Chromatography Part 1B PDF

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Summary

MLSC-3111, Clinical Biochemistry IIColumn Chromatography- Substances to be separated are introduced into a column packed with an absorbent (SP) and pass through the column at different rates depending on the affinity of each substance for the SP and for the MP flowing through the column. - Used in g...

Description

MLSC-3111, Clinical Biochemistry II Column Chromatography -

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Substances to be separated are introduced into a column packed with an absorbent (SP) and pass through the column at different rates depending on the affinity of each substance for the SP and for the MP flowing through the column. Used in gas and liquid chromatography.

Components of Column Chromatography -

Column, acts as the SP. Gas or Liquid, acts as the MP. Must be extremely pure to avoid contaminants from producing unstable base lines and peaks. Means to Apply Sample, sample must be injected into the MP. Means to Allow MP Flow, to allow movement of the MP. Detector, to detect substances. Stationary Phase, the column contains packing material coated with absorbent. The material can be either beads or an absorbent lining. Chromatogram, each peak zone represents a separated solute. Produces a graphical picture called the chromatogram.

Sample Preparation -

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Samples are rarely analyzed as submitted due to impurities, solute interactions and saturation of detection. Because of this, pre-treatment of samples is very common for column chromatography. Samples can be processed by: o Centrifugation o Protein Precipitation o Extraction o Filtration Derivatization (GC), chemical modification of a molecule by replacement of functional groups with other entities to make it volatile. Samples my need to be converted into a gas for chromatography.

Gas Chromatography (GC) -

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Used to separate compounds that can be vaporized without decomposition (volatile compounds). The higher the vapour pressure of a liquid at a given temperature, the higher its volatility (ability to vaporize) and the lower the normal boiling point will be. Gas-Solid Chromatography, uses a solid SP with uncoated particles with large absorbent surface area. Gas-Liquid Chromatography, SP particles coated with non-volatile liquid. Requires a carrier gas that carries the vaporized components through the column. Most common carrier gasses are Nitrogen, Argon, Helium and Hydrogen.

MLSC-3111, Clinical Biochemistry II -

First, the sample mixture in injected into a heated block and vaporized. Next, the vapour is swept by a stream of carrier gas into the system to the column (SP). Components will be adsorbed onto the SP at the head of the column. Components are then gradually desorbed by stream of fresh carrier gas. Partitioning between two phases occurs repeatedly as carrier gas carries the components toward the column outlet. The detector will then finally detect the components exiting the column to form the chromatograph.

Gas Chromatography Characteristics -

Highly volatile products will have a lower boiling point and a high vapour pressure. They will mainly stay in the gas mobile phase and travel faster and elute out faster. Less volatile compounds will have a higher boiling point and a low vapour pressure. They will stay in the gas mobile phase less and travel slower and elute out slower. Eluent passes through detector and produces electrical signal proportional to concentration of volatile components.

Injection -

Samples are injected into the system via a glass micro syringe. Typical volumes range from 0.5 – 5.0 uL. Injector has heating block set to 30-50 degrees higher than the column temperature. This allows the immediate vaporization of the sample before it enters the column. The sample can enter the column by two manners: o Split Injection, an excess volume of sample is injected, but the injector only allows a pre-determined volume to enter the column. The excess goes into the waste. o Split-less Injection, the entire injected sample enters the column with none going into the waste. These are the ones used in our lab, as we inject the entire sample directly onto the head of the column.

Column -

The column has a microscopic layer of liquid/polymer on an inert solid support inside of a column. There are two types of columns: o Capillary, has a liquid layer coated on the walls of the column. It is a very thin column. These columns are usually extremely long. o Packed, the column wall has inert particles such as a porous polymer or glass beads coated with non-volatile liquid (SP). Usually 1/8 to ¼ inch wide and 3-12 feet long.

MLSC-3111, Clinical Biochemistry II Oven -

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Serves to house the column and maintain the temperature of the column required for vaporization and separation. The rate that the sample passes through the column is directly proportional to the temperature of the column. The higher the temperature, the faster the components move through the column. Column temperature also affects the component retention (sticking time) on the column. Too hot columns cause peaks to bunch together at the beginning of the chromatogram resulting in poor resolution. Too cold columns cause some components to not elute in time resulting in components never making it to the detector for plotting on the chromatogram (Components do not elute before the program end time)....