation
 Rate zonal separation
 Isopycnic separation

58. 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.

60. 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.

61. 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.

62. 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.

64. 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

65. 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.

67. 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

68. 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.

69. 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

70. 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.

71. 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.

72. 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.

73. 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.

74. 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.

75. 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.

76.  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.

77.  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.

78.  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.

79.  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.

80. Thank you