Column Chromatography
What is Column Chromatography used for?
Column Chromatography that like to make it for though finding mixture obtaining and product of high purity is on differences between the tensions of the mixture components by the column. Generally this station reface used to build up the column is silica the mobile phases very often the mixture in non-polar solvents such as hexene and acetate
Preparing a Column
Quantity and integrity of the column ascension and termination the efficiency and accuracy of separation in your column. where you screw should in obtaining pure components this can be done in the following weight plug in the glass column with a small amount of gotten with around one point five to ten centimeters height as you are around one hundred twenty five to one hundred fifty milliliters veterans of silica and two hundred fifty milliliters to beaker use should enough to produce of around twenty to twenty five center meters high prepare your solvent mixture and mix large chronicler flask around two hundred meters be enough to start off with prepare vary slow adding a sample of the eluent to a silicon mixed rapidly with a glass rod and poor gently into the glass column open the column allowed to solve into stocks flowing into a flask below
Start slapping the blast and family carefully to help backing and to ensure absence of air bubbles. Keep lowing silica slowing why you do this. You can prepare column separate by cutting a short piece of rubber tubing and tying the cable tie. Once all the slow has been below eroded round the solvent through the crowded around five to ten times using hands pump to compress the silica column.
Once this is done you can of the test solvent to column issue goods backing by generally brought onto the top of the column. Taking care not to dissolve the surface.
Loading your Sample
Run the solvent down to the surface of the column and close the important not to expose the silica to air of solvent at just about the surface using the pipette connected back to gently add your sample the surface of the column by pipette include mixture along the sides of the column glass. Keep a smiling on the mounting your sample through veterans at this point was all your sample has been added the pipette a minute amount of eluent to issue are all you sample has reached the surface.
Run down just a small amount to the solvent into the column by the opening the tap. When the solvent has enter the column surface but there is still a minute layoffs solvent above and another pipette of eluent. Run down the solvent slightly refer to you should all the sample has into the column. Next begins pipette to eluent amount of solvent has been added of the column with the rest of the eluent
Running the column
Prepare a few black with concerns about amount of clean test tubes. Open it up adjust the flow rate to give a regular flow not to false and not to slow around five to seven minutes for test tubes be sufficient. Felling them through around two quarters of the capacity is enough.
Once around test tubes are filled prepare width to see plate and mark starting points for each test tubes another spot for your clue mixture. Spot samples from each test tubes and run TLC. Once develop this determine whether factional your mixture and using out or not. He can also analyze samples to GC for accurate determination of purity the arms of each factions should ideally be checking this way. One series of test tubes would your production faction solute selected and you are sure that the content appeal. Pool the test tube a large down the bottom flask evaporates off most of the solvent. You can then subsequently transfer to smaller flasks as necessary as you go along.
Precautions
Always ensure your column is well packed before starting the run. Do this by slapping the column well during preparation and testing the surface gently with a flattened rod.
Be careful to remove any air bubbles from within the column before starting the run. Never let the column run dry at any point as this will lead to infiltration of air and rapid cracking of the column.
Take care to maintain the integrity of the column surface. Be gentle when pouring solvents by pouring against a glass rod along the sides of the glass column. If the surface is disturbed before having started the run you can create a vortex with a glass rod and re-pack the top by slapping and compression.
Alternatively, you can use some sand to cover the top and allow for better protection.
Ensure that all your loaded sample enters the column before refilling the column with eluent. This would otherwise lead to spreading of the crude mixture throughout the entire portion of eluent added, and lead to both loss of product and poor separation.
Make sure not to let test tubes overflow during the run. This can not only cause a mess but also result in loss of your product if the product fraction is eluting out.