Chromosome Structure and Function

Chromatography Basics

• Chromatography involves separating a mixture as it moves through a "column," leading to interaction between the mixture's components and the Stationary Phase (SP).
• Interactions between components and the SP can be due to Surface Adsorption, Partition (liquid x liquid immiscible), Ion exchange, or Exclusion.
• Exclusion Chromatography separates molecules based on size/shape; "chemically or physically" different molecules or "two isomers" can be separated.
• The distinct shapes of molecules can influence their behavior during separation, affecting the "fraction force" (resistance) they experience as they move.
• Substances with similar properties are harder to separate.
• Chromatography is a "trial technique," meaning experiments are repeated under varying conditions (different SP, MP) to achieve optimal separation.

Factors Influencing Separation

• "Overlap" between components can occur, making individual reception difficult, therefore "efficiency" and "resolution" are prioritized.
• Resolution in general, the higher resolution, the better the separation.
• Resolution is the shortest distance between two points that remain distinguishable.
• Distinctly separated colors indicate successful separation ("as two separate spots"), otherwise "overlap" occurs.

Chromatography Types

• Chromatography exists in two modes; Column chromatography and Planar Chromatography
• Column Chromatography (Preparative) involves large quantities of material that are separated.
• The material is placed in a column with "adsorbent."
• Planar Chromatography (Analytical) is performed on a solid support.
• A "inert" material like glass or aluminum foil is used as a support for a "thin layer" of "adsorbent"
• A line is drawn 0.5 cm from the end of a "plate" for the "start" line using a pencil.
• A "thin film" on a glass plate comprised of "fine powder" is created by spreading a thin layer of adsorbent.
• The surface area is increased by decreasing particle size. Further, the adsorbent in the stationary phase is combined with a binder to provide "mechanical strength."
• Following this, a second line is drawn 0.5 cm from the other end to act as the solvent front.

Process of Planar Chromatography

• Plate preparation involves applying a Staionary Phase (SP) mixed with a "binder" to an inert plate to create a thin film.
• Applying a small amount of mixture is required when applying a SP as a thin layer, due to limited adsorption sites
• Small applications are called "spots".
• A developing solvent, called the MP (Mobile phase), is used in a container, ensuring the MP height is below the sample origin line (Start).
• A lower level of MP than Spot keeps the components from dissolving, as compounds will move with the MP. Each substance in a mixture has varying affinities towards the Mobile Phase and Stationary Phase and moves until it reachees the "solvent front."
• Compounds separate through different affinites with the Mobile Phase and Stationary Phase. The solvent front is the furthest the MP can go.
• The planar analytical mode may then be used after the MP reaches the end.

Planar Analyitical Mode

• Planar Analytical Mode is when the plate is removed from the container.
• The sample's components are identified by the location of "spots" on the plate, indicating the quantity of product produced by the reaction.
• A Chemical test is used to tell whether the reaction is over, and to determine which compounds are present as long as a substance is suspected.

Chemical Reactions

• Chemical reactions can be monitored using chromatography to track reactants and products.
• Spots are produced where different products and reactants are located.

Analysis

• To identify a compound, a plate is prepared with a dot of a substance.
• By placing this dot on the start line, it is determined is this is the product of interest.
• If the spot is in the start line, the product is located.
• Key Parameters: How materials move within the MP and SP.
• Different compounds will move with different affinity with both Stationary and Mobile Phases.
• Distribution Coefficient ("K") indicates a substances affinity
  • This value is hard to measure; K= (Concentration of SP) / (Concentration of MP)
• Replace the k with a value called Rf, where the distances are calculated.
• Rf= (distance from substance)/(distance from solvent front)
  • Distance from start to the center of the spot/ Distance from the start to the solvent front.
  • The component indicates each element.
• Values are between 0 and 1.
• Cannot be zero, as there is no movement.
• Will be less than 1 because you can't go further than you started, its dependent on solvent/materials.

Components

• Components type may be determined by the amount of separated components.
• Composition may be learned, only if the compound is suspected, this may be "Tabulerked".

Important SP factors

• Can use components to classify the type of Separated compounds the type of compound.
• RR is measured, and calculated.
• The drawing process can begin, with cellulose fiber.
• Polarity can be high, the separation can then take place, there is Thin Layer Chromatography (TLC), has SP is looked at Visually, to differentiate by spots.
  • If you see the SP you are looking at colored spots - it will identify now colors are being separated
• Important SP elements.
  • Particle size must be near uniform.
  • Surface area is raised more.
  • Size is roughly recommended between 10-25Mm
• Always make sure to use Homogeneity
  • The particle size cannot be uniform.
  • It is very hard to get the particles together correctly.
  • It is difficult to move particles, particle size will be smaller later.

Column Chromatography

• Volume is same with Planar as the ones used for Planar.
  • Particle size is about [10-25 Mm)
  • Large area is bigger than around 150mm the particle will effect Surface size "Surface area".
  • There are two main Comparisons: The area is small the flow rate is high.
  • Particle size can increase. Small amounts of things flow with the Mobile Phase- MP
    • Area will decrease with Particle size, high amount needs a area to enter through - must rely on Thin - layered structure.

Adsorbents

• Possible to get polluted by the Environment - "Impurities''
  • Very polarized, and is usually with water-
• Active Sites can block. Occupying spaces.
  • When something Chemisorts in it will be a Monclayer
  • Solutions will be heated.
• Put in the oven for near 150 - 180 degrees
  • The small traces mainly occupy the Water molecules of the surface.

Applications

• Adsorbints are used to dissolve and separate
• Examples include: Liquid is still used to separate Alkaline, Amino acids, Inorganic Anions, lipids/oils, acids, steriods. Alliumna can be used to separate amino acids.
• Steroids, Vitamins amino acids. -Starxh, Cellulose powder seperates amino acids alkaloids, dyes,

Carbohydrates

• Known as sugars. and made through dehydration.
• Contain Hydrogen atoms -- Monomer turns to glucose.

Silica and Aluminium

• Silica Gell gets prepared with HO, it gets prepared with Sodium Sillicate- or hydrolysis
  • condensation polymerization occurs to for poly Silica Gell.
• • Polymers made with Hydroxl atoms.
• Alluminium Hydroxide/oxide also occcurs, with with hydrolysis- or also can occur with polymerization
• These molecules absorb Aluminium.

Planar chromotography specifics

• Thin layer chromatography means that the Chemisorpition is not working.
• Silica gel is the common used Studies for TIC- is prepared Hydrolysis
  • Mchenical Strength has a adhesive affect on Inert Plate
  • Need to change binders on the plate.
• Binders help with Adhesion: usually Calcium Sulfates, to have higher levels of mechanization .

Applications

• Binders enhance adhesion -- Visual detection depends on if the colored compounds are able to be distinguished - or able/cant tell apart the compounds. -- Can distinguish the different elements being added.

SP distinguishment

• Silica Gels contain indicator to fluoresce. -- Fluorescent: A form of omission, where a element can display certain emission- for higher energy elements. ------ Phosphorescence - can lead to different lifespans where elements have different behaviors from fluorescence.

Emission of Light

• Materials emit a type of light based on its molecules (Milli, nano.femto, micro)
• • Lifespan will change if its fluorescence or or phosphorescncence is large.
• UV.Infared

Luminescence

• indicator: high levels of energy, will produce less emission.
• • The plants that activated zinc. the light can adsorb.
  • Plates will appear with UV.

Applications with indicator

• Bind SP with solvents and a fluorescent.
• Look for points and areas where mixture travels along, can't distinguish what point has. - Need to apply UV show the distinct points on what is being shown.
  • There will be spots that show if something isn't working correctly, the elements are being rejected/or they are not showing up properly.