Column chromatography (Silica or alumina) is one of the conventional methods, globally accepted for purifying of different class of organic compounds. As everyone is aware of, it includes glass column, silica or alumina stationary phase, eluting solvent mixture with different polarity. I have some interesting experience with this purification technique in my professional career, that I am going to share in this write-up.
Although my first experience with column chromatography was in my master course, but that was just for training/education purpose, where in I had little chance to learn or experience much.
Till now, I worked in almost seven professional research laboratories and among them, six laboratories I worked were pure organic synthesis labs (Including academic and industrial). Every time I joined the new laboratory, I have to face new challenge as the method followed in each lab for this technique was totally different. In the first laboratory (Bharavi), where I first got the professional experience, the method used was, for 1 g crude material 10 g silica was standard and if the spots are very near on TLC, for 1 g crude material 20-30 g silica. But the particle size of silica usually used was 60-120 mesh. Based on the quantity of silica used, column width and length was considered giving enough space for solvent above silica bed. My second job at NCL, Pune was again different from first one. Here the diameter of column was main factor. For 1-2 g crude, column dia should be 2-3 cm and length approximately 30 cm. For 5 g and above, the dia should be 10 cm, etc. Here again, the silica particle size was 60-120 mesh. Then I moved to Japan for PhD course at Nishino Laboratory at Kyushu Institute of technology, headed by Prof. Norikazu Nishino. He was typical Japanese Professor, very particular about the techniques and methods. He made a rule that, we have to use formula of volume of cylinder to measure the amount of silica, where in diameter and heights were variables based on quantity of crude material. So, we used to measure the silica quantity in ML in conical flask based on calculation. Here also for normal separation 20 cm height and for critical separation 30-40 cm height. Diameter again was same as in NCL, method. Here since the compounds were amino acid and peptide based polar compounds, dichloromethane and methanol were usual solvents, but silica particle size was fixed as 230-400 mesh. After finishing PhD from Japan, I moved to USA to work as postdoc in Holton laboratory at Florida State University. Here, like many US laboratories, Prof. Holton made an SOP of Flash column chromatography to purify the compounds. Obviously, the solvent selection was based on polarity of molecule (Hexane/Ethyl acetate or DCM/Methanol). Flash here means, silica particle size should be 230-400 mesh and slurry should be made in solvent mixture which give 0.2 Rf value for desired spot. Once the crude is made slurry by adsorbing on silica, it is loaded on stationary bed and then pressure is applied from top using nitrogen, so that the column should complete maximum in one hour. After moving to second postdoc at Kyushu Institute of Japan again to work in Hayase laboratory. In this lab, I had some freedom to follow my own method, as I was the senior organic chemist there. We here were working mostly on planar conjugated dyes, so there was possibility of forming aggregates, hence separation was tedious. I mixed up all my previous experiences and adopted my own method by using 230-400 mesh silica particle size and 30 cm length column, using DCM/Methanol solvent mixture (Polar compounds), but only difference is, I failed to make slurry for these dyes in silica, so I used to dissolve the crude in minimum DCM and load on stationary bed and drain. I also used to apply pressure if needed using nitrogen gas. Having spent in those previous labs, I returned back to India and started my industrial career in reputed MNC, Sigma-Aldrich (Now Merck), I again have to face new method of column technique which was as per SOP. In industries, following SOP is mandatory and its matter of compliance. Here, although the methods were somewhat matching some or other previously learnt methods, but difference here was, we use to use 250 ml, 500 ml, 1 lire and 2 liter columns volume based on the quantity of crude material and height was normally 30 cm. Since most of the compounds were moderately polar, dealing with hexane and ethyl acetate was routine business. Rarely we used to use DCM/methanol solvent mixture (Particularly for acids, amides etc). Although majority of us used 60-120 mesh particle size silica, but there were some fascinate people with lots of patience, who used to use 230-400 mesh particle size silica, with pressure applying from top by nitrogen gas.
Column chromatography, theoretically works on same principle (Adsorption and polarity), but every lab I joined gave me different experience, due to different technique practiced in that particular lab. In one lab, if silica is measured in grams, in other lab it was measured in ML. Height of the stationary phase was dependent on space between spots on TLC. If one lab followed gravity column, others followed pressure column to save time. Whatever may be the technique practiced, the purpose was same, that is purifying the compound to achieve highest purity.
To conclude with, what I can say is, column chromatography although looks simple, but there is no fixed method followed globally. It is a lab specific method, and purely depends on individual skill set. Every lab follows that particular skill set, that is comfortable to purify the compound. Not only column chromatography, there are many such techniques followed in organic synthesis labs, which as purely skill based, which makes organic chemistry interesting experimental science.
