CENTRIFUGATION AND CHROMTOGRAPHY

 Centrifugation:

Centrifugation is a mechanical process that uses centrifugal force to separate components of a mixture based on their size, shape, density, medium viscosity, and rotor speed.

Principle:

The principle behind centrifugation is sedimentation. In a mixture, particles with a higher density tend to settle at the bottom due to gravity. A centrifuge accelerates this separation by spinning the mixture at high speeds, creating a much stronger "effective gravitational force" known as centrifugal force.

Process:

The Rotor: The rotor holds the sample containers (e.g., tubes or bottles) in place.

Centrifugal Force: As the rotor spins, it generates centrifugal force, an outward push acting radially from the center of rotation.

Sedimentation: This force causes denser particles in the mixture to migrate away from the axis of rotation and settle at the bottom of the tube, forming a pellet.

Supernatant: The less dense components remain in the liquid above the pellet, which is called the supernatant.

The efficiency of a centrifuge is based on 

• Relative Centrifugal Force (RCF) or g-force.
• Rotor Speed (RPM).
• Particle Size, Shape, and Density.
• Medium Viscosity.
• Temperature.

Types of Centrifugations:

Differential Centrifugation: It involves multiple rounds of centrifugation at progressively higher speeds. After each spin, the supernatant is transferred to a new tube and spun again, allowing for the separation of particles of decreasing size and density (e.g., separating cell organelles).

Density Gradient Centrifugation: A density gradient is created in the centrifuge tube using solutions of varying concentrations (e.g., sucrose or cesium chloride). The sample is layered onto this gradient, and during centrifugation, particles migrate through the gradient until they reach a point where their density matches that of the surrounding solution, forming distinct bands.

Rate Zonal Centrifugation: Particles separate into zones based on their sedimentation coefficient through a pre-formed density gradient.

Isopycnic Centrifugation: Particles separate and form bands at their equilibrium buoyant density within a self-generating density gradient.

Application:

• Molecular Biology and Genomics.
• Preparation of Platelet-Rich Plasma (PRP).
• Cell Isolation and Purification.
• Cell and Tissue Culture.
• Vaccine and Pharmaceutical Production.

Low-speed centrifuge:

It operates at lower rotational speeds compared to high-speed or ultracentrifuges, making it ideal for gentler separations where the integrity of delicate particles needs to be maintained.

Key Characteristics:

Speed Range: Typically operates at a maximum speed of 4000-6000 RPM.

Relative Centrifugal Force (RCF): The RCF (or g-force) generated by low-speed centrifuges typically ranges from a few hundred to approximately 5,000 x g.

Temperature Control: Many low-speed centrifuges operate at room temperature without specific temperature control.

Rotor Types: They commonly utilize both fixed-angle rotors and swinging-bucket rotors.

Capacity: Low-speed centrifuges vary in capacity, ranging from small benchtop models that handle a few milliliters to larger floor-standing units capable of processing several liters.

Cost and Ease of Use: They are generally less expensive and simpler to operate than high-speed or ultracentrifuges.

High-speed centrifuge:

A high-speed centrifuge is a laboratory instrument designed to achieve significantly higher rotational speeds and, consequently, greater centrifugal forces than low-speed centrifuges.

Speed Range: High-speed centrifuges typically operate at speeds ranging from 15,000 RPM up to 30,000 RPM.

Relative Centrifugal Force (RCF): They can generate RCF values in the range of tens of thousands of x g, sometimes exceeding 100,000 x g.

Temperature Control: Due to the high speeds and the sensitivity of many biological samples, most high-speed centrifuges are refrigerated.

Fixed-angle rotors are very common for high-speed applications, particularly for pelleting small particles, as they offer efficient sedimentation.

Swinging-bucket rotors are also used, especially for density gradient centrifugations where distinct banding of components is desired.

Capacity: They generally handle a range of sample volumes, from microliters (in microcentrifuges, some of which are classified as high-speed) to several liters (in larger floor-standing models).

Ultra centrifuge:

An ultracentrifuge is the most powerful type of centrifuge, designed to generate extremely high centrifugal forces to separate the smallest and most delicate components of a mixture, often at a molecular or macromolecular level.

 Extremely High Speeds: Ultracentrifuges spin at incredibly high speeds, typically ranging from 60,000 RPM up to 150,000 RPM.

Massive Relative Centrifugal Force (RCF): The RCF generated by ultracentrifuges can reach astonishing levels, from 200,000 x g to over 1,000,000 x g. 

Vacuum System: A crucial feature of ultracentrifuges is the vacuum system. At such high speeds, air friction with the rotor would generate enormous heat, damaging samples and potentially the instrument.

Refrigeration: Like high-speed centrifuges, ultracentrifuges are always refrigerated to maintain precise temperature control and protect heat-sensitive biological samples.

 

Fixed-angle rotors: Ideal for pelleting small particles quickly.

Swinging-bucket rotors: Excellent for density gradient centrifugations where distinct bands of separated components are desired.

Vertical rotors: Designed for very rapid separations, often used in isopycnic centrifugation.

Chromatography:

Chromatography is a powerful and versatile laboratory technique used for separating, identifying, and purifying the components of a mixture. The name "chromatography" literally means "color writing" (from Greek chroma for color and graphein for to write), stemming from its initial discovery by Russian botanist Mikhail Tsvet in 1903, who used it to separate plant pigments into distinct colored bands.

Principle:

Stationary Phase: This phase is fixed in place and can be a solid material, a liquid coated on a solid support, or a gel. The specific properties of the stationary phase are crucial for separation.

Mobile Phase: This phase is a fluid (liquid or gas) that moves through or over the stationary phase, carrying the sample mixture with it.

Types of Chromatography:

• Liquid Chromatography (LC).
• Gas Chromatography (GC).
• Adsorption Chromatography.
• Partition Chromatography.
• Ion-Exchange Chromatography (IEX).
• Size Exclusion Chromatography (SEC) / Gel Filtration Chromatography.
• Affinity Chromatography.
• Hydrophobic Interaction Chromatography (HIC).

Thin layer chromatography:

Thin-Layer Chromatography (TLC) is a simple, fast, and cost-effective planar chromatographic technique used for separating components of non-volatile mixtures.

Stationary Phase: This is a thin, uniform layer of an adsorbent material (typically silica gel, but also alumina, cellulose, or modified silica such as C18) coated onto an inert, rigid support plate, such as glass, plastic, or aluminum foil. The stationary phase is usually polar.

Mobile Phase: This is a liquid solvent or a mixture of solvents (often organic solvents like ethyl acetate, hexane, methanol, etc.) that moves up the stationary phase by capillary action. The mobile phase's polarity is crucial for effective separation.

  

RF value:

The migration of each compound is characterized by its retention factor (Rf), which is a unique and reproducible value for a given compound under specific TLC conditions (stationary phase, mobile phase, temperature).

Rf= Distance travelled by the spot/Distance travelled by solvent front​

The Rf value is always between 0 and 1. 

Detection methods:

• UV Light (Ultraviolet Light).
• Iodine Vapor.
• Chemical Spray Reagents (Stains).
• Charring with Sulfuric Acid.

Column chromatography:

Stationary Phase: A solid adsorbent material packed into a cylindrical column. The most common stationary phases are silica gel (${\text{SiO}}_2$) and alumina (${\text{Al}}_2{\text{O}}_3$), both of which are polar. The choice of the stationary phase depends on the properties of the compounds to be separated.

Mobile Phase (Eluent): A liquid solvent or a mixture of solvents that is passed through the column, carrying the sample mixture. The mobile phase is chosen based on its ability to dissolve the sample and its elution strength (how well it moves compounds through the column).

 Detection Methods:

• UV/Visible Absorbance Detector.
• Refractive Index (RI) Detector.
• Evaporative Light Scattering Detector (ELSD).
• Mass Spectrometry (LC-MS / GC-MS).
• Fluorescence Detector (FLD).

Gas chromatography:

Principle:

Volatility: For a compound to be analyzed by GC, it must be volatile (easily vaporized) and thermally stable (not decompose at the high temperatures used in the injector and column).

Mobile Phase (Carrier Gas): This is an inert gas (e.g., Helium, Nitrogen, Hydrogen) that flows continuously through the system. It acts as the carrier for the vaporized sample, pushing it through the column.

Stationary Phase: This is a thin layer of a liquid (most common, Gas-Liquid Chromatography, GLC) or a solid (less common, Gas-Solid Chromatography, GSC) coated on the inside of the column or on solid support particles within the column.

Separation and Detection: Due to these differential interactions and varying speeds, the components of the mixture separate into distinct "bands" as they travel through the column. As each separated component exits the column, it passes through a detector, which generates an electrical signal that is proportional to the amount of the compound. This signal is then plotted against time to produce a chromatogram.

Detection Methods: 

 

• Flame Ionization Detector (FID).
• Thermal Conductivity Detector (TCD).
• Electron Capture Detector (ECD).
• Nitrogen-Phosphorus Detector (NPD) / Thermionic Detector.
• Flame Photometric Detector (FPD).
• Mass Spectrometry (GC-MS).

Ion-exchange:

 Principle: 

Stationary Phase (Ion Exchange Resin/Matrix):

• This consists of an insoluble polymer matrix (often made of polystyrene, cellulose, agarose, or polyacrylamide) to which charged functional groups are permanently attached.

Mobile Phase: This is a buffer solution, typically with a controlled pH and ionic strength.

Binding: Molecules in the sample that possess a net charge opposite to that of the stationary phase will bind to the resin by displacing the exchangeable counter-ions.

Elution: The bound molecules are then eluted by changing the properties of the mobile phase, which reduces the electrostatic interaction between the bound molecules and the resin.

Regeneration: After elution, the column is regenerated by washing it with a high concentration of salt or a buffer at a specific pH to restore the resin to its initial state, ready for the next sample.

Applications:

• Protein and Enzyme Purification.
• Nucleic Acid Separation and Purification.
• Amino Acid and Peptide Analysis.
• Food and Beverage Industry.
• Pharmaceuticals.
• Nuclear Industry.