KROMATOGRAFI LAPIS TIPIS

KROMATOGRAFI LAPIS TIPIS TLC Persiapan KLT Proses spo/ng dan developing Spot detec7on Prepara7ve TLC Bagaimana mengecek Senyawa yang terpisah di KLT? 1.  Dengan penyinaran lampu UV (254 dan 366 nm) 2.  Dengan reaksi pewarnaan 2008 was the year where I became an amino acid chemist and as a consequence the Ninhydrin Stain has become my new friend. Many of the amino acids I handle these days are not UV ac7ve but fortunately the Ninhydrin Stain is great for visualising free amines and primary amines. Most of my amines are BOC-­‐
protected but they s7ll develop nicely with ninhydrin because the BOC group is cleaved upon treatment with the stain. Ninhydrin Stain Recipe 100 ml container 0.2 g Ninhydrin 0.5 ml Ace7c acid 100 ml n-­‐Butanol 4.5 ml Water AWer treatment with a heat gun Ninhydrin Stain tends to give brightly coloured pink to purple spots as shown above. The spots can fade rather fast so record the result immediately. In the past I have been using a Ninhydrin Stain that didn't contain water, however adding a bit of water seems to improve the result a fair bit PMA Stain is a highly versa7le general stain for developing TLC plates. The stain is easily prepared by dissolving 10 wt. % phosphomolybdic acid in ethanol. More PMA can be used however as stains go PMA is rela7vely expensive. Phosphomolydic acid (PMA) Stain Recipe 100 ml container 100 ml Ethanol 10 g Phosphomolybdic acid, H3[P(Mo3O10)4] ·∙ xH2O TLCs developed with PMA stain upon hea7ng give green/dark green/dark blue spots on a yellow/green background (see picture). Hanessian's Stain Don't you just love that feeling when you are checking your reac7on mixture by TLC and no maeer which stain you use nothing appears on the plate. If your compound also isn't UV ac7ve you've got a real problem. Well the good news is that there is one stain that will do the job for you -­‐ Hanessian's Stain! Hanessian's stain is an excellent mul7-­‐purpose stain that when used the right way usually gives blue spots (TLC plate A). Since it is a water based stain it requires vigorous hea7ng for development. However, if you overdo it the en7re plate goes dark blue (TLC plate B). Hanessian's Stain Recipe 1 100 ml container 90 ml Water 5 g Ammonium molybdate, (NH4)6Mo7O24-­‐4H20 1 g Cerium sulfate, Ce(SO4)2 10 ml Concentrated sulfuric acid -­‐ Hanessian's Stain Recipe 2 100 ml container 90 ml Water 2.5 g Ammonium molybdate, (NH4)
6Mo7O24-­‐4H20 1 g Cerium ammonium sulfate, Ce(NH4)4(SO4)
4-­‐2H2O 10 ml Concentrated sulfuric acid This is my favourite all purpose stain and aWer a quick look under the UV lamp this is my next stop. It virtually always works well to give spots in a wide variety of colours which can be handy for spo/ng what your aWer when you have mul7ple spots Vanillin Stain Recipe: 100 ml container 6 g Vanillin 1.5 ml Conc. sulfuric acid 95 ml 96% Ethanol Add the vanillin to your container followed by ethanol to give a clear solu7on. Carefully add the sulfuric acid. The final product is a clear colourless solu7on. However, aWer some dipping of plates it will quickly become a clear yellow solu7on (see picture above). How to use the vanillin stain: Run your TLC and let it dry. If you want to check for UV ac7vity you should do so now and not later. Dip the TLC plate in your vanillin stain and heat it using a heat gun (gently at first so that you don't spray the stuff all over your hood) un7l coloured spots appear. Remember to put the lid back on 7ghtly otherwise it'll dry out. To the leW there's a picture of a TLC plate that I developed today using the vanillin stain. Kromatografi kolom