Difference between revisions of "Centrifugation"

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{{more footnotes|date=November 2010}}
 
{{more footnotes|date=November 2010}}
'''Centrifugation''' is a process that involves the use of the [[centrifugal force]] for the [[separation process|sedimentation of mixture]]s 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 [[gravitation]]al 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 "supernate" or "[[supernatant|supernatant liquid]]". The supernatant liquid is then either quickly [[decantation|decanted]] from the tube without disturbing the precipitate, or withdrawn with a [[Pasteur pipette]]. {{citation needed|date=December 2010}}
+
'''Centrifugation''' is a process that involves the use of the [[centrifugal force]] for the [[separation process|sedimentation of heterogeneous mixtures]] with a [[centrifuge]], used in industry and in laboratory settings. This process is used to separate two immiscible liquids. 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 [[gravitation]]al 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 "supernate" or "[[Precipitation (chemistry)|supernatant liquid]]". The supernatant liquid is then either quickly [[decantation|decanted]] from the tube without disturbing the precipitate, or withdrawn with a [[Pasteur pipette]].{{citation needed|date=December 2010}}
  
The rate of centrifugation is specified by the [[angular velocity]] measured in [[revolutions per minute]] (RPM), or acceleration expressed as [[g-force|''g'']]. The conversion factor between RPM and ''g'' depends on the [[radius]] of the sample in the centrifuge [[wikt:rotor|rotor]]. The particles' [[settling|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]].
+
The rate of centrifugation is specified by the [[angular velocity]] measured in [[revolutions per minute]] (RPM), or acceleration expressed as [[g-force|''g'']]. The conversion factor between RPM and ''g'' depends on the [[radius]] of the sample in the centrifuge [[wikt:rotor|rotor]]. 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]]. The most common application is the separation of solid from highly concentrated suspensions, which is use in the treatment of sewage sludges for dewatering where less consistent sediment is produced.<ref>Article on "Centrifugation" retrieved on 15th October 2013 from http://www.lenntech.com/library/clarification/clarification/centrifugation.htm</ref>
  
 
In the chemical and food industries, special centrifuges can [[continuous process|process a continuous stream]] of particle-laden liquid.
 
In the chemical and food industries, special centrifuges can [[continuous process|process a continuous stream]] of particle-laden liquid.
Line 9: Line 9:
  
 
==Centrifugation in biological research==
 
==Centrifugation in biological research==
 
 
===Microcentrifuges===
 
===Microcentrifuges===
Microcentrifuges are used to process small volumes of biological molecules, [[cell (biology)|cells]], or [[Cell nucleus|nuclei]]. Microcentrifuge tubes generally hold 0.5 - 2 mL of liquid, and are spun at maximum angular speeds of 12,000–13,000 rpm. Microcentrifuges are small enough to fit on a table-top and have rotors that can quickly change speeds. They may or may not have a [[refrigeration]] function.
+
Microcentrifuges are used to process small volumes of biological molecules, [[cell (biology)|cells]], or [[Cell nucleus|nuclei]]. Microcentrifuge tubes generally hold 0.5 - 2.0 mL of liquid, and are spun at maximum angular speeds of 12,000–13,000 rpm. Microcentrifuges are small enough to fit on a table-top and have rotors that can quickly change speeds. They may or may not have a [[refrigeration]] function.
  
 
===High-speed centrifuges===
 
===High-speed centrifuges===
High-speed or superspeed centrifuges can handle larger sample volumes, from a few tens of millilitres to several litres. Additionally, larger centrifuges can also reach higher angular velocities (around 30000 rpm). The rotors may come with different adapters to hold various sizes of [[test tubes]], bottles, or [[microtiter plate]]s.
+
High-speed or superspeed centrifuges can handle larger sample volumes, from a few tens of millilitres to several litres. Additionally, larger centrifuges can also reach higher angular velocities (around 30,000 rpm). The rotors may come with different adapters to hold various sizes of [[test tubes]], bottles, or [[microtiter plate]]s.
  
===Fractionation Process===
+
===Fractionation process===
Method of fractionation procedure:
+
General method of fractionation:
 
Cell sample is stored in a suspension which is:       
 
Cell sample is stored in a suspension which is:       
# Buffered - neutral pH, preventing damage to the structure of proteins including enzymes (which could affect ionic bonds)
+
#Buffered - neutral pH, preventing damage to the structure of proteins including enzymes (which could affect ionic bonds)
# Isotonic (of equal water potential) - this prevents water gain or loss by the organelles
+
#Isotonic (of equal water potential) - this prevents water gain or loss by the organelles
# Cool - reducing the overall activity of enzyme released later in the procedure
+
#Cool - reducing the overall activity of enzyme released later in the procedure
* Cells are homogenised in a blender and filtered to remove debris
+
*Cells are homogenised in a blender and filtered to remove debris
* The homogenised sample is placed in an ultracentrifuge and spun in low speed - nuclei settle out, forming a pellet
+
*The homogenised sample is placed in an ultracentrifuge and spun in low speed - nuclei settle out, forming a pellet
* The supernatant (suspension containing remaining organelles) is spun at a higher speed - chloroplasts settle out
+
*The supernatant (suspension containing remaining organelles) is spun at a higher speed - chloroplasts settle out
* The supernatant is spun at a higher speed still - mitochondria and lysosomes settle out
+
*The supernatant is spun at a higher speed still - mitochondria and lysosomes settle out
* The supernatant is spun at an even higher speed - ribosomes, membranes settle out
+
*The supernatant is spun at an even higher speed - ribosomes, membranes settle out
  
 
The ribosomes, membranes and Golgi complexes can be separated by another technique called density gradient centrifugation.
 
The ribosomes, membranes and Golgi complexes can be separated by another technique called density gradient centrifugation.
Line 32: Line 31:
 
===Ultracentrifuges===
 
===Ultracentrifuges===
 
{{main|Differential centrifugation|Isopycnic centrifugation|ultracentrifugation}}
 
{{main|Differential centrifugation|Isopycnic centrifugation|ultracentrifugation}}
[[Ultracentrifugation]] makes use of high centrifugal force for studying properties of biological particles. Compared to microcentrifuges or high-speed centrifuges, ultracentrifuges can isolate much smaller particles, including ribosomes, proteins, and viruses. Ultracentrifuges can also be used in the study of membrane fractionation. This occurs because ultracentrifuges can reach maximum angular velocities in excess of 70000 rpm. Additionally, while microcentrifuges and supercentrifuges separate particles in batches (limited volumes of samples must be handled manually in test tubes or bottles), ultracentrifuges can separate molecules in batch or continuous flow systems.
+
[[Ultracentrifugation]] makes use of high centrifugal force for studying properties of biological particles. Compared to microcentrifuges or high-speed centrifuges, ultracentrifuges can isolate much smaller particles, including ribosomes, proteins, and viruses. Ultracentrifuges can also be used in the study of membrane fractionation. This occurs because ultracentrifuges can reach maximum angular velocities in excess of 70,000 rpm. Additionally, while microcentrifuges and supercentrifuges separate particles in batches (limited volumes of samples must be handled manually in test tubes or bottles), ultracentrifuges can separate molecules in batch or continuous flow systems.
  
 
In addition to purification, analytical ultracentrifugation (AUC) can be used for determination of the properties of macromolecules such as shape, mass, composition, and conformation. Samples are centrifuged with a high-density solution such as [[sucrose]], [[caesium chloride]], or [[iodixanol]]. The high-density solution may be at a uniform concentration throughout the test tube ("cushion") or a varying concentration ("[[gradient]]"). Molecular properties can be modeled through [[sedimentation]] velocity analysis or sedimentation equilibrium analysis. During the run, the particles or molecules will migrate through the test tube at different speeds depending on their physical properties and the properties of the solution, and eventually form a pellet at the bottom of the tube, or bands at various heights.
 
In addition to purification, analytical ultracentrifugation (AUC) can be used for determination of the properties of macromolecules such as shape, mass, composition, and conformation. Samples are centrifuged with a high-density solution such as [[sucrose]], [[caesium chloride]], or [[iodixanol]]. The high-density solution may be at a uniform concentration throughout the test tube ("cushion") or a varying concentration ("[[gradient]]"). Molecular properties can be modeled through [[sedimentation]] velocity analysis or sedimentation equilibrium analysis. During the run, the particles or molecules will migrate through the test tube at different speeds depending on their physical properties and the properties of the solution, and eventually form a pellet at the bottom of the tube, or bands at various heights.
 
==Centrifugation analysis==
 
 
===Lamm equation===
 
Particle dispersion and sedimentation can be analyzed using the [[Lamm equation]]. The calculation of the sedimentation coefficient and diffusion coefficient is useful for determining the physical properties of the molecule, including shape and conformational changes. However, the Lamm equation is most ideal for modeling concentrations of ideal, non-interacting solutes. Chemical reactions are unaccounted for by this equation. Additionally, for large molecular weight particles, sedimentation is not always smooth. This may lead to the overestimation of the diffusion coefficient, or oscillation effects at the bottom of a solution cell.
 
 
===Sigma analysis===
 
[[Sigma analysis]] is a useful tool determining centrifuge properties. It is similar to the continuity equation that relates volumetric flow rate Q, fluid velocity u, and flow path cross-sectional Area A:
 
 
<math>Q = uA</math>
 
 
In the case of sigma analysis, u is replaced by v<sub>g</sub>,the settling velocity at centripetal acceleration of g (9.81&nbsp;m/s<sup>2</sup>), Σ replaces area, and is a property of the type of centrifuge, and Q is the input fluid flow rate. Σ has the same units as area.
 
 
<math>Q =2 v_g\Sigma</math>
 
  
 
==Other applications==
 
==Other applications==
*Remove fat from milk to produce skimmed milk
+
*Removing fat from milk to produce skimmed milk
*Separating [[textile]].
+
*Separating [[textile]]s
 
*Removing water from lettuce after washing it in a [[salad spinner]]
 
*Removing water from lettuce after washing it in a [[salad spinner]]
*Separating particles from an air-flow using [[cyclonic separation]].
+
*Separating particles from an air-flow using [[cyclonic separation]]
 
*The [[clarification and stabilization of wine]]
 
*The [[clarification and stabilization of wine]]
*It is used in washing machines to separate water particles from clothes while drying in washing machines
+
*Separation of water particles from clothes while spin-drying in washing machines
 +
*Separation of urine components and blood components in forensic and research labs
  
 
==History==
 
==History==
By 1923 [[Theodor Svedberg]] and his student H. Rinde had successfully analyzed large grained sols terms of their gravitational sedimentation.<ref name="VanHolde">Van Holde, K. E. (1998). Analytical ultracentrifugation from 1924 to the present: A remarkable history. Chemtracts – Biochemistry and Molecular Biology. 11:933-943</ref> Sols consist of a substance evenly distributed in another substance, also known as a [[Colloid]].<ref name="Svedberg">Svedberg, T. (1927). The Ultracentrifuge Nobel Lecture</ref> However, smaller grained sols, such as those containing gold, could not be analyzed.<ref name="VanHolde"/> To investigate this problem Svedberg developed an analytical centrifuge, equipped with a photographic absorption system, which would exert a much greater centrifugal effect.<ref name="VanHolde"/> In addition, he developed the theory necessary to measure molecular weight.<ref name="Svedberg"/> During this time, Svedberg’s attention shifted from gold to proteins.<ref name="VanHolde"/>  
+
By 1923 [[Theodor Svedberg]] and his student H. Rinde had successfully analyzed large-grained sols in terms of their gravitational sedimentation.<ref name="VanHolde">Van Holde, K. E. (1998). Analytical ultracentrifugation from 1924 to the present: A remarkable history. Chemtracts – Biochemistry and Molecular Biology. 11:933-943</ref> Sols consist of a substance evenly distributed in another substance, also known as a [[colloid]].<ref name="Svedberg">Svedberg, T. (1927). The Ultracentrifuge Nobel Lecture</ref> However, smaller grained sols, such as those containing gold, could not be analyzed.<ref name="VanHolde"/> To investigate this problem Svedberg developed an analytical centrifuge, equipped with a photographic absorption system, which would exert a much greater centrifugal effect.<ref name="VanHolde"/> In addition, he developed the theory necessary to measure molecular weight.<ref name="Svedberg"/> During this time, Svedberg’s attention shifted from gold to proteins.<ref name="VanHolde"/>
  
By 1900, it was generally accepted that proteins were composed of amino acids; however, whether proteins were colloids or [[macromolecules]] was still under debate.<ref name="Tanford">Tanford, C., and Reynolds, J. 2001. Nature’s robots: A history of proteins. Oxford University Press. pp. 303-305</ref> One protein being investigated at the time was [[Hemoglobin]]. It was determined to have 712 carbon, 1,130 hydrogen, 243 oxygen, two sulfur atoms, and at least one iron atom. This gave hemoglobin a resulting weight of approximately 16,000 Da but it was uncertain whether this value was a multiple of one or four (dependent upon the number of iron atoms present).<ref name="Simoni">Simoni, D. S., Hill, R. L., and Vaughan, M. (2002). The structure and function of hemoglobin: Gilbery Smithson Adair and the Adair equations. The Journal of Biological Chemistry. 277(31): e1-e2</ref> In regards to Svedberg’s studies, hemoglobin was the main protein of interest. 
+
By 1900, it was generally accepted that proteins were composed of amino acids; however, whether proteins were colloids or [[macromolecule]]s was still under debate.<ref name="Tanford">Tanford, C., and Reynolds, J. 2001. Nature’s robots: A history of proteins. Oxford University Press. pp. 303-305</ref> One protein being investigated at the time was [[hemoglobin]]. It was determined to have 712 carbon, 1,130 hydrogen, 243 oxygen, two sulfur atoms, and at least one iron atom. This gave hemoglobin a resulting weight of approximately 16,000 Da but it was uncertain whether this value was a multiple of one or four (dependent upon the number of iron atoms present).<ref name="Simoni">Simoni, D. S., Hill, R. L., and Vaughan, M. (2002). The structure and function of hemoglobin: Gilbery Smithson Adair and the Adair equations. The Journal of Biological Chemistry. 277(31): e1-e2</ref>
  
Through a series of experiments, utilizing the [[sedimentation equilibrium]] technique, two important observations were made: hemoglobin has a molecular weight of 68,000 Da, suggesting that there are four iron atoms present rather than one, and that no matter where the hemoglobin was isolated from, it had exactly the same molecular weight.<ref name="VanHolde"/><ref name="Svedberg"/> How something of such a large molecular mass could be consistently found, regardless of where it was sampled from in the body, was unprecedented and favored the idea the proteins are macromolecules rather than colloids.<ref name="Tanford"/> In order to investigate this phenomenon, a centrifuge with even higher speeds was needed, and thus the [[ultracentrifuge]] was created to apply the theory of sedimentation-diffusion.<ref name="VanHolde"/> The same molecular mass was determined, and the presence of a spreading boundary suggested that it was a single compact particle.<ref name="VanHolde"/> Further application of centrifugation showed that under different conditions the large homogenous particles could be broken down into discrete subunits.<ref name="VanHolde"/> The development of centrifugation was without a doubt an important turning point in protein science.
+
Through a series of experiments utilizing the [[sedimentation equilibrium]] technique, two important observations were made: hemoglobin has a molecular weight of 68,000 Da, suggesting that there are four iron atoms present rather than one, and that no matter where the hemoglobin was isolated from, it had exactly the same molecular weight.<ref name="VanHolde"/><ref name="Svedberg"/> How something of such a large molecular mass could be consistently found, regardless of where it was sampled from in the body, was unprecedented and favored the idea the proteins are macromolecules rather than colloids.<ref name="Tanford"/> In order to investigate this phenomenon, a centrifuge with even higher speeds was needed, and thus the [[ultracentrifuge]] was created to apply the theory of sedimentation-diffusion.<ref name="VanHolde"/> The same molecular mass was determined, and the presence of a spreading boundary suggested that it was a single compact particle.<ref name="VanHolde"/> Further application of centrifugation showed that under different conditions the large homogeneous particles could be broken down into discrete subunits.<ref name="VanHolde"/> The development of centrifugation was a great advance in experimental protein science.
  
 
==Sources==
 
==Sources==
Line 73: Line 59:
 
*Howlett, G.J., Minton, A.P., Rivas, G. ''Analytical Ultracentrifugation for the Study of Protein Association and Assembly''. Current Opinion in Chemical Biology, Vol. 10, 2006. pp.&nbsp;430–436.
 
*Howlett, G.J., Minton, A.P., Rivas, G. ''Analytical Ultracentrifugation for the Study of Protein Association and Assembly''. Current Opinion in Chemical Biology, Vol. 10, 2006. pp.&nbsp;430–436.
 
*Dam, J., Velikovsky, C.A., Mariuzza R.A., et al. ''Sedimentation Velocity Analysis of Heterogeneous Protein-Protein Interactions: Lamm Equation Modeling and Sedimentation Coefficient Distributions c(s)''. Biophysical Journal, Vol. 89, 2005. pp.&nbsp;619–634.
 
*Dam, J., Velikovsky, C.A., Mariuzza R.A., et al. ''Sedimentation Velocity Analysis of Heterogeneous Protein-Protein Interactions: Lamm Equation Modeling and Sedimentation Coefficient Distributions c(s)''. Biophysical Journal, Vol. 89, 2005. pp.&nbsp;619–634.
*Berkowitz, S.A., Philo, J.S. ''Monitoring the Homogeneity of Adenovirus Preparations (a Gene Therapy Delivery System) Using Analytical Ultracentrifugation''. Analytical Biochemistry, Vol. 362, 2007. pp.&nbsp;16–37.
+
*Berkowitz, S.A., Philo, J.S. ''Monitoring the Homogeneity of Adenovirus Preparations (a Gene Therapy Delivery System) Using Analytical Ultracentrifugation''. Analytical Biochemistry, Vol. 362, 2007. pp.&nbsp;16–37.
{{refend}}.
+
{{refend}}
  
 
==References==
 
==References==
Line 80: Line 66:
  
 
[[Category:Centrifugation]]
 
[[Category:Centrifugation]]
 
[[ar:تثفيل]]
 
[[ca:Centrifugació]]
 
[[cs:Centrifugace]]
 
[[et:Tsentrifuugimine]]
 
[[el:Φυγοκέντρηση]]
 
[[es:Centrifugación]]
 
[[fr:Centrifugation]]
 
[[id:Sentrifugasi]]
 
[[kk:Центрифугтау]]
 
[[ku:Perewnawîtî]]
 
[[ml:അപകേന്ദ്രണം]]
 
[[ja:遠心分離]]
 
[[om:Centrifugation]]
 
[[pl:Wirowanie frakcjonujące]]
 
[[pt:Centrifugação]]
 
[[ru:Центрифугирование]]
 
[[sl:Centrifugiranje]]
 
[[sr:Centrifuga]]
 
[[fi:Sentrifugointi]]
 
[[sv:Centrifugering]]
 
[[uk:Центрифугування]]
 

Latest revision as of 18:18, 21 January 2014

Template:More footnotes Centrifugation is a process that involves the use of the centrifugal force for the sedimentation of heterogeneous mixtures with a centrifuge, used in industry and in laboratory settings. This process is used to separate two immiscible liquids. 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 "supernate" or "supernatant liquid". The supernatant liquid is then either quickly decanted from the tube without disturbing the precipitate, or withdrawn with a Pasteur pipette.{{ safesubst:#invoke:Unsubst||date=__DATE__ |$B= {{#invoke:Category handler|main}}{{#invoke:Category handler|main}}[citation needed] }}

The rate of centrifugation is specified by the angular velocity measured in revolutions per minute (RPM), or acceleration expressed as g. The conversion factor between RPM and g depends on the radius of the sample in the centrifuge rotor. 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. The most common application is the separation of solid from highly concentrated suspensions, which is use in the treatment of sewage sludges for dewatering where less consistent sediment is produced.[1]

In the chemical and food industries, special centrifuges can process a continuous stream of particle-laden liquid.

Centrifugation is the most common method used for uranium enrichment, relying on the slight mass difference between atoms of U238 and U235 in uranium hexafluoride gas.{{ safesubst:#invoke:Unsubst||date=__DATE__ |$B= {{#invoke:Category handler|main}}{{#invoke:Category handler|main}}[citation needed] }}

Centrifugation in biological research

Microcentrifuges

Microcentrifuges are used to process small volumes of biological molecules, cells, or nuclei. Microcentrifuge tubes generally hold 0.5 - 2.0 mL of liquid, and are spun at maximum angular speeds of 12,000–13,000 rpm. Microcentrifuges are small enough to fit on a table-top and have rotors that can quickly change speeds. They may or may not have a refrigeration function.

High-speed centrifuges

High-speed or superspeed centrifuges can handle larger sample volumes, from a few tens of millilitres to several litres. Additionally, larger centrifuges can also reach higher angular velocities (around 30,000 rpm). The rotors may come with different adapters to hold various sizes of test tubes, bottles, or microtiter plates.

Fractionation process

General method of fractionation: Cell sample is stored in a suspension which is:

  1. Buffered - neutral pH, preventing damage to the structure of proteins including enzymes (which could affect ionic bonds)
  2. Isotonic (of equal water potential) - this prevents water gain or loss by the organelles
  3. Cool - reducing the overall activity of enzyme released later in the procedure
  • Cells are homogenised in a blender and filtered to remove debris
  • The homogenised sample is placed in an ultracentrifuge and spun in low speed - nuclei settle out, forming a pellet
  • The supernatant (suspension containing remaining organelles) is spun at a higher speed - chloroplasts settle out
  • The supernatant is spun at a higher speed still - mitochondria and lysosomes settle out
  • The supernatant is spun at an even higher speed - ribosomes, membranes settle out

The ribosomes, membranes and Golgi complexes can be separated by another technique called density gradient centrifugation.

Ultracentrifuges

{{#invoke:main|main}} Ultracentrifugation makes use of high centrifugal force for studying properties of biological particles. Compared to microcentrifuges or high-speed centrifuges, ultracentrifuges can isolate much smaller particles, including ribosomes, proteins, and viruses. Ultracentrifuges can also be used in the study of membrane fractionation. This occurs because ultracentrifuges can reach maximum angular velocities in excess of 70,000 rpm. Additionally, while microcentrifuges and supercentrifuges separate particles in batches (limited volumes of samples must be handled manually in test tubes or bottles), ultracentrifuges can separate molecules in batch or continuous flow systems.

In addition to purification, analytical ultracentrifugation (AUC) can be used for determination of the properties of macromolecules such as shape, mass, composition, and conformation. Samples are centrifuged with a high-density solution such as sucrose, caesium chloride, or iodixanol. The high-density solution may be at a uniform concentration throughout the test tube ("cushion") or a varying concentration ("gradient"). Molecular properties can be modeled through sedimentation velocity analysis or sedimentation equilibrium analysis. During the run, the particles or molecules will migrate through the test tube at different speeds depending on their physical properties and the properties of the solution, and eventually form a pellet at the bottom of the tube, or bands at various heights.

Other applications

  • Removing fat from milk to produce skimmed milk
  • Separating textiles
  • Removing water from lettuce after washing it in a salad spinner
  • Separating particles from an air-flow using cyclonic separation
  • The clarification and stabilization of wine
  • Separation of water particles from clothes while spin-drying in washing machines
  • Separation of urine components and blood components in forensic and research labs

History

By 1923 Theodor Svedberg and his student H. Rinde had successfully analyzed large-grained sols in terms of their gravitational sedimentation.[2] Sols consist of a substance evenly distributed in another substance, also known as a colloid.[3] However, smaller grained sols, such as those containing gold, could not be analyzed.[2] To investigate this problem Svedberg developed an analytical centrifuge, equipped with a photographic absorption system, which would exert a much greater centrifugal effect.[2] In addition, he developed the theory necessary to measure molecular weight.[3] During this time, Svedberg’s attention shifted from gold to proteins.[2]

By 1900, it was generally accepted that proteins were composed of amino acids; however, whether proteins were colloids or macromolecules was still under debate.[4] One protein being investigated at the time was hemoglobin. It was determined to have 712 carbon, 1,130 hydrogen, 243 oxygen, two sulfur atoms, and at least one iron atom. This gave hemoglobin a resulting weight of approximately 16,000 Da but it was uncertain whether this value was a multiple of one or four (dependent upon the number of iron atoms present).[5]

Through a series of experiments utilizing the sedimentation equilibrium technique, two important observations were made: hemoglobin has a molecular weight of 68,000 Da, suggesting that there are four iron atoms present rather than one, and that no matter where the hemoglobin was isolated from, it had exactly the same molecular weight.[2][3] How something of such a large molecular mass could be consistently found, regardless of where it was sampled from in the body, was unprecedented and favored the idea the proteins are macromolecules rather than colloids.[4] In order to investigate this phenomenon, a centrifuge with even higher speeds was needed, and thus the ultracentrifuge was created to apply the theory of sedimentation-diffusion.[2] The same molecular mass was determined, and the presence of a spreading boundary suggested that it was a single compact particle.[2] Further application of centrifugation showed that under different conditions the large homogeneous particles could be broken down into discrete subunits.[2] The development of centrifugation was a great advance in experimental protein science.

Sources

Template:Refbegin

  • Harrison, Roger G., Todd, Paul, Rudge, Scott R., Petrides D.P. Bioseparations Science and Engineering. Oxford University Press, 2003.
  • Dishon, M., Weiss, G.H., Yphantis, D.A. Numerical Solutions of the Lamm Equation. I. Numerical Procedure. Biopolymers, Vol. 4, 1966. pp. 449–455.
  • Cao, W., Demeler B. Modeling Analytical Ultracentrifugation Experiments with an Adaptive Space-Time Finite Element Solution for Multicomponent Reacting Systems. Biophysical Journal, Vol. 95, 2008. pp. 54–65.
  • Cole, J.L., Hansen, J.C. Analytical Ultracentrifugation as a Contemporary Biomolecular Research Tool. Methods and Reviews, 1999/2000.
  • Howlett, G.J., Minton, A.P., Rivas, G. Analytical Ultracentrifugation for the Study of Protein Association and Assembly. Current Opinion in Chemical Biology, Vol. 10, 2006. pp. 430–436.
  • Dam, J., Velikovsky, C.A., Mariuzza R.A., et al. Sedimentation Velocity Analysis of Heterogeneous Protein-Protein Interactions: Lamm Equation Modeling and Sedimentation Coefficient Distributions c(s). Biophysical Journal, Vol. 89, 2005. pp. 619–634.
  • Berkowitz, S.A., Philo, J.S. Monitoring the Homogeneity of Adenovirus Preparations (a Gene Therapy Delivery System) Using Analytical Ultracentrifugation. Analytical Biochemistry, Vol. 362, 2007. pp. 16–37.

Template:Refend

References

  1. Article on "Centrifugation" retrieved on 15th October 2013 from http://www.lenntech.com/library/clarification/clarification/centrifugation.htm
  2. 2.0 2.1 2.2 2.3 2.4 2.5 2.6 2.7 Van Holde, K. E. (1998). Analytical ultracentrifugation from 1924 to the present: A remarkable history. Chemtracts – Biochemistry and Molecular Biology. 11:933-943
  3. 3.0 3.1 3.2 Svedberg, T. (1927). The Ultracentrifuge Nobel Lecture
  4. 4.0 4.1 Tanford, C., and Reynolds, J. 2001. Nature’s robots: A history of proteins. Oxford University Press. pp. 303-305
  5. Simoni, D. S., Hill, R. L., and Vaughan, M. (2002). The structure and function of hemoglobin: Gilbery Smithson Adair and the Adair equations. The Journal of Biological Chemistry. 277(31): e1-e2