introduction of Pipettes , centrifugation , centifuge. principle of centrifuge and pipettes. different types of centrifugation, centrifuge and pipettes. handling of pipettes and centrifuge, calibration of pipettes and centrifuge.

1. CENTRIFUGATION
AND PIPETTES
Hari Sharan Makaju
M.Sc. Clinical Biochemistry
1st Year

2. INTRODUCTION
Centrifugation
• Centrifugation is the process of using centrifugal force to separate the
lighter portions of a solution, mixture, or suspension from the heavier
portions.
Centrifuge
• Is a device by which centrifugation is effected. Or the instrument used
centrifugation is called centrifuge.
• Centrifugation is a key technique
• For isolating and analyzing cells, sub cellular fractions, supramolecular
complexes and isolated macromolecules such as proteins or nucleic
acids.

3. HISTORY
• In 1864 ,Antonin Prandtl, who developed the first dairy centrifuge
for the purpose of separating cream from milk.
• In 1869, Miescher used a crude centrifuge system to isolate a cell
organelle.
• Svedberg in late 1920s (Nobel prize – 1926) developed first
analytical ultracentrifuge .
• Technical refinement of the preparative centrifugation technique
by Claude and colleagues in 1940s .
3

4. PRINCIPLE
• The centrifuge involves principle of sedimentation, where the
acceleration at centrifugal force causes denser substances to
separate out along the radial direction at the bottom of the
tube.
• By the same concept lighter objects will tend to move to the
top of the tube; in the rotating picture, move to the center.
• In a solution, particles whose density is higher than that of the
solvent sink(sediment), and particles that are lighter than it
float to the top.
• The greater the difference in density, the faster they move
4

5. CALCULATION
• The rate of sedimentation is dependent upon
• the applied centrifugal field (cm s2), G,
• that is determined by the radial distance, r, of the particle from the axis of
rotation (in cm) and
• the square of the angular velocity, ω, of the rotor (in radians per second):
G = ω2r
• The average angular velocity of a rigid body that rotates about a fixed
axis is defined as the ratio of the angular displacement in a given time
interval.
ω= 2π s/60
5

6. • A more common measurement of F, in terms of Gravitational
force g, is Relative Centrifugal Force (RCF), Is given as
• RC F = 1.118 × 10−5 × r × rpm2
Where,
• 1.118 × 10 −5 is an empirical factor
• r = radius in centimeters from the center of rotation to the bottom
of the tube in the rotor cavity or bucket during centrifugation
• rpm = the speed of rotation of the rotor in revolutions per minute
• The RCF of a centrifuge may also be determined from a
nomogram distributed by manufacturers of centrifuges.

7. NOMOGRAPH
• A nomograph is used for the convenient conversion between
relative centrifugal force and speed of the centrifuge at different
radii of the centrifugation spindle to a point along the
centrifuge tube.
• A nomograph consists of three columns representing
• the radial distance (in mm),
• the relative centrifugal field and
• the rotor speed (in r.p.m.).
7

8. 8

9. INSTRUMENTATION
• The basic centrifuge consists of mainly two components,
• Electric motor : with drive shaft to spin the sample
• Rotor : to hold the tubes or other containers of the sample
• also include
• A power switch :
• allows current supply
• Timer:
• allows a rotor to reach a preprogrammed speed under optimized
conditions and then decelerate without braking after the set time
• Speed control :
• by potentiometer, which changes the voltage supplied to the motor
• Tachometer :
• indicates the speed of the rotor (rpm)
• Brake:
• operates by reversing the polarity of the current to the motor
9

10. OTHER COMPONENTS
• Protective shield: to minimize aerosol production if a
tube is broken
• Refrigerator: to reduce the temperature within the
chamber
• Audible or visible alarms: to indicate malfunctions such
as imbalance of the rotor
• Cushioning pads: to lessen the possibility of tube
breakage during centrifugation
• Drive motor
• large centrifuge - heavy duty DC electrical motor
• small centrifuge – AC motors
10

11. TYPES OF CENTRIFUGE
• Depending on the particular application , speed, types of rotor,
different types of centrifuge are available which differ in their
overall design and size.
• The most obvious differences between centrifuges are:
• the maximum speed at which biological specimens are subjected to
increased sedimentation;
• the presence or absence of a vacuum;
• the potential for refrigeration or general manipulation of the temperature
during a centrifugation run;
• the maximum volume of samples and capacity for individual
centrifugation tubes.
11

12. TYPES OF ROTOR
Vertical
tube
rotors
Swinging-
bucket rotors
Fixed
angle
rotors

13. Fixed angle rotors
• Tubes held at a fixed angle – between 14 ̊ and 40 ̊ to the
vertical axis
• Hence, centrifugal field is exerted at an angle
• Particles – move radically outwards, have to travel short
distance i.e. across the column of liquid to the side of
container
• Isopycnic banding may also be routinely performed
• ideal tool for pelleting during the differential separation of
biological particles where sedimentation rates differ
significantly
13

14. Fixed angle rotors

15. SWINGING BUCKET ROTOR
• Sample tubes are loaded into individual buckets that hang
vertically while the rotor is at rest.
• When the rotor begins to rotate the buckets swing out to a
horizontal position. Useful when samples are to be resolved in
density gradients with maximum resolution of banding zones.
• The particles being sedimented must travel the entire length of
the column of liquid to reach the bottom of the tube
• The longer path length permits better separation of individual
particle types from a mixture.
• This rotor is relatively inefficient for pelleting .
15

16. 16
Swinging bucket rotor

17. VERTICAL TUBE ROTORS
• Tube containing the sample solution are placed vertically
parallel to the axis of rotation
• Samples are not separated down the length of the
centrifuge tube but across the diameter of the tube,
separation time is shorter
• Reduced angle results in much shorter run times as
compared to fixed angle rotors
• Types: True vertical rotors and near-vertical rotors
17

18. 18
Vertical tube rotors

19. Fixed Angle Swinging
bucket
Vertical
Path length Short Long Shortest
Separation time Short Long Fastest
Type of separation Pelleting Rate Zonal Isopycnic
Tube position held at an angle to
the axis of rotation
swing out to a
horizontal
position
held parallel
to the
axis of
rotation
19

20. TYPES OF CENTRIFUGE
• Accor. to the speed of the rotor, centrifuge can be classified
into:
a) Low speed centrifuge
b) High speed centrifuge
c) Ultra-centrifuge

21. Low speed Centrifuge
• Can provide centrifugal fields of maximum 10 000 g
• General laboratory centrifuge e.g. bench top centrifuge,
microfuge
• small volume to large volume centrifuge are available.
• Used for routine laboratory process e.g.. Serum/plasma
separation, urine sedimentation
• Two types of rotors are used :
• fixed angle
• swinging bucket
• steel or brass rotors are generally used 21

22. SMALL BENCH TOP CENTRIFUGE
With or without refrigeration
Slow speed (e.g. up to 4000
RPM)
Common in clinical lab
(blood/plasma/serum
separation)
 Can take approx (up to) 100
tubes, depending on diameter
22

23. SMALL MICROFUGE
 work with speed: 8000- 13000 rpm
& RCF10000g
• for rapid sedimentationof small
volumes (1-2 min)
• Eg: Blood , Synaptosomes ( effect
of drugs on biogenic amines)

24. HIGH SPEED CENTRIFUGE
 Maximum speed of 25000rpm,
providing 90000g centrifugal forces.
 Equipped with refrigeration to remove
heat generated.
 Temperature maintained at 0-40C by
means of thermocouple.
 hree types of rotors are available for
high speed centrifugation.
 a. Fixed Angel rotor.
 b. Swinging –bucket rotors
 c. vertical rotor
 Used to collect microorganism, cell
debris, cells, large cellular organelles,
precipitates of chemical reactions.
 Also useful in isolating the sub- cellular
organelles (nuclei, mitochondria,
lysosomes
24
Product
Specifications
Labnet
Maximum
Speed
30,000 rpm
Maximum RCF 65,390 x g
Maximum
Capacity
6 x 250ml
Temperature
Range
-20 to 40°C

25. ULTRACENTRIFUGE
Can be operated at relative centrifugal fields of
up to 900 000 g.
Rotor chamber is sealed and evacuated by pump
to attain vacuum.
Refrigeration system (temp 0-40C).
Rotor chamber is always enclosed in a heavy
armor plate.
Centrifugation for isolation and purification of
components is known as preparatory
centrifugation, while that carried out with a
desire for characterization is known as analytical
centrifugation. 25

26. ULTRACENTRIFUGE
Product
Specifications
Becman coulter
Maximum Speed 100000 rpm
Maximum RCF 80200 x g
26

27. 27

28. TYPES OF CENTRIFUGATION
1) Preparative centrifugation
a) Differential centrifugation
b) Density gradient centrifugation
i. Rate zonal centrifugation
ii. Isopycnic centrifugation
c) Continuous flow centrifugation
2) Analytical centrifugation
28

29. PREPARATIVE CENTRIFUGATION
• This technique is concerned with the actual separation,
isolation, and purification of for example whole cell, sub
cellular organelles, plasma membranes, polysomes ,
ribosomes , chromatin , nucleic acids, lipoprotein and viruses
for subsequent biochemical investigation.
• designed for sample preparation.
• also commonly used for quantitative estimations of
sedimentation coefficients of particles in a density gradient
• Types:
• Differential centrifugation
• Density gradient centrifugation and
• Continuous flow centrifugation
29

30. DIFFERENTIAL CENTRIFUGATION
• based on the differences in the sedimentation rates of
particles in samples of different size in different centrifugal
force
• If a suspension of particles is centrifuged in a tube without a
density gradient, each particle will move toward the bottom
of a tube.
• In this case, the rate of sedimentation ,v, is dependent upon
particle size ‘s’
• since ‘s’ is mostly a function of particle size, the rate of
sedimentation is proportional to particle size.
• two fractions can be obtained from a solution of particles:
• A pellet containing sediment particles
• A supernatant solution
30

31. • Crude tissue homogenates are divided into different fractions by
the stepwise increase of the applied centrifugal field, in a medium
of uniform density
• Under each centrifugal field, particles are sequentially separated
based upon their sedimentation rate
31
Increasing Speed

32. APPLICATION
SUBCELLULAR FRACTIONATION
32

33. • At the end of each stage, the pellet and supernatant are
separated
• Pellet is washed several times by re-suspension in the
homogenization medium followed by re-centrifugation under
the same condition
• This procedure minimizes cross contamination, improves
particle separation and eventually gives a fairly pure
preparation of pellet fraction
33

34. CONT..
• The major problem with differential centrifugation is that to
separate one particle from another effectively, a 10-fold
difference in mass is usually required.
• Thus, this centrifugation is recommended for the separation of
for eg. proteins from large particles such as cells or organelles.
• However, it cannot be used for the isolation of one protein from
another
34

35. DENSITY GRADIENT CENTRIFUGATION
 To further separate biological
particles based on size and density.
 It is the preferred method to purify
sub cellular organelles and
macromolecules.
 can be carried out in a solution of an
inert substance eg. sucrose in which
the concentration increases from the
top to the bottom of the centrifuge
tube, i.e. density increases from top
to bottom.
35

36. CONT..
A mixture of particles to be separated is layered on the top of a
preformed liquid density gradient
various components will separate according to size or densities,
and form bands or zones of particles with similar densities.
the use of such density gradients greatly enhances the resolving
power.
Properties
• Stabilizes the liquid column
• Prevents mixing of separated particles
• Improves the resolution of separated particles
36

37. CRITERIA FOR DENSITY
GRADIENT MATERIAL
• stable, non-toxic, non inflammable and sterile
• Should not absorb light during monitoring
• Have negligible osmotic pressure
• Provide minimum change in pH, isotonic strength and viscosity
• Gradient material used are:
• Sucrose (66%, 50C) Sodium Bromide
• Silica sols Sodium Iodide
• Glycerol Rubidium chloride
• Cesium chloride CsCl Metrizamide
• Sorbitol Cs Acetate
• Polyvinylpyrrolidone
37

38. PREPARATION OF
DENSITY GRADIENT
• Step gradient:
Separation of serum lipoprotein fractions such as VLDL,
LDL and HDL
• Continuous linear gradient:
Separate ribosomal subunits, polyribosomes and viruses
38

39. 39
Step gradient Continuous linear gradient:

40. DENSITY GRADIENT
CENTRIFUGATION
• Types
1. Rate zonal centrifugation
2. Isopycnic centrifugation
40

41. Rate zonal centrifugation
• Take advantage of particle size and
mass instead of particle density for
sedimentation.
• Preformed step gradient is used
• During centrifugation, particles
move through the gradient at their
characteristic sedimentation rates,
forming zones that can be
recovered at the end of the run
41

42. CRITERIA FOR SUCCESSFUL RATE-
ZONAL CENTRIFUGATION:
• Density of sample solution must be less than that of the lowest
density portion of the gradient.
• Density of sample particle must be greater than that of highest
density portion of the gradient.
• Path length of 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.
42

43. Isopycnic centrifugation
• Equilibrium gradient centrifugation
• Particles are separated based on the density
of the molecules
• Molecules move to the position where their
density is same as the gradient material
(Isopycnic position)
• Particle of a particular density will sink during
centrifugation until a position is reaches
where the density of the surrounding
solution is exactly the same as the density of
the particle.
• Self forming gradient is used
• Ex: separation of Nucleic acid in CsCl
(Caseium chloride) gradient 43

45. Rate Zonal Isopycnic
Synonym S-zonal, sedimentation velocity Density equilibrium, sedimentation
equilibrium
Gradient •Shallow,
•Maximum gradient density
less than the least dense
sedimenting specie,
•Gradient continuous.
•Steep,
•Maximum gradient density greater than
that of the most dense sedimenting specie,
•Continuous or discontinuous
gradients.
Centrifuga-
tion
•Incomplete sedimentation,
•Low speed,
•Complete sedimentation till
equilibrium is achieved,
•Short time •High speed,
• Long time.
Separation RNA- DNA hybrids,
ribosomal subunits, etc.,
DNA, plasma lipoproteins,
lysosomes, mitochondria,

47. CONTINUOUS FLOW CENTRIFUGATION
 In this process, large volumes of material can be centrifuged at
high centrifugal forces without having to fill and decant a large
number of centrifuge tubes or frequently stop and start the
rotor.
 The combination of high throughput and high centrifugal force
makes continuous flow processing especially useful for:
• Pelleting of subcellular fractions
• Sedimentation of bacteria
• Large-scale collection of viruses for commercial vaccine
preparation or for research purposes
47

48. ANALYTICAL CENTRIFUGATION
• Has a device by which the sedimentation rate of molecules can be
optically measured during centrifugation and can be used to
obtain data on the sedimentation properties of particles.
• Used for characterization of biological samples rather than
separation
• It operates at a very high speed of 70000rpm
• refrigerated to reduce heat generation and evacuated to reduce
friction
48

49. ANALYTICAL CENTRIFUGATION
• Analytical ultracentrifugation is most often employed in
• the determination of the purity of macromolecules;
• the determination of the relative molecular mass of solutes in
their native state;
• the examination of changes in the molecular mass of
supramolecular complexes;
• the detection of conformational changes; and in
• ligand-binding studies

50. CALIBRATION OF CENTRIFUGE
• This procedure provides accurate rotation speed, timer verification and
centrifuges that are temperature controlled in a laboratory environment.
• Centrifuges used in the laboratory are to be considered as contaminated
and should only be handled with gloves and other personal protective
equipment and/or thoroughly disinfected before calibration verification.
50

51. VERIFICATION OF
ROTATION SPEED:
Place a small section of black and white reflective tape that comes with
the tachometer on the center spindle of the test centrifuge.
In order to measure the rotation speed, there must be a viewing port in the
top cover that will allow the tachometer line of sight to the reflective tape.
Place a normal well-balanced load using specimen covers into the
centrifuge.
Start the centrifuge and allow it come to equilibrium at a normal operating
speed.
Use the tachometer through the viewing port above the reflective tape to
take a rotation rate reading.
Record the rotation speed indicated by the centrifuge either by the dial
setting or by a built in tachometer on the centrifuge . 51

52. VERIFICATION OF TIMER :
Set the centrifuge timer at a setting frequently used in procedures,
and start the stopwatch simultaneously.
Stop the stopwatch at the same time as the centrifuge timer ends.
Calculate the difference between the two times .
52

53. • Verification of rotation speed:
If the difference between the test centrifuge and the certified
tachometer is ± 5% of the procedure speed, then the test
centrifuge rotation calibration is verified as acceptable.
• Verification of timer:
Acceptable difference between the test timer and the certified
timer must be ±2% of the total test timer setting.
53

54. MAINTENANCE OF
CENTRIFUGE
Daily maintenance
 Wipe the inside of the bowl with disinfectant solution and rinse thoroughly.
 The centrifuge must not be used if the interior is hot, if unusual vibrations or
noises occur, or if deterioration (corrosion of parts) is detected.
 A qualified service technician should be contacted.
 Most vibrations are due to improper balancing and can be corrected by
rebalancing the buckets and tubes.
Monthly maintenance
 Clean the centrifuge housing, rotor chamber, rotors and rotor accessories
with a neutral cleaning agent.
 Clean plastic and non-metal parts with a fresh solution of 0.5% sodium
hypochlorite . 54

55. OPERATIONS
Tubes recommended by their manufacturer should be used.
Top of tube should not protrude so far above the bucket.
Properly balanced- weight of racks, tubes, and content on
opposite side of a rotor should not differ by more than 1%.
(Centrifuges auto balance are available).
Cleanliness –minimizing the possible of spread of infection .
Spillage and break of tube should be considered as the
bloodborne pathogen hazard.
Speed of centrifuge should be checked .
Centrifuge timer to be checked per week.

56. APPLICATION
In the clinical laboratory, centrifugation is used to
1. Remove cellular elements from blood to provide cell-free plasma
or serum for analysis .
2. Concentrate cellular elements and other components of biological
fluids for microscopic examination or chemical analysis.
3. Remove chemically precipitated protein from an analytical
specimen.
4. Separate protein-bound or antibody-bound ligand from free ligand
in immunochemical and other assays .
5. Extract solutes in biological fluids from aqueous to organic
solvents.
6. Separate lipid components such as chylomicrons from other
components of plasma or serum, and lipoproteins from one
another . 56

57. SUMMARY
• Centrifugation techniques has wide application so it plays an
important role in separation , preparation and analysis.
• Today, centrifugation techniques represent a critical tool for
modern biochemistry and are employed in almost all invasive
subcellular studies.
• Further development of more sophisticated centrifuge helps in
advanced molecular studies.
57

59. PIPETTES
• Used for the transfer of a volume of liquid from one
container to another.
• Designed either:
• to contain (TC) a specific volume of liquid
• or to deliver (TD) a specified volume.
• Pipettes used in Clinical, molecular diagnostic, and
analytical laboratories includes :
• (1) Manual transfer and measuring pipettes,
• (2) Micropipettes, and
• (3) Electronic and mechanical pipetting devices

60. CLASSIFICATION OF PIPETTES
1. Manual Pipettes
i. Transfer Pipettes (volumetric)
ii. Measuring Pipettes
2. Micropipettes
i. Air displacement
ii. Positive displacement
iii. Dilutor or dispenser
3. Mechanical devices
Sub-classification
a) TC
b) TD
c) TD/Blow out

61. TRANSFER PIPETTE
Transfer pipette
• Designed to transfer a known volume of liquid.
• Include both
• Volumetric and Ostwald-Folin pipettes
Volumetric transfer pipette
• Calibrated to deliver accurately a fixed volume of a dilute aqueous solution.
• The reliability of the calibration of the volumetric pipette decreases with
decreased size, and therefore special micropipettes have been developed.
Ostwald-Folin pipettes
• Similar to volumetric pipettes but have the bulb closer to the delivery tip
• Used for accurate measurement of viscous fluids, such as blood or serum.
• In contrast to a volumetric pipette, an Ostwald-Folin pipette has an etched
ring near the mouthpiece, indicating that it is a blow-out pipette.

62. Volumetric ( transfer ) Ostwald-folin pipettes

63. MEASURING PIPETTES
Measuring Pipettes
• second principal type of pipette
• This is a piece of glass tubing that is drawn out to a tip and graduated
uniformly along its length.
• Two types :
1. Mohr pipette
• is calibrated between two marks on the stem
• Require controlled delivery of the solution between the calibration
marks
2. Serologic pipette
• Has graduated marks down to the tip.
• Must be blown out to deliver the entire volume of the pipette and has
an etched ring (or pair of rings) near the bulb end of the pipette
• Serologic pipettes have a larger orifice than do Mohr pipettes
• thus drain faster.

64. In practice, measuring pipettes are used principally for measurement of
reagents
 and generally are not considered sufficiently accurate for measuring
samples and calibrators.

65. PIPETTING TECHNIQUE
• Pipetting bulb should always be used
• Held vertical
• Read at eye level
• Lowest part of meniscus at line level
• In volumetric pipettes
• The flow of liquid should be unrestricted
• The tips should be touched to the inclined
surface of the receiving
container for 2 seconds after the liquid has
ceased to flow Inclined container and touch tip
• In Serologic pipettes:
• First, the pipette is allowed to drain,
• Then the remaining liquid is blown out.

66. MICROPIPETTES
Micropipettes
• Used for the measurement of microliter volumes.
• Most micropipettes are calibrated to contain (TC) the stated
volume rather than to deliver it (TD).
• Volumes are expressed in microliters ( µL)
• 1-1000µl
• 0.5µl-20ml
• Fixed or adjustable

67. MICROPIPETTES
Advantages
• Adjustable
• Ergonomic design
• Piston driven
• Stability
• Safety
• Ease of use
• Disposable tips
• No washing or drying
• Save time
• Avoid cross contamination
• Improve precision

68. TYPES OF MICROPIPETTES
1. Air displacement pipettes
• Accurate & precise
• Relies on piston for suction creation
• Air cushion between piston and disposable tip
• No contact of piston and sample
• Can be used as
• Forward Pipetting
• Reverse Pipetting

69. Air displacement pipette

70. FORWARD PIPETTING
• Preparation
• Hold in vertical position.
• Depress the plunger to first stop position
• Aspiration
• Immerse tip in the liquid.
• move plunger smoothly to the rest position
• Distribution
• Place tip at an angle (10 to 45°) against the inside wall of the
receiving vessel.
• Depress plunger to the first stop position.
• Purge
• depress the plunger to the second stop position (“blow-out” )
• Remove pipette tip end from sidewall by sliding it up the wall
• Home
• Allow the plunger to move up to the rest position

72. REVERSE PIPETTING
• Preparation
• Hold in vertical position.
• Depress the plunger to second stop position
• Aspiration
• Immerse the pipette tip in the liquid.
• Allow the plunger to move up smoothly to the rest position
• Distribution
• Place tip at an angle (10 to 45°) against the inside wall of the
receiving vessel.
• Depress plunger to the first stop position
• Complete Purge
• Wait one second and purge.
• If the pipette tip is not to be re-used, depress the plunger to
purge position over an appropriate waste container and then
eject the tip.

74. TYPES OF
MICROPIPETTES
Positive displacement
pipettes
• Moving piston in pipet tip
• Carry over concern
• Rinsing and blotting between
samples
• Used to accurately pipette
very viscous, volatile, hot or
cold, or corrosive samples

75. Positive displacement pipettes

77. TYPES OF MICROPIPETTES
• Dispenser ordilutor
• To dispense repeatedly specified volume
• Attach to reagent bottle directly
• Depression of plunger dispense specified
volume
• Error rate 1%
• Precision rate 0.1%
• Useful for serial dispensing

78. MECHANICAL DEVICES
• Semiautomatic and Automatic Pipettes and Dispensers.
• Single well or multiple wells
• Use disposable tips or washing out
• Programmable
• 96 or 384 wells

79. GENERAL GUIDELINES
• Check at the beginning of work, wipe with 70%
ethanol. Set the volume.
• Fluid and pipet tip on same temp. Recommended tip
for pipet.
• High-quality tips of contamination-free polypropylene.
Tip for single use.
• Avoid inverting pipette when liquid in the tip.
• Avoid fingers contamination by using the tip ejector
and gloves. Use Pipet stands
• Check calibration regularly.
• Follow the instructions for recalibration by the
manufacturer.

80. QUALITY CONTROL
• General
• Check accuracy and
precision
• Depend amount of use
• At least once or twice per
year
• Routine maintenance
• Air displacement
– Stroke length
– Air seal
• Positive displacement
– Spring check
– Replacement of Teflon tip

81. QUALITY CONTROL
VALIDATION
Methods
• Gravimetric
• Spectrophotometric

82. GRAVIMETRIC METHOD
 Gravimetric measurement of dispense aliquot
of water with density correction
 Procedure
1. Water, weighing vial &pipet at temp room.
2. Record temp of pure water.
3. Weigh empty stoppered vial. (wv)
4. Dispense sample of water in weighed vial.
5. Re-stoppered, reweigh and record.(wf)

83. • Refer to table “true capacity of glass vessels from the weight of
contained water when weighed in air” to obtain correction
factor(Ft)
• Calculate volume delivered(VD) as
• VD, ml =(wf –wv )xFt
• Example for 10ml pipet:
• wf =31.9961g
• wv =22.391g
• T = 24°C
• Ft = 1.003771
• VD = 31.9961- 22.0391 x 1.003771
= 9.9945mL
• Deviation or error
(10-9.9945)/10 x 100 = 0.055%
• 0.1% error is acceptable.

85. SPECTROPHOTOMETRIC
METHOD
• Alternative to gravimetric
• Use compound that absorb light
• p-nitrophenol

86. Reagents requirement
• Na0H 0.01 mol/L
• p-nitriphenol 105mg/dl
• Preparation of reagent
• Dissolve 105mg of p-nitriphenol in 100mL deionized water
Preparation of dilutions
• Reference dilution
• Fill three 250mL volumetric flasks with 0.01 mol/L NaOH.
• Than add to each 1.0mL p-nitriphenol using different pipet each time.
• Test dilution
• Arrange 5 test tubes, using calibrated pipet add 2.5mL NaOH to each to
each add 10µL p-nitriphenol using test micropipet.
• read absorbance of each reference and test dilution at 401nm in 10mm
cuvet.
Procedure for calibration of 10µL pipet

87. • p-nitriphenol in NaOH give reading 0.550
• Average the readings of three reference dilution (A1) should
be equal to 0.550
• Average the five test dilutions (A2)
• Calculation :
• VD(µl) = A2/A1 x D x V
• Where
• D is dilution of test dilution (1/251 here)
• V is final volume in microliters of test dilution (2510µL)
• So if
• A1 =0.550
• A2 =0.561
• Volume delivered is 10.20µL. Error is 2%.
• Normal capacity is 0.5%-1%.

90. REFERENCES
• Wilson Keith. Walker John. Principles and Techniques of
Biochemistry and Molecular Biology .7th
edition.Cambridge University Press.2010
• Burtis CA, Ashwood ER, Burns DE. Tietz Textbook of Clinial
Chemistry and Molecular Diagnostics. 5th ed. United
Stated of America: Elsevier; 2012.
• http://www.gilson.com/literature/pipetting
• www.thermoscientifi.com/finpipette
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