The Basic Separation Technique

CENTRIFUGATION

Centrifugation is a basic separation technique. A centrifuge is a device for separating particles in an applied centrifugal field in a solution.

There are two different forces act on an object moving in a circular motion.

Centrifugal force:

Force directed outward from the center. E.g. While turning a bus in twist way, the passengers strike on the bus wall is due to centrifugal force.

Centripetal force:

The force exerted towards the center is now as centripetal force. E.g. the force acts on passengers by the turning car.

Now, suppose a particle is exerted to sediment by centrifugal force, then

The rate or velocity at which it sediments is proportional to the force applied

• Sedimentation is more rapid when the force applied is greater than the gravitational force of the Earth
• Basis of separation is to exert a larger force than does the Earth’s gravitational force.

Basic Principle of Sedimentation

The particles to be separated are suspended in a specific liquid media, held in tubes or bottles which are located in rotor in centrifuge machine, positioned centrally to the drive shaft. These particles are differing in size, shape and density.

As we have already mentioned that,

The rate of sedimentation is dependent upon the applied centrifugal field (G)

G          = W²R …………………………equ (i)

Where

W:        Angular velocity of revolving particle (Remember: one revolution of the rotor is equal to 2 radians)

R:         Radial distance from axis of rotation

In terms of revolution per minute, we have W= 2p rev min^-1/ 60

Therefore:

G          = W²R

           

It is expressed as a multiple of the earth’s gravitational field (g=981 cm s^-2).

Hence

RCF, Relative Centrifugal Field

= G / g

=

RCF      = 1.119 x 10^-5(rev min^-1)² R …………………………….equ (ii)

= x g unit (number times g)

It means, RCF is the ratio of the weight of the particle in the applied centrifugal field to the weight of the same particle when acted by gravity alone. Therefore the rotor speed, radial dimensions and time of the rotor must be quoted during the centrifugation.

However:

This is not the only case in Biochemical experiments as biological samples are always found in dissolved or suspended form in a solution. Thus, the rate of sedimentation not only depends on the centrifugal field but also on

1. Mass of particle

2. Density of particle

3. Density and viscosity of the medium used

4. The extent to which its shape deviates from spherical

Now according to Newton’s Second law of Motion, the centrifugal force (F) exerted on particle is

= M. a

= M. W²R ……………………………….equ (iii)

Where:

M: mass of particle

a: acceleration while in angular motion= W²R

Increasing the sharpness of a turn, w and r decreases. Since r is linear, w has greater effect on the particle.

It causes the molecules to sediment down the centrifuge tube. They start to move downward to sediment; however they encounter opposing force, a frictional resistance in their movement.

Frictional force          = f

                             = 6p. h. R_p. ) ……………………………equ (iv)

Where:

f:          Frictional force

dr/dt: Rate of sedimentation expressed as the change in radius with time (velocity v)

h:         Viscosity coefficient of medium

R_p:        Radius of sedimenting particle

The sedimenting molecule must also displace the solvent into which it sediments and give rise to a buoyant force

Buoyant force            = mass x a

= V. d_m W²R ……………………..equ (v)

Where:

V:         Specific volume of the molecule

d_m:       Density of the medium

While sedimenting, the velocity of the particle increases until it equals the frictional force resisting its motion through the medium. This is an equilibrium state when the particles stop to move or sediment. From equations iii, iv and v.

Centrifugal force       = Frictional force + Buoyant force

M. W²R           = 6p. h. R_p. ) + V. d_m W²R

  pR_p³ d_p W²R             = 6p. h. R_p. ) +   pR_p³ d_m W²R   

  pR_p^3 (d_p - d_m) W²R       = 6p. h. R_p. )

                  =  h R_p^{2  (}d_p - d_m) W²R 

v          =  h R_p^{2  (}d_p - d_m) W²R  ……………………………..equ (vi)

Where:

dr/dt:   v, is the velocity of the sedimenting particle

Mass:   Density x Volume

d_p:        Density of particle

d_m:       Density of medium

From above equation, it seems clear that velocity is proportional to its size, to the differences in density between the particle and medium and to the applied centrifugal field. It is zero when the density of the particle and medium are equal. It decreases when the viscosity of the medium increases.

Since the R_p is in square form, the size of particle has greater influence on velocity.

For a particle, h, R_p^, d_p^, d_m and W all are constants

                        t           =   In

Where

t:          The sedimentation time in seconds

R_t:        Radial distance from the axis of rotation to liquid meniscus

R_b:        Radial distance from the axis of rotation to bottom of tube

It is now clear that a mixture of heterogeneous approximately spherical particles can be separated by centrifugation on the basis of their densities, their sizes and etc.

t           µ          

It means, higher the size particles, faster is the sedimentation (Short time for sedimentation) of it and smaller the size slower is the sedimentation (takes longer time).

CENTRIFUGATION: RCF CALCULATION

The relative centrifugal force (RCF) can be calculated from the following equation:

RCF = (1.119 x 10-5) (rpm)²(r)

Where rpm is the speed of rotation expressed in revolutions per minute and r (radius) is the distance from the axis expressed in cm. The RCF units are "x g" where g represents the force of gravity. RCF can also be determined from the NOMOGRAPH below. Place a straight edge to intersect the radius and the desired RCF to calculate the needed rpm. Alternatively place the straight edge on the radius and the rpm to calculate the g-force. For example, spinning a sample at 2500 rpm in a rotor with a 7.7 cm radius results in a RCF of 550 x g.

 

Figure 1: Nomograph showing relationship between RCF, RPM and Radius
Centrifuges and their uses

1.      Low  Speed Centrifuge

·         Least expensive and simplest in many design

·         Maximum rotor speed of 4000-6000rpm (3000-7000 X g)

a) Small bench centrifuges

·         To collect small amounts of materials (250mm³⁾ that is rapidly sediment (1-2 min)

·         No special cooling system

·         Ambient air flows around the rotor to cool the system

·         Use to rapid sedimentation of blood samples

b) Large capacity refrigerated centrifuges

·         Refrigerated rotor chambers for cooling the sample

·         Large volumes 10, 50 and 100 cm³ processing depending upon the rotors and tubes

·         Maximum capacity of 1.25 dm³

·         Rotors are mounted on a rigid suspension

·         Erythrocytes, coarse or bulky precipitates, yeast cells, nuclei and chloroplasts

2.      Microcentrifuge

·         Maximum rotor speed of 12000rpm with RCF of 10000g

·         Have total capacity of 1.5ml over very short time (0.05-5 min)

·         Use to sediment large particles like cell ppt 

3.      High speed refrigerated centrifuge

·         Maximum rotor speed of 25000rpm with RCF of 60000g

·         Have total capacity of 1.25 dm³

·         Interchangeable fixed angle and swinging buckets rotors

·         Use to collect microorganisms, cellular debris, larger cellular organelles and proteins precipitates by ammonium sulphate

·         Not use for viruses and smaller organelles like ribosome

4.      Continuous flow centrifuge

·         Relatively simple and high speed centrifuge

·         Special design rotor (long and tubular) with non interchangeable system

·         Have total capacity of 1-1.25 dm³/min with continuous flow

·         Particles sediment at wall and excess clarified medium overflows through an outlet port

·         Use to collect bacterial and yeast cells from their mass culture of about 100-500 dm³

5.      Ultracentrifuge

·         Powerful with speed

·         2 types

a) Preparative ultracentrifuge

• Maximum rotor speed of 30000-80000 rpm with RCF of 600000 x g
• Highly sophisticated with refrigerated, sealed and evacuated to minimize excess heat generate
• More sophisticated temperature monitoring system employing an infrared temperature sensor
• Overspeed control system to prevent operation of rotor above its max rated speed
• Vibration minimize system (a flexible drive shaft system) during unequal loading of the centrifuge tubes
• Enclosed in heavy armour plating
• Airfuse for some biochemical applications requiring high centrifugal force
• Use for sediment macromolecule/ligand binding kinetic studies, steroid hormone receptor assays, separation of major lipoprotein from plasma and deproteinisation of physiological fluids for amino acid analysis

b) Analytical ultracentrifuge

• Maximum rotor speed of 70000 rpm with RCF of 500000 x g
• Highly protective chambers with refrigerated and evacuated system also have an optical system to enable the sedimenting material to be observed throughout the process.
• Three types of optical system, a light absorption system, alternative Schlieren system and Rayleigh interferometric system (both measures refractive index of solution)

Classification of Microorganisms

PHYLOGENY: THE STUDY OF EVOLUTIONARY RELATIONSHIPS OF LIVING ORGANISMS

§  Over 1.7 million different organisms have been identified to date.

§  Many similarities among living organisms:

·         Made up of cells surrounded by a plasma membrane.

·         Use ATP as energy source.

·         Store genetic information as DNA.

·         Ribosomes are the site of protein synthesis.

§  Both differences and similarities among organisms are caused by natural selection (Darwin, 1858).

§  Organisms can be classified into taxonomic categories (taxa), based on the differences and similarities among them.

§  Ancient Greeks (on to 1700’s) classified all living organisms into two groups:

1.           1. Kingdom Plantae

2.           2. Kingdom Animalia

§  1735---Linnaeus also used 2 kingdom systems, but established other groups (taxa) and classified most known organisms into all his groups. Microorganisms didn’t get a clear place in this system, but they were fairly new and not well known anyway.

§  In 1850s bacteria and fungi were incorrectly placed in the Plant Kingdom.

§  In 1860s Kingdom Protista was proposed to include bacteria, fungi, algae, and protozoa, but many scientists’ still classified bacteria and fungi as plants.

§  In 1866, Haeckel proposed adding the kingdom Protista for all microorganisms.

Three Kingdom Systems

1.      Plant

2.      Animal

3.      Protista

§  Intense disagreement over classification of bacteria and fungi persisted over 100 years.

§  In 1930s electron microscopy made it clear that bacterial cells lacked a nucleus. The term procaryote was introduced in 1937.

§  In 1959 Kingdom Fungi was established.

§  In 1961 the current definition of the term procaryote was established.

§  In 1968 the Kingdom Procaryotae was accepted by biologists.

§  In 1969 Robert Whitaker proposed a five-kingdom system of biological classification for all living organisms.

Five Kingdom Systems Proposed in 1969 by Robert Whitaker:

1.      Kingdom Procaryotae (Monera): Oldest known cells. Lived over 3.5 billion years ago. Lack a nucleus and membrane bound organelles.

The other four kingdoms are eucaryotes. Have a true nucleus and membrane bound organelles.

2.      Kingdom Protista: Mostly unicellular, lack tissue organization. Most have flagella during life.

3.      Kingdom Fungi: May be unicellular (yeasts) or multicellular (molds). Many are saprotrophs.

4.      Kingdom Plantae: Multicellular, photosynthetic.

5.   Kingdom Animalia: Multicellular, heterotrophs that ingest food through a mouth or oral cavity.

Differences between Eucaryotic and Procaryotic Cells

	Procaryotes	Eucaryotes
Cell size	0.2-2 um in diameter	10-100 um in diameter
True Nucleus	Absent	Present
Membranous Organelles	Absent	Present
Cell Wall	Chemically complex When present	simple
Ribosome	Smaller (70S)	Larger (80S) in cell 70S in organelles
DNA	Single circular	Multiple linear chromosome chromosomes (histones)
Cell Division	Binary fission	Mitosis
Figure	Figure a	Figure b

Figure A: Detail cell structure of Procaryotic cell

Figure B: Detail cell structure of Eucaryotic cell

The Three Domain System:

Domain: In 1978 Carl Woese proposed this level of classification above kingdom.

There are three domains based on the following distinguishing criteria:

·         Cell wall composition

·         Membrane lipids

·         RNA sequence

·         Protein synthesis

·         Antibiotic sensitivity

I. Domain Eubacteria: “True bacteria”.

II. Domain Archaeabacteria: “Ancient bacteria”

III. Domain Eucarya: All eucaryotes: Protista, Fungi, Plantae, and Animalia.

Recent developments in molecular biology and biochemistry have revealed that there are two types of procaryotic cells, based on differences in their ribosomes, cell walls, and metabolism.

1. Eubacteria: “True bacteria”.

·         Cell wall contains peptidoglycan.

·         Sensitive to antibiotics.

2. Archaeabacteria: “Ancient bacteria”

• Differences in structure of membrane lipids
• Differences in RNA
• Cell wall structure (NO peptidoglycan)
• Unusual metabolic processes-----the ability to metabolize unusual substrates and the production of unusual end products
• Ability to thrive in extreme physical conditions

·         The archae are placed on to three groups:

1. Methanogens: Strict anaerobes that produce methane. E.g.     Methanococccus

2. Extreme Halophiles: Require high salt concentrations. E.g. Halobacterium

3. Thermoacidophiles: Live in hot, acidic environments. E.g. Acidocaldarius

3. Eucarya: It contains four kingdoms

• kingdom:  protista---unicellular eukaryocytes. in the future, this kingdom may be split.
• kingdom:  fungi---unicellular yeasts, multicellular molds, macroscopic fungi--these all absorb organic matter through their plasma membranes
• kingdom:  plantae---plants--macroscopic algae, mosses, ferns, conifers, flowering plants--all are multicellular, all carry on photosynthesis
• kingdom:  animalia---animals--sponges, worms, insects, vertebrates--all ingest nutrients 

Figure C: The Three Domain System

The two empire system:

The two-empire system (two-superkingdom system) was the top-level biological classification system in general use before the establishment of the three-domain system. It classified life into Prokaryota and Eukaryota. When the three-domain system was introduced, some biologists preferred the two-superkingdom system, claiming that the three-domain system overemphasized the division between Archaea and Bacteria. However, given the current state of knowledge and the rapid progress in biological scientific advancement, especially due to genetic analyses, that view has all but vanished.

		Two superdomains	Three domains	Six kingdoms
Biota / Vitae life	Acytota / Aphanobionta (Viruses, Viroids, Prions?, ...) non-cellular life
	Cytota cellular life	Prokaryota / Procarya (Monera)	Bacteria	Eubacteria
			Archaea	Archaebacteria
		Eukaryota / Eukarya		Protista
				Fungi
				Plantae
				Animalia

 Figure D: The Two Empire System