Sample centrifugation: principles and types
— Equipo JP Selecta
Sample centrifugation is one of the most widely used separation techniques in clinical, research and industrial laboratories. It makes it possible to concentrate particles, clarify suspensions or isolate specific fractions in minutes, where gravity sedimentation would take much longer.
Its usefulness depends on applying the right force, time, temperature and rotor to each sample. It is not enough to insert a tube and select a speed. Centrifugation alters the distribution of a sample's components and therefore determines the quality of the fraction recovered.
Understanding what each parameter measures and which technique meets each objective helps you choose a centrifuge that is compatible and a protocol that is reproducible.
The centrifugation process applied to the laboratory
Sample centrifugation is a physical process in which a rotor spins the sample to accelerate the sedimentation of its components.
Larger or denser particles tend to move towards the outer part of the tube and form a pellet, while the less dense fractions remain in the supernatant or are distributed in layers. The effective intensity is expressed as relative centrifugal force (RCF), in × g.
RCF, and not just rpm, is the value that should guide a protocol because it depends on the rotor radius: two centrifuges can spin at the same revolutions per minute and apply different forces.
From there, time, temperature and rotor type determine the resolution of the separation. The aim is not to use the maximum force available, but to reach the validated condition that preserves the sample and allows the target fraction to be recovered.
Blood centrifugation: the most common application
When whole blood is centrifuged, its components are arranged by density.
- Top layer: the plasma. It accounts for approximately 55% of blood volume.
- At the interface, a thin layer of leucocytes and platelets appears, known as the buffy coat.
- The erythrocytes are concentrated at the bottom, around 45%.
This separation makes it possible to prepare plasma or serum depending on the tube and the protocol, to measure haematocrit or to work with specific cell fractions.
Blood centrifugation parameters must always follow the analytical method, the tube type and the manufacturer's instructions. As a reference, many clinical procedures use approximately 1,500–3,000 × g for about 10 minutes, but this is not a universal rule.
For heat-sensitive samples or protocols sensitive to platelet activation and haemolysis, temperature control is decisive. For blood processing, at J.P. Selecta we offer refrigerated centrifuges such as the Macrofriger-BLT GR-50T, designed for blood banks and clinical analysis, and haematology equipment for the subsequent processing of the samples.
Types of centrifugation by technique
The type of sample centrifugation is selected according to the separation objective: obtaining a pellet, enriching a cell population, isolating a subcellular fraction or concentrating a small volume.
The same centrifuge can give very different results if the rotor, RCF, time or suspension medium changes.
Differential centrifugation
Differential centrifugation separates the components of a suspension through successive steps at increasing forces. After each step, the pellet or the supernatant is recovered before continuing.
Thus, an initial gentle centrifugation can remove intact cells, cell debris or nuclei; intermediate forces make it possible to enrich fractions such as mitochondria; and higher forces concentrate smaller particles.
It is a practical technique for cell fractionation and sample preparation, although the fractions are not completely pure: particles of similar density or size can sediment together. For this reason, when the assay requires higher resolution, differential centrifugation is usually used as a preliminary step before density gradient separation.
Density gradient centrifugation
In density gradient centrifugation, the sample is layered onto a medium of increasing density, either continuous or in layers, for example a sucrose solution, Ficoll or another medium compatible with the protocol. During the spin, each component migrates according to its density and forms distinct bands, which makes it easier to recover more clearly defined populations than with differential separation.
The composition of the gradient must be adapted to the object of study. A medium suitable for subcellular fractions will not necessarily preserve the viability of a cell population, and an incorrect concentration can prevent the bands from resolving. Acceleration and braking also matter: an abrupt stop can disturb the gradient and remix the separated fractions.
Cytocentrifugation
Cytocentrifugation, usually carried out with cytocentrifuges, concentrates cells suspended in a liquid onto a defined area of a microscope slide. Its purpose is not to obtain a pellet for resuspension, but to prepare a uniform cell smear that can be fixed, stained and observed under the microscope.
It is used, for example, with low-cellularity biological fluids, bronchoalveolar lavages, urine or effusion samples. The force and time must be sufficient to deposit the cells without deforming or rupturing them. An overly aggressive preparation can produce artefacts and hinder morphological interpretation; an overly gentle one yields an unrepresentative sample.
Microcentrifugation
Microcentrifugation works with small volumes, normally in microtubes, and makes it possible to rapidly concentrate DNA, RNA, proteins, precipitates or cells. It is a routine operation in molecular biology because it combines high RCF with compact formats and short run times, provided that the tube, volume and rotor are compatible.
Its main advantage is processing many aliquots quickly and reducing sample handling.
Even so, tubes should be balanced correctly and the rotor's load limit respected. An imbalance can affect the quality of the separation, generate vibrations and compromise both the sample and the safety of the centrifuge.
For microtubes and microhaematocrit tests, J.P. Selecta offers the Centrolit II-BL microcentrifuge, with an electromagnetic safety lid lock.
Ultracentrifugation
Ultracentrifugation is reserved for very small particles, macromolecules or subcellular fractions that require very high forces. It is used in research to separate, among other objectives, protein complexes, vesicles or certain membrane fractions. It can be performed as preparative ultracentrifugation, to recover material, or analytical ultracentrifugation, to study the properties of the sample.
It is a technique that requires rotors, tubes and procedures specifically designed for its working range. Selection should not be based on speed alone: the RCF limit, the rotor radius, the density of the medium and the integrity of the material are critical. For this reason, an ultracentrifugation protocol must not be extrapolated to a conventional centrifuge.
In sample centrifugation, the differential technique prioritises stepwise separation; the gradient technique provides greater resolution; cytocentrifugation prepares cells for observation; microcentrifugation handles small aliquots; and ultracentrifugation addresses very small-scale particles. The choice depends on the sample type, the volume, the required RCF and whether temperature control is needed.
At J.P. Selecta we offer universal centrifuges, benchtop centrifuges such as the Centromix BLT and refrigerated centrifuges to adapt the rotor, capacity and working conditions to each application.
Explore our range of laboratory centrifuges.