Editing Centrifugation (section)

==Centrifugation in biological research==
===Microcentrifuges===
Microcentrifuges are specially designed table-top models with light, small-volume rotors capable of very fast acceleration up to approximately 17,000 rpm. They are lightweight devices which are primarily used for short-time centrifugation of samples up to around 0.2–2.0 mL. However, due to their small scale, they are readily transportable and, if necessary, can be operated in a cold room.<ref name="Graham"/> They can be refrigerated or not. The microcentrifuge is normally used in research laboratories where small samples of biological molecules, [[cell (biology)|cells]], or [[Cell nucleus|nuclei]] are required to be subjected to high RCF for relatively short time intervals.<ref name="Graham">{{Cite book |publisher = BIOS Scientific Publishers |isbn = 978-1-85996-037-0 |last1 = Graham |first1 = J.M. |last2 = Rickwood |first2 = D. |title = Biological Centrifugation |date = 2001}}</ref> Microcentrifuges designed for high-speed operation can reach up to 35,000 rpm, giving RCF up to 30000×g, and are called high-speed microcentrifuges.<ref name="Khandpur">{{Cite book| publisher = John Wiley & Sons| isbn = 978-1-119-28812-1| last = Khandpur| first = Raghbir Singh| title = Compendium of Biomedical Instrumentation, 3 Volume Set| date = 2020-02-25}}</ref>

===Low-speed centrifuges===
Low-speed centrifuges are used to harvest chemical precipitates, intact cells (animal, plant and some microorganisms), nuclei, chloroplasts, large mitochondria and the larger plasma-membrane fragments. Density gradients for purifying cells are also run in these centrifuges. Swinging-bucket rotors tend to be used very widely because of the huge flexibility of sample size through the use of adaptors.<ref name="Graham"/> These machines have maximum rotor speeds of less than 10 000 rpm and vary from small, bench-top to large, floor-standing centrifuges.<ref>{{cite book |last1=Ford |first1=T. C. |last2=Graham |first2=John M. |title=An Introduction to Centrifugation |year=1991 |publisher=Bios |isbn=978-1-872748-40-5 |language=en}}</ref>

===High-speed centrifuges===
High-speed centrifuges are typically used to harvest microorganisms, [[virus]]es, [[mitochondria]], [[lysosomes]], [[peroxisomes]] and intact tubular Golgi membranes. The majority of the simple pelleting tasks are carried out in fixed angle rotors. Some density-gradient work for purifying cells and organelles can be carried out in swinging-bucket rotors, or in the case of [[Percoll]] gradients in fixed-angle rotors.<ref name="Graham"/> 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.

===Ultracentrifugations===
[[Ultracentrifugation]] makes use of high centrifugal force for studying properties of biological particles at exceptionally high speeds. Current [[ultracentrifuge]]s can spin to as much as 150,000 rpm (equivalent to 1,000,000 x g).<ref>{{cite web |last1=Mendes |first1=Adélia |title=Ultracentrifugation basics and applications |url=https://conductscience.com/ultracentrifugation-basics-and-applications/ |website=Conduct Science |date=2 March 2020}}</ref> They are used to harvest all membrane vesicles derived from the plasma membrane, [[endoplasmic reticulum]] (ER) and [[Golgi membrane]], [[endosomes]], [[ribosomes]], ribosomal subunits, [[plasmids]], [[DNA]], [[RNA]] and proteins in fixed-angle rotors.<ref name="Graham"/> Compared to microcentrifuges or high-speed centrifuges, ultracentrifuges can isolate much smaller particles and, additionally, whilst 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.{{cn|date=June 2024}}

Ultracentrifugation is employed for separation of macromolecules/ligand binding kinetic studies, separation of various lipoprotein fractions from plasma and deprotonisation of physiological fluids for amino acid analysis.<ref name="Fischer">{{cite web|title=Centrifugation Theory|url=https://www.fishersci.se/se/en/scientific-products/centrifuge-guide/centrifugation-theory.html|website=Fischer Scientific|publisher=Thermo Fisher Scientific|access-date=9 March 2018|archive-url=https://web.archive.org/web/20190820084129/https://www.fishersci.se/se/en/scientific-products/centrifuge-guide/centrifugation-theory.html|archive-date=20 August 2019|url-status=dead}}</ref>

They are the most commonly used centrifuge for the density-gradient purification of all particles except cells, and, whilst swinging buckets have been traditionally used for this purpose, fixed-angle rotors and vertical rotors are also used, particularly for self-generated gradients and can improve the efficiency of separation greatly. There are two kinds of ultracentrifuges: the analytical and the preparative.

====Analytical ultracentrifugation====
Analytical ultracentrifugation (AUC) can be used for determination of the properties of macromolecules such as shape, mass, composition, and conformation. It is a commonly used biomolecular analysis technique used to evaluate sample purity, to characterize the assembly and disassembly mechanisms of [[biomolecular complex]]es, to determine subunit [[stoichiometries]], to identify and characterize macromolecular conformational changes, and to calculate equilibrium constants and thermodynamic parameters for self-associating and hetero-associating systems.<ref>{{cite journal |last1=Cole |first1=JL |last2=Hansen |first2=JC |title=Analytical ultracentrifugation as a contemporary biomolecular research tool. |journal=Journal of Biomolecular Techniques |date=December 1999 |volume=10 |issue=4 |pages=163–76 |pmid=19499023 |issn=1524-0215 |pmc=2291609}}</ref> Analytical ultracentrifuges incorporate a scanning [[Visible light|visible]]/[[ultraviolet light]]-based optical detection system for real-time monitoring of the sample’s progress during a spin.<ref name="compare">{{cite web |title=Analytical and Preparative Ultracentrifuges |url=https://www.labcompare.com/10-Featured-Articles/135690-Analytical-and-Preparative-Ultracentrifuges/ |website=www.labcompare.com |language=en}}</ref>

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 particle 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.

====Preparative ultracentrifugation====
Preparative ultracentrifuges are often used for separating particles according to their densities, isolating and/or harvesting denser particles for collection in the pellet, and clarifying suspensions containing particles. Sometimes researchers also use preparative ultracentrifuges if they need the flexibility to change the type of rotor in the instrument. Preparative ultracentrifuges can be equipped with a wide range of different rotor types, which can spin samples of different numbers, at different angles, and at different speeds.<ref name="compare"/>

===Fractionation process===
In biological research, [[cell fractionation]] typically includes the isolation of cellular components while retaining the individual roles of each component. Generally, the 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)
*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

Centrifugation is the first step in most [[fractionation]]s. Through low-speed centrifugation, cell debris may be removed, leaving a supernatant preserving the contents of the cell. Repeated centrifugation at progressively higher speeds will fractionate homogenates of cells into their components. In general, the smaller the subcellular component, the greater is the centrifugal force required to sediment it.<ref>{{cite book |last1=Alberts |first1=Bruce |last2=Johnson |first2=Alexander |last3=Lewis |first3=Julian |last4=Raff |first4=Martin |last5=Roberts |first5=Keith |last6=Walter |first6=Peter |title=Molecular biology of the cell |date=2002 |publisher=Garland Science |location=New York |isbn=0-8153-4072-9 |edition=4th |url=https://www.ncbi.nlm.nih.gov/books/NBK26936/ |language=en |chapter=Fractionation of Cells}}</ref> The soluble fraction of any [[lysate]] can then be further separated into its constituents using a variety of methods.

===Differential centrifugation===
[[Differential centrifugation]] is the simplest method of fractionation by centrifugation,<ref name="Graham"/> commonly used to separate organelles and membranes found in cells. Organelles generally differ from each other in density and in size, making the use of differential centrifugation, and centrifugation in general, possible. The organelles can then be identified by testing for indicators that are unique to the specific organelles.<ref name="Ballou"/> The most widely used application of this technique is to produce crude subcellular fractions from a tissue homogenate such as that from rat liver.<ref name="Graham"/> Particles of different densities or sizes in a suspension are sedimented at different rates, with the larger and denser particles sedimenting faster. These sedimentation rates can be increased by using centrifugal force.<ref>{{cite web |last1=Frei |first1=Mark |title=Centrifugation Separations |url=https://www.sigmaaldrich.com/technical-documents/articles/biofiles/centrifugation-separations.html |website=Sigma-Aldrich |access-date=23 November 2020}}</ref>

A suspension of cells is subjected to a series of increasing centrifugal force cycles to produce a series of pellets comprising cells with a declining sedimentation rate. Homogenate includes nuclei, mitochondria, lysosomes, peroxisomes, plasma membrane sheets and a broad range of vesicles derived from a number of intracellular membrane compartments and also from the plasma membrane, typically in a buffered medium.<ref name="Graham"/>

===Density gradient centrifugation===
[[Density gradient centrifugation]] is known to be one of the most efficient methods for separating suspended particles, and is used both as a separation technique and as a method for measuring the density of particles or molecules in a mixture.<ref name="Brakke1951">{{cite journal|last1=Brakke|first1=Myron K.|title=Density Gradient Centrifugation: A New Separation Technique|journal=J. Am. Chem. Soc. |date=April 1951|volume=73|issue=4|pages=1847–1848|doi=10.1021/ja01148a508|bibcode=1951JAChS..73.1847B }}</ref>

It is used to separate particles on the basis of size, shape, and density by using a medium of graded densities. During a relatively short or slow centrifugation, the particles are separated by size, with larger particles sedimenting farther than smaller ones. Over a long or fast centrifugation, particles travel to locations in the gradient where the density of the medium is the same as that of the particle density; (ρp – ρm) → 0. Therefore, a small, dense particle initially sediments less readily than a large, low density particle. The large particles reach their equilibrium density position early, while the small particles slowly migrate across the large particle zone and ultimately take up an equilibrium position deeper into the gradient.<ref>{{cite book|last=Price|first=C. A.|chapter=1 - Particle Abstraction in Biology—An Introduction |title=Centrifugation in Density Gradients |year=1982 |pages=1–11 |doi=10.1016/B978-0-12-564580-5.50006-4 |chapter-url=https://www.sciencedirect.com/science/article/pii/B9780125645805500064 |publisher=Academic Press|isbn=978-0-12-564580-5}}</ref>

A tube, after being centrifuged by this method, has particles in order of density based on height. The object or particle of interest will reside in the position within the tube corresponding to its density.<ref name="Oster1963">{{cite journal|last1=Oster|first1=Gerald|last2=Yamamoto|first2=Masahide|title=Density Gradient Techniques|journal=Chem. Rev.|date=June 1963|volume=63|issue=3|pages=257–268|doi=10.1021/cr60223a003}}</ref> Nevertheless, some non-ideal sedimentations are still possible when using this method. The first potential issue is the unwanted aggregation of particles, but this can occur in any centrifugation. The second possibility occurs when droplets of solution that contain particles sediment. This is more likely to occur when working with a solution that has a layer of suspension floating on a dense liquid, which in fact have little to no density gradient.<ref name="Brakke1951"/>