Centrifugation. Downstream processing assignment. Iida Mäkeläinen Henna Penttinen Jenni Heikkinen Vera Kutiavina

Size: px

Start display at page:

Download "Centrifugation. Downstream processing assignment. Iida Mäkeläinen Henna Penttinen Jenni Heikkinen Vera Kutiavina"

Deirdre Norton
5 years ago
Views:

1 Aalto University The University of the chemical technology CHEM-E Bioprocess technology II Centrifugation Downstream processing assignment Iida Mäkeläinen Henna Penttinen Jenni Heikkinen Vera Kutiavina

2 1 Abstract Centrifugation is widely utilized and important separation technique in many industrial fields such as food, pharmaceutical, biotechnical and chemical industries. The operating principle of centrifuges is based on centrifugal force that facilitates the separation of different components in the mixture based on their sedimentation features. The applied force on particles depends on the rotation speed and the radius of centrifuge rotor. Thus, centrifugal force causes denser and larger components to migrate outward from the axis of rotation. Generally industrial centrifuges are distributed into two different classes according to their feeding type and operational principle. Filtration centrifuges differ from sedimentation centrifuges by utilizing filtration material e.g. slots, holes, filtration cloth to filtrate liquid material. The type of centrifuge that is chosen for industrial process is dependent on the process and separable components. Background Centrifugation technique has been widely used in industrial and laboratory settings for over 100 years. The first studies utilizing centrifugation technique are from the beginning of the 1800s [1]. The first centrifuge for industrial use was presented by Antonin Prandtl in 1864 for milk processing [2]. Later in 1878 Gustaf de Laval introduced continuous centrifuge for industrial applications, enabling usage of centrifuges in wider range [1]. Currently centrifuges are regularly utilized in different fields in laboratory-scale settings as well as in larger-scale industrial processes. Centrifugation is a separation technique where different components of mixture are separated based on their density or particle size. The separation of different substances is based on centrifugal force that is produced by high speed rotation. Centrifugal force causes denser components to be directed away from the axis of rotation whereas lighter components migrate towards it. [3] Centrifugation is generally utilized with separation of insoluble particles from liquids, but it is utilized as well in the separation of two immiscible liquids. For example, in industrial milk processing the centrifugal separation of the cream from milk is based on the density differences of the components. Centrifuges that are utilized in industrial setting, can be classified into two different categories: sedimentation and filter centrifuges. The classification is based on the general operation principles of the centrifuges, but the continuous and batch feeding type of centrifuge is also taken into consideration in classification. [5] Sedimentation centrifuges separate insoluble particles from liquids based on different sedimentation features, such as size, shape, density and centrifugal force [6]. Sedimentation is a process where molecules or particles sediment downward due to gravitational force. Larger and denser particles sediment faster. Centrifugation facilitates sedimentation with centrifugal force, separating the particles into solid pellet (precipitate) and liquid solvent (supernatant). [7] In comparison to sedimentation centrifuges, centrifugal filtration utilizes filtration material to separate the components from the mixture [5].

3 2 Theory and mechanism The working principle is based on centrifugal force that makes particles move toward bowl wall of the centrifuge. This force is usually expressed with number of earth gravities (g). The process of particles sedimentation depends on several factors. There is a direct correlation between rate of sedimentation and size of particles, speed of centrifugation, density difference between liquid and solid phase of solution and an inverse relation with fluid viscosity. [5] Figure 1. Particle in rotation. Figure 1 shows how the particles move in rotation. The centrifugal acceleration of the particles means that the force initiates particle movement outward from the axis of rotation. Centrifugal acceleration occurs the radial position (r) of the particles and the square of the angular velocity ( ). In figure 1 shows that angular velocity is calculated using the tangential velocity (u θ ) and radial position. The force acting on a particle increases, when the acceleration increasing. That cause a sedimentation of the particles at the axis of the rotation. [8, 9, 10] Figure 2. The measurements of the centrifuge. Radius is depending of the shape and type of the rotor of the centrifuge. Basically, if centrifuges rotor is fixed angle rotor as is shown in figure 2, there are two different values for radius. Fixed angle rotor means that tubes are in same position during centrifugation and the rotational speed cannot affect them. The other options are a swinging bucket centrifuge, where tubes hanging in the rotor. In that kind of centrifuge radius is depending on rotational speed as is shown in figure 3. When rotational speed higher, also the radius is increasing. Centrifuges with tubes are generally used in laboratory scale, not in downstream processing. [9]

4 3 Figure 3. Radius varies with rotation speed. Calculations Relatively centrifugal force (RFC) is the unit, which is used when is question of centrifugal force. Generally used revolutions per minute (RPM) is not a valid unit, because the force depends on the radius of the centrifuge. If the RPM is known and also the radius of the rotor, is calculation of the RFC easy using the equation: RFC = 1,118 r (!"#!""" )!, where the unit of the radius of the rotor (r) is millimetres. The unit of RFC is g as an Earth s gravitational force. [6, 9] As has been mentioned before, sedimentation rate is directly related to the features of particles, namely size and mass. Particles that are bigger in size than 5 µm accumulate at the bottom due to gravitational force, whereas if the size is smaller the process can be described with Brownian motion. That is why the smaller particles are, the stronger centrifugal force is required for sedimentation. In that case, the required force can be described with equation: F =!!!,! where m - mass of the particle, v - velocity of the particle, r - rotation circle radius. The ratio between centrifugal force and gravitational force represents the centrifugal effect: C =!! =!!!", where G - gravitational force

5 4 Classification All modifications of centrifuges can be distinguished into two main types: sedimentation and filtering centrifuges. In sedimentation centrifuges the centrifugal force is used to separate solids from liquids or two liquids with different densities. Sedimentation centrifuges include decanter, disk-stack, solid-bowl basket and tubular bowl centrifuges, which will be described below. Filtering centrifuges use centrifugal force to pass a liquid through a filtration media, such as a screen or cloth while solids are captured by the filtering media. [5] Sedimentations centrifuges There are many different types of sedimentation centrifuges and the best centrifuge type can be chosen on the basis of application and specificity of the process. Disc bowl centrifuge or disc stack centrifuge was first invented in order to separate cream in milk production industry. These centrifuges usually used for separation of two liquids or liquid and relatively low suspended solid phase. This centrifuge is composed by a vertical rotor with several conical discs on it. These systems of conical spacers allow to increase the sedimentation area. During rotation, centrifugal forces make denser solids move towards the bowl of the centrifuge where they then can be collected. [5, 13] Decanter centrifuge is sedimentation centrifuge, which consists out of a horizontally oriented bowl in a shape of a cone with a conveying scroll inside. The main working principle is related to the differences of densities of the liquids as denser liquids will drop to the bottom wall of the centrifuge. Conveying scroll creates a liquid pool, solids settle to the bowl wall and then transported by a conveying scroll to the end of a bowl where they are collected, whereas less denser liquid returns back to the pool. To prevent accumulation of solid particles on the scroll and reach optimal retention time for the separation, difference in rotational speeds of bowl and scroll is used in decanter centrifuges. [5] Hydrocyclone is one type of sedimentation centrifuges. It differs from other sedimentation centrifuges so that it does not have any rotation parts. The centrifugal force form, when suspension is pumped to colon with high speed. Because of the shape of the colon the suspension start to swirl, which occur the separation of the solid particles to the walls of the colon. The liquid material stays in the centre of the colon. The solid particles are removed of the bottom of the colon and clarified liquid material in the top. Hydrocyclones is generally used in continuous processes. [5] Solid-bowl centrifuge is a good example about the batch-type centrifuge. It consists of the one basket and it is generally used of collection particles of low concentration suspensions. In addition, it is also a good method to separate fine particles from liquid material, because of large diameter and high g forces. [5] Filtration centrifuges Filtration centrifuges similar as sedimentation centrifuges, but they are based on the filtration of the liquid material. Mechanism of the filtration centrifuge is the same as in other

6 5 centrifuges thus the separation is based on the centrifugal force. The difference is that filtration centrifuges has the filtration material, for example slots, holes, a porous membrane or filtrate cloth depending of the application and the purpose of the process. Filtration centrifuges are in different types as sedimentation centrifuges. [5] Filtration centrifuges can work as in continuous process as in batch process depending of the type of the centrifuge. Generally, filtration centrifuges are used in continuous processes, because the higher throughput capacity is often needed in industrial processes. Filtration centrifuges can be used for separation of the suspension with high solid material concentration, because of the filtration material. Solid particles form a cake on the filtration material. The solid material can be washed in centrifuge with water if centrifuge is equipped with rinsing system. That help for example to get all residual material out of solid material. [5] In addition to centrifugal force, many other things affect to the centrifugation of the material with filtration centrifuge, for example cake thickness, viscosity, screen area and the filtration material. [1, 5] If the cake growth too big on the filtration material, it can be removed during process. Batch process is more flexible in that case, but in some filtration centrifuges is possible to cut the cake decreasing rotation speed during continuous process when solid material drop to the bottom of centrifuge. [5] Importance Centrifugation is one of the most important and widely applied research techniques in biochemistry, cellular and molecular biology, and in medicine. Current research applications rely on isolation of cells, subcellular organelles, and macromolecules, often in high yields. [11] Today, centrifuges are routinely used in a variety of disciplines ranging from large-scale commercial applications to laboratory-scale scientific research. The number of centrifuge designs and configurations used in the mineral, petrochemical, chemical, medical, pharmaceutical, municipal/industrial waste, dairy, food, polymer, energy and agricultural industries (to name a few) seem almost as numerous as the applications themselves. [12] Industrially, centrifuges are used for a variety of purposes related to separation of materials on the basis of density. Generally, centrifuges are used throughout many manufacturing industries e.g. food and agri business, pharmaceutical/biotechnology, environmental industries and chemical industries. It appears in heavy industrial uses such as sugar and oil refining and in light industrial separations such as dewatering of vegetables. [5] Centrifugation and membrane filtration are the only techniques used for large-scale cell harvesting. Centrifugation has advantages for dense (density > 1.03 g/cm 3 ) and large (diameter > 2 µm) microorganisms. For example, centrifugation is very efficient for harvesting yeast. [14]

6 Case study: Pickering Emulsion as an Efficient Platform for Enzymatic Reactions without Stirring A Pickering emulsion is an interesting new method to process enzymatic reactions without stirring

7 6 Case study: Pickering Emulsion as an Efficient Platform for Enzymatic Reactions without Stirring A Pickering emulsion is an interesting new method to process enzymatic reactions without stirring and immobilization. Stirring and shaking can cause the damage to enzymes structures. Because of high price enzymes should be efficiently recyclable. Immobilization of enzymes is one possible solution for these problems, but sometimes it leads to a decrease in enzyme activity. [15] Figure 5 shows how a conventional biphasic enzymatic reaction can be converted into a water-in-oil Pickering emulsion system by adding a small amount of solid particle emulsifier. Pickering emulsions that are stabilized by solid particles have advantages over the conventional surfactant-stabilized emulsions such as the ease of product particle separation and low toxicity of particles. The large oil water interfacial area allows biphasic reaction systems to access high efficiency, which has been extensively demonstrated in hydrogenation, oxidation, as well as enzymatic reactions. Pickering emulsion systems were found to enable biphasic reactions to proceed efficiently through autodiffusion of molecules without need for stirring due to large interface area and short molecule diffusion distance. [15] Figure 5. Schematic figure of the Pickering emulsion method for stirring-free biphasic enzymatic reactions. [15] At the end of reaction, the emulsion is returned to a biphasic system by centrifugation, where the solid particle emulsifier is picked for recycling. After the upper layer of organic product is

8 7 removed through liquid transfer, the enzyme and emulsifier in the water phase can be directly used for the next emulsion reaction. [15] Conclusion Although centrifugation technique is over a hundred years old it is still an important method in the broad range of scientific, medical and industrial applications. Centrifuges are widely used in a variety of disciplines ranging from laboratory-scale scientific research to largescale commercial applications. A centrifuge is used to separate particles or even macromolecules based on size, shape or density. Centrifugal separation is used when gravity separation (settling, sedimentation or flotation) is too slow and the particles do not settle readily or at all. A centrifuge can generate centrifugal acceleration which is much greater than the Earth s gravity. The centrifuges in industrial use can be divided into two categories sedimentation and filter centrifuges which of each can be subdivide into continuous or batch feeding type. After fermentation, the biomass is commonly harvested by a centrifugation process to separate insoluble particles, which are usually cells, subcellular organelles, viruses and large molecules.

9 8 References [1] Wilson, Ian D. Poole, Colin F.. (2009). Handbook of Methods and Instrumentation in Separation Science, Volume 1 - Centrifugation to Covalent Chromatography. Elsevier. Online version available at: [2] Holtbrügge, J., Kunze, A. K., Niesbach, A., Schmidt, P., Schulz, R., Sudhoff, D., & Skiborowski, M. (2016). Reactive and Membrane-Assisted Separations. P. Lutze, & A. Górak (Eds.). Walter de Gruyter GmbH & Co KG. [3]JoVE Science Education Database. General Laboratory Techniques. An Introduction to the Centrifuge. JoVE, Cambridge, MA, doi: /5019 (2016), [4] Anonymous, RPM vs. RCF, [5] Beveridge, T. (2000), Large-Scale Centrifugation, Minister of Public Works and Government Services Canada [6] Rickwood, D.,Centrifugation Techniques, (2001) [7] Creighton, Thomas E.. (2010). Physical and Chemical Basis of Molecular Biology Sedimentation by Centrifugation. Helvetian Press. Online version available at: [8] Centrifugation: Theory, Sedimentation Rate, Coefficient and Other Details, webpage, [9] Clarke, P. 2009, pdf, Theory of sedimentation and centrifugation, [10] Fundamental of particle technology, pdf, Chapter 8: Centrifugal separation, [11] Cole-Parmer: Basics of Centrifugation [12] Handbook of Methods and Instrumentation in Separation Science page 49

10 9 [13] Conical Plate centrifuge, Wikipedia, webpage, [14] Harrison R., Todd P., Rudge S., Petrides D. (2015). Bioseparations Science and Engineering. Oxford University Press, 547 s. [15] Pickering Emulsion as an Efficient Platform for Enzymatic Reactions without Stirring Lijuan Wei, Ming Zhang, Xiaoming Zhang, Hongchuan Xin, Hengquan Yang, School of Chemistry and Chemical Engineering, Shanxi University, Taiyuan , China American Chemical Society, September 28, 2016,

Instrumental technique. Manju C K

Instrumental technique. Manju C K Instrumental technique Manju C K 03.09.201 6 CENTRIFUGE A centrifuge is a piece of equipment that puts an object in rotation around a fixed axis (spins it in a circle), applying a potentially strong force