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Analytical ultracentrifuge

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Analytical ultracentrifuge

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In This presentation you will learn about centrifugation and its principle and main tpic of this ppt is AUC(Analytical ultracentrifugation) and its pricliple and analysis process which are perform in it and Application of AUC

so enjoy learning

You can share your thougths on my mail pmo.afak313@gmail.com

In This presentation you will learn about centrifugation and its principle and main tpic of this ppt is AUC(Analytical ultracentrifugation) and its pricliple and analysis process which are perform in it and Application of AUC

so enjoy learning

You can share your thougths on my mail pmo.afak313@gmail.com

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Analytical ultracentrifuge

  1. 1. Analytical ultracentrifuge Name: Mohammadafak Patel Sub: biomedical Technology Paper: 403-Microscopy & Clinical techniques Unit: ii-Electrophoresis and Centrifugation
  2. 2. Contain  About Centrifugation  Principle of Centrifugation  Type of Centrifugation and over view of it & Comparison  Introduction about Analytical ultracentrifugation (AUC)  History of AUC  Analytical ultracentrifuge & its principle  Design of AUC & Instrumentation  Type of analysis performed with analytical centrifuge  Application of AUC
  3. 3. About Centrifugation • Is a separation technique which use the • particles whose density is higher than the solvent sediment(sink) and particles that are lighter than it float to the top. • Factors that affect the sedimentation: the shape and size of particles, the density of particles, molecular mass (under the influence of the gravity) • Gravity can be replaced with the much more powerful centrifugal force (provided by a centrifuge) • Applied in industrial chemistry, biochemistry, cellular and molecular biology, environmental technology For the sedimentation centrifugal force
  4. 4. Centrifuge Machine • A device for separating particles from the solution according to their size, shape, density, viscosity of the medium and rotor speed • Basic Part of centrifuge are two (2) 1. An electric motor . 2. Rotor (which hold the tube). Type of basic centrifuge(based on rpm) Low speed centrifuge High speed centrifuge ultracentrifuge
  5. 5. Principle of Centrifuge • Centrifuge Work on a • Under the influence of gravitational force, substances separate according to their density • Sedimentation depends upon the applied centrifugal field (G) • It determined by the given equation  Here G is centrifugal field  𝜔 is angular velocity of rotor  r is radial distance principle of sedimentation 𝑮 = 𝝎𝟐 𝒓
  6. 6. Types of Centrifugation techniques:
  7. 7. Centrifugation technique Preparative Centrifugation technique Analytical Centrifugation technique • Concerned with the actual separation, isolation, purification (eg. Whole blood cells, Subcellular organelles, Plasma membrane, Polysomes, Ribosomes, Chromatin, Nucleic acid, Lipoproteins and Viruses). • This is most commonly used technique. • Concerned with the study of sedimentation characteristics of biological macromolecules and molecular structures. • Devoted to the study of pure macromolecules or particles.
  8. 8. Comparison between preparative and analytical centrifugation Analytical ultra-centrifugation  Uses small sample size (less than 1ml)  Built in optical system (to analyze the process)  Uses relative pure sample  Used to precisely determine sedimentation coefficient and MW of molecules Preparative centrifugation  Larger sample size can be used  No optical read-out during the process.  Less pure sample can be used  Used to estimate sedimentation co-efficient Molecular weight
  9. 9. Analytical Centrifugation technique Introduction • Concerned with the study of sedimentation characteristics of biological macromolecules and molecular structures • Devoted to the study of pure macromolecules or particles. • Require small amount of material, utilize specially design rotors and detector system to continuously monitor the process of separation of the material in the centrifugal field. • Used to deduce the purity, relative molecular mass and shape of material. • Analytical Centrifuge is Capable of speed approaching 70,000 rpm. Spins a rotor at an accurately controlled speed and temperature. • provided with protective chamber, refrigeration and optical system to observe sedimentation of the material throughout the duration of centrifugation. • The concentration of the sample is determined at known times using absorbance measurements.
  10. 10. History • The first AUC was built by Theodor Svedberg in the year of 1920s • he used it to characterize proteins and other molecules in solution, demonstrating the individual character of biological macromolecules. • He get the Nobel prize in Chemistry (1926). • The technology was commercialized by Spinco the Specialized Instrument Company in 1946, in the form of the Model E analytical ultracentrifuge. • Now a part of Beckman Coulter, Inc., it is the sole manufacturer of AUC equipment today. • Modern versions of AUC make use of a variety of optical systems and use an external computer for facilitated data acquisition and analysis.
  11. 11. Analytical Centrifuge (AUC) Inside of AUC
  12. 12. Principle of AUC: • The basic principle remains same as a other centrifuges(sedimentation principle)  Sedimentation coefficient: 𝑮 = 𝝎𝟐𝒓 Sedimentation depends upon the applied centrifugal field (G) 𝐬 = 𝒗 𝝎𝟐𝒓  The velocity of the particle per unit centrifugal field  Sedimentation constant in in seconds usually around 10-13  Which is celled svedberg unit (S)  1 S = 10-13 s.
  13. 13. AUC: Design • Analytical centrifuge = preparative ultracentrifuge + optical detecting system • Centrifugation parameters and data acquisition under computer control Rotate at 1,20,000 to 1,50,000 rpm Measure the sample concentration inside the cell during or after sedimentation
  14. 14. Instrumentation of AUC The component part of analytical centrifuge (AUC) are:  A rotor and cells  An optical system  A data acquisition and analysis system
  15. 15. Analytical ultracentrifuge:
  16. 16. A rotor and cells: • Rotor is a solid with the holes to hold the sample cell • Each cell contains the center piece with channel to hold liquid sample • Centerpieces are available that hold either 4 to 8 cell • a typical AUC rotor generate a centrifugal field in the cell of about 2,50,000g under such condition 1 g of partical experience an apparent weight of 250kg. • The rotor must be capable to allow the passage of light through the spinning sample for temperature measurement. • The rotor is equipped with four holes, which is required for the counterbalance
  17. 17. • A cell is very important component • It contains sample and control sector • one contains solution with solvent and analyte and other just solvent • the sectors are trapezoidal
  18. 18. optical system • As the rotor spins each cell passes through the optical path of detectors capable of measuring the concentration of molecule at known time • There are three commercially available optical detectors to measure the concentration: A. Absorbance spectrophotometer B. Rayleigh interometer C. Florescence detector A. Absorbance spectrophotometer:  Usable over wavelength range 190 to 800nm (Beer-Lambert low)  Absorbance is directly proportional to the solute  concentration
  19. 19. B. Rayleigh interometer • Signal consist of equally spaced horizontal fringes and does not rely on chromophore, hance colorless compunds can be characterized • Example polysaccharides and lipids C. Florescence detector • laser light and suitable lable are used in this • A detector collects light absorption data, which a computer digitize and records.
  20. 20. The two most common type of analysis performed with analytical centrifuge: Analysis Sedimentation velocity experiment Sedimentation equilibrium experiment
  21. 21. Sedimentation velocity experiment • By this experiment we can determine the size and shape of the molecule, polydispersity, conformation change. • Performed at very high speed to overcome the effect of diffusion. • This experiment is performed using 2 sector cells(shown in fig.) • An initially uniform solution is placed in a cell and a high angular velocity is applied to cause rapid sedimentation
  22. 22. Sedimentation equilibrium experiment • Requires lower rotor speed than sedimentation experiment. • In this experiment we used 6 sector centerpieces • In sedimentation equilibrium, equilibrium is established between sedimentation away from the center of rotation and diffusion towards the center of rotation. • Its provides the same type of information about the solution molecular mass, stoichiometries, association constants. • This experiment is actually used for the calculation of hydrodynamic information • By this experiment we can also determined : 1. Sedimentation coefficient s 2. Diffusion constant D or friction factor f 3. Molecular mass 4. Estimation of molecular shape in solution
  23. 23. Application of analytical ultracentrifugation(AUC): • With the AUC the the following charactiristics of molecules can be deternied: 1. Molecular mass 2. Stoichiometry 3. Association 4. Assembly models 5. Ligand binding 6. Conformation & shape
  24. 24. Application of AUC: 1. Molecular Weight Determination:  measurement of molecular weights of solutes in the native state.  determination of average molecular weight by sedimentation equilibrium, 2. Stoichiometry:  the stoichiometry of a molecule can be calculated from the determined MW  with high quality data it can be easily established whether the native conformation of a protein is a hexa or heptamer 3. Analysis of Associating Systems:  Sedimentation equilibrium give an idea about the wide range of interactions, binding of small molecules and ions to macromolecules, the selfassociation of macromolecules by sedimentation velocity and sedimentation equilibrium experiments.
  25. 25. 4. Assembly models:  The assembly of protein complex can be calculated from the determined molecular mass  A thermodynamic analysis may be made using sedimentation equilibrium method 5. Ligand binding :  this can also be analyzed using sedimentation velocity method if the ligand and acceptor differ greatly in their sedimentation coefficient 6. Conformation & shape:  Information about the shape and the conformation of the protein as well as the intraction between macromolecule can be obtained from a sedimentation velocity experiment  Sedimentation coefficients are particular useful for monitoring changes in conformation of protein
  26. 26. References • Wilson, K. walker Principle and technique of practical biochemistry.(7th edition 2010) • Analytical ultra-centrifugation - YouTube • Analytical centrifugation (slideshare.net) • Analytical ultracentrifugation - Wikipedia
  27. 27. Long Questions 1. Write about the analysis processes which are carried out in analytical centrifuge(AUC) 2. Give the detailed note on instrumentation of analytical centrifuge
  28. 28. Short Questions: 1. Give the application of AUC 2. Write down the two basic part of centrifuge 3. give the type of centrifugation process 4. What is svedberg unit. 5. give the type of optical system which are used in AUC
  29. 29. MCQs 1. After centrifugation of milk, the supernatant is a) fat b) whey c) casein d) water 2. Which of the following statements about the basic principle of sedimentation is False? a) The denser a biological structure is, the faster it sediments in a centrifugal field. b) The more massive a biological particle is, the slower it moves in a centrifugal field. c) The denser the buffer system is, the slower the particle will move in a centrifugal field. d) The greater the centrifugal force is, the faster the particle sediments.
  30. 30. 3. Which of the following is/are the application of analytical centrifugation? I. the determination of the purity of macromolecules. II. the determination of the relative molecular masses of solutes in their native state. III. the examination of changes in the molecular mass of supramolecular complexes. IV. the detection of conformational changes. A. i only. B. i and ii only. C. i, ii, and iii only. D. All of these.

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