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24_Chromatography - Chromatography “color” and “to...

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Chromatography - “color” and “to write” Originally described by Tswett - 1906 He devised a method to separate plant pigments using a tube filled with CaCO 3 . After adding a plant extract, he was able to produce several colored bands by washing the extract through the column with an organic solvent. response sample volume Approach can be used to evaluate relative retention. Not useful as a method of separation. Materials move down a column by being displaced by a more highly retained solute. Example - ion exchange / water softeners You can’t achieve complete resolution. Making the column longer has no effect
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A solute partitions between two phases (equilibrium). Separation is based on relative retention. Making the column longer will increase the degree of separation. In plate theory, we treat our chromatographic column as though it was a ‘static’ system in equilibrium. Each species exhibits an equilibrium between the mobile and stationary phase. A mobile phase A stationary phase K = For a separation, assume that: The column is of fixed length. Flow is held constant. We can then describe several terms based on how our average sample species performs. The first thing to do is measure how long it takes for our solute to travel through our column.
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where V R = t R x flowrate V R or t R The average linear rate for the solute is: v = L / t R where L is the column length. And for the mobile phase it is: u = L /t R This is simply the time required for a non- interacting substance to pass through the column. v = u x f f is the fraction of time that the solute spends in the mobile phase. f = n solute M / n solute S Since both the mobile and stationary phases have known volumes (V M , V S ), we can now determine K - our partition coefficient. v = u = u = u C M V M C M V M +C M V S 1 1 + C S V S C M V M 1 1 + KV S V M ( ( ) ) v = u ( ) This shows the factors required to have a component elute and how two materials can be separated. Each material has its own K. As K increases, elution takes longer. Other terms affect the overall separation. 1 1 + K V S / V M Raising V S General increase in retention V M General decrease in retention u Increases speed of separation. V S and V M can be altered by changing column diameter and length for a specific column packing. u can be altered by changing the flowrate. All terms can be found by knowing how the column was prepared.
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L t R L t M 1 1 + k’ x = k’ = t R - t M t M We now have a simple method to determine k values based on elution times. ! = K B K A We can also use: ! = = You can either: Determine ! based on the retention times for you solutes or Estimate if your species are separable based on ! . k’ a k’ b t R B - t M t R A - t M Mobile phase volume is proportional to column length so retention is also increased for longer columns. However, as peaks travel through the column, they broaden. Width increases with the square root of column length.
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