Which type of liquid is forced to the top of a red top tube after being spun in a centrifuge?

1.18.7.3 Centrifugation

Centrifugation has been used to separate colloids from aqueous solution on the basis of particle size and density. The samples are prefiltered to remove particulate material (by definition through a 0.45-μm filter paper) and then placed in centrifuge tubes. Samples usually undergo centrifugation at 25,000 rpm for a minimum of 45 min. This is repeated until the conductance and surface tension correspond to that of pure water.42 Again it takes protracted time scales to process samples, making the method unsuitable for marine and estuarine solutions where large sample volumes are required. However, it is suggested that centrifugation is far more effective than diafiltration and ion exchange at removing LMW molecules, and that centrifugation tubes are readily decontaminated and sterilized. The main disadvantage of centrifugation is therefore the limited sample capacity of the centrifuge.

6.4.1.2 Centrifugation

Centrifugation is the simplest approach to extract ENPs from aqueous samples. It requires high centrifugal speeds and long times to separate ENPs from supernatants, which may not guarantee complete separations (Laborda et al., 2016). Moreover, some unwanted super molecules, such as organic matters and proteins, may accompany with ENPs. Thus preparative centrifugation alone is not considered as effective technique for the fractionation of ENPs (Tsao et al., 2011). The directly available pool of ENPs in soils can be collected from soil pore water with subsequent characterization of ENPs. Centrifugation is the most commonly used method to extract pore water from soils (Rodrigues et al., 2016).

3.36.2.2.2 Centrifugation

Centrifugation methods employ centrifugal force to achieve separation of particles from a liquid medium. Centrifuges are generally classified according to their rotors, e.g., swing-bucket rotors, fixed-angle rotors, and vertical rotors. Different types of rotor are used for different applications. For example, the fixed-angle rotor is commonly used for environmental water samples due to its excellent ability in pelleting. Bacteria cells can usually be pelleted with a centrifugation speed of 8000 × g, which is equivalent to a centrifugal force of 8000 times greater than the Earth’s gravitational force. In general, the speed of centrifugation is decided by the pelleting applications.100

The cost of centrifugation involves only the initial investment of the centrifuge. Routine monitoring is suitable, as this method does not require any chemical reagent or other expensive consumable. In addition, centrifugation methods are not constrained by the turbidity of the water sample. As a result, large volumes of water can be processed in a relatively short period of time. The larger volume of water can better represent the entire sample matrix, increase the concentration factor, and subsequently increase the sensitivity of the downstream detection method. However, concentration through centrifugation has the tendency to simultaneously concentrate other particles including nontargeted cells, especially for water samples with high levels of sediments. The presence of these additional particles complicates the downstream analysis and detection, and also creates a bias toward particle-associated bacteria. Another limitation is the tendency of bacterial losses when decanting the supernatant.

3.05.12.6.1 Centrifugation

During centrifugation, the components of blood will separate into layers. Erythrocytes will condense to the bottom layer and plasma will be on the top layer of the tube. The buffy coat will form the interface between plasma and erythrocytes. A small volume of whole blood is typically centrifuged at 1200–1500 × g for 5–10 min.13

Small volumes of whole blood should be centrifuged at 1500 × g for 15 min to obtain platelet-poor plasma (platelet content less than 10 × 109/l).9 Plasma should be aspirated off with a transfer pipette, while taking care not to remove any of the other layers in the tube. To prevent contamination from the cellular components in the tube, no more than about three-quarters of the plasma layer should be extracted. It should also not be poured off unless there is a gel layer separating it from the rest of the blood components. Serum is separated in the same manner of centrifugation. As with plasma, it should not be poured off unless there is a gel separation layer in the tube.

The method of centrifugation can also be used to harvest the buffy coat.13 After centrifuging a tube of blood, the top layer of plasma should be aspirated off with a transfer pipette. The buffy coat is then carefully removed again using a pipette. At this point there will be contamination of the buffy coat with RBCs, platelets, and some plasma. Depending on what the buffy coat or components of the buffy coat is used for, further separation of the cells may be necessary. Other methods will be described following this section.

Centrifugation is also a widely accepted method for the isolation of erythrocytes for both laboratory analysis and blood transfusion purposes. Following centrifugation of whole blood, a transfer pipette is used to aspirate only the erythrocytes from the bottom of the centrifugation tube into a clean aliquot tube. It is a good idea to wash the RBCs two to three times in a buffered isotonic saline solution to remove excess plasma and proteins. In the aliquot tube, the buffered saline should be added in a volume of two to three times the volume of red cells. Resuspend the RBCs in the saline solution and centrifuge as above. Following centrifugation, the red cells will form a pellet at the bottom of the tube. Aspirate off the saline and repeat the resuspension and centrifugation process with fresh saline. After the process is done two to three times, the red cells are ready for manipulation and analysis.

Centrifuge methods for isolating platelets have also been employed. Upon gentle centrifugation (approximately 350 × g for 15 min), platelets are suspended in the plasma layer, resulting in platelet-rich plasma. Care must be taken in the preparation of platelet isolates as they are susceptible to activation during any manipulation process. Excessive manipulation, agitation, or centrifugation may result in their activation.

With larger volumes of whole blood (such as transfusion sized units), centrifugation at 5000 × g for 5–7 min yields platelet-poor plasma.15 Platelet-rich plasma is prepared by a lighter centrifugation at 2000 × g for 3 min. Plasma is removed from a centrifuged unit of blood by means of a plasma extractor and clamps. WBCs may be filtered by means of a leukoreduction filter. Isolation of blood components for clinical transfusion is a standardized process and is detailed in the AABB technical manual.

Introduction

Centrifugation is a mechanical process that utilizes a spinning medium to separate one or more components of a sample according to density or size. While gaseous or immiscible liquids can be separated by centrifugation, the majority of applications involve sedimentation of solid particles in a liquid medium. Centrifugal separations may be classified as either analytical or preparative. In ‘analytical centrifugation’, the objective is to monitor particle sedimentation behavior in order to characterize particle properties, e.g., molecular weight, shape, and association. In ‘preparative centrifugation’, the objective is to separate and recover one or more components from a sample mix. Preparative centrifugation encompasses the vast majority of centrifugal applications.

While centrifugal techniques have been in use for at least a thousand years, the first recorded scientific study using a centrifuge did not appear until 1806, when Thomas Knight reported differences in the orientation of roots and stems of seedlings when placed in a rotating wheel. Some of the more significant developments since that time have included: the first commercial centrifuge, a hand-cranked cream separator, introduced in 1878 by the Swedish inventor, DeLaval; introduction of the analytical centrifuge for viewing particle sedimentation in 1923 by Theodor Svedberg; the isolation of subcellular components by centrifugal techniques in the 1940s; and the first use of a density-gradient medium by Edward Pickels in 1943. More recent advances have been characterized by significant improvements in materials and equipment and a broadening of applications.

Today, centrifuges are routinely used in a variety of disciplines including the medical, pharmaceutical, mineral, chemical, dairy, food, and agricultural industries. Available centrifuge designs and configurations seem almost as numerous as the applications themselves. An in-depth description of centrifuge designs and applications is beyond the scope of this treatise. Instead, this article will present the reader with an introduction to the theory of centrifugation, an overview of the various types of preparative centrifugal separations, and a description of some of the more common centrifuge and rotor designs along with their more common applications.

1.10.4.5 Sedimentation Equilibrium in a Density Gradient

Centrifugation of a mixed solvent, containing components having different densities, generates a density gradient. At SE a solute collects in a band about the point in the density gradient which corresponds to its buoyant density. The width of the band depends on the molecular weight of the solute, being broader for lower M. The density gradient technique can be employed qualitatively to distinguish between species having different partial specific volumes139, 140 and can be useful for the detection of microgel.139 Quantitatively, it may be used for the determination of molecular weight139, 141, 142 and, for some copolymers, in compositional analysis.143 Density gradient SE has also been utilized in studies of preferential absorption.144

2.56.3.3 Centrifugation

Centrifugation takes advantage of differences in particle size and density and is commonly used to remove cells and cell debris in the initial stage of many purification schemes. Flocculants (such as PEI) or low-pH conditioning may be required to enhance separation of debris from the product protein. The high throughputs, excellent separation, and closed configuration afforded by continuous disk stack centrifuges make them a robust starting point for a platform purification process. Hermetic machines with ‘soft’ entry zones are ideal for fragile CHO cells, and can minimize unwanted cell lysis and release of intracellular components that could degrade the product. Continuous disk stack machines can incur high yield losses in high biomass fermentations, but decanters are well suited to handling the high solids content of microbial or fungal fermentations.

24.3.1.4 Centrifugation

The centrifugation of particles under a force field is well known and has been used extensively to determine the molecular weights of biological macromolecules such as proteins.99, 100 This molecular weight is derived from the measurement of the radius of the sedimenting particles. The increase in radius of a colloidal particle caused by an adsorbed layer can thus be measured, as demonstrated by Garvey et al.101 with poly(vinyl alcohol) adsorbed on to polystyrene latices. Alternatively, slow-speed centrifugation of colloidally stable particles leads to a hexagonally close-packed sediment, giving a measure of the particle radius and thus a thickness of the adsorbed layer.102

3.42.3.5 Centrifugation

Centrifugation is a process where the sample is spun at a particular force (g) for a specific time to force particles in the solution to the bottom of the centrifuge tube.

Centrifuges vary in number, size, and volume capacity of tubes and in speed. The power of a centrifuge is determined as the relative centrifugal force (RCF),11 which is measured in times gravity (×g), and is a function of the radius of the centrifuge arm, R, between the center and the position of the sample (in cm) and the speed of rotation, S, in revolutions per minute (rpm), and is governed by the Equation 3:

(3)RCF(x g)=1.1118×10−5 RS2

When adapting a method from a previous method to your centrifuge, check the RCF of the centrifuge of the original method to ensure equivalence.

For efficient operation it is vital that the centrifuge be balanced – that is equal number and weight of samples around the circumference of the rotors. ‘Dummy’ tubes containing just water can be added to ensure that the centrifuge is balanced prior to operation.

Centrifuging with tubes of liquids of known density can be used to separate mixtures of different densities.

Centrifugation can also be used to separate components with different molecular weights. In this process, the separating tubes have a filter of specific size. A material is placed in the top of the tube, which is then placed in a centrifuge tube and spun down. The material that is smaller than the pore size is collected from the bottom of the tube and the material that is larger than the pore size is recovered from the top by inverting the filter in a second tube and spinning or washing it out.12 The process can then be repeated with tubes of different pore sizes. A recent example of their use in a forensic investigation is the separation of semen into fractions of >300 kDa, 300–100 k, 100–50 k, 50–30 k, 30–10 k, and <3 kDa size to investigate the relative fluorescent intensities of the size fractions and to determine the composition of the fractions by subsequent chromatographic separation of suitable fraction.13

A. Description of the centrifugation technique

Centrifugation consists of substituting natural gravity with a radial centrifugal force reaching several thousand times the gravitational force. Products to be separated are placed in a vessel known as a bowl, which is subjected to a high-speed rotation. The application of the dynamic fundamental law (F=mΓ) makes it possible to express the force that acts on any particle of mass m (and/or on any elementary volume of liquid) and to accelerate phase separation. In this case, Γ is the central (or radial) acceleration of a steady circular movement, its modulus being V2/R (V is the tangential velocity of the particle and R its distance from the rotation axis). Since the tangential velocity is equal to the product of the angular speed ω by the radius R, the acceleration and the force can be written as follows:

and

In practice, this means that if we want, for example, to generate an acceleration of 5,000g at a distance of 0.4 m from the rotation axis, the rotation speed is calculated as follows (ω) = 2πN):

Radial acceleration = 5,000×9.81 m/s2 = 4&PI;2N2R, hence N = 55.8 rev/s or 3,345 rev/min.

Remark. In the movement of particles, the influence of gravity is quite negligible compared to the force due to radial acceleration.