Separation of major unsaturated fatty acids in plant seeds (inverse paper layering)

in paper analysis, the phase is usually supported by water , the moving phase is
organic
solvent. However,
compounds such as fatty acids
should be used with organic solvents as the support phase and water-containing solvents as mobile phases, so as to better distribute, that is, the so-called "inverse paper layering method." Because different fatty acids have different distribution coefficients in the two phases, there is a difference in the rate of movement on the
filtration paper
in the analysis, so that the mixed fatty acids are separated. Then treated with copper acetate, the fatty acids into the corresponding copper salts, and then with red ammonia color, you can get a blue-gray dot-like layering map. , materials, equipment and
reagentsmaterials: rapeseed.
(ii) instruments and equipment: 1. chromosome paper; 2. layering cylinder; 3. layering frame; 4. color plate; 5.
Oven
or hair dryer; 6. Microscope.
(III). Reagents. Three, experimental steps
(i) sample preparation cutting samples: a
driding
rape seeds, with a blade in parallel with the umbilical cord to cut a part of the outer leaves (note not to damage the embryo), into the small finger tube (or with a cave white porcelain plate instead), can also use the whole seed. Esterification: Add 0.1 ml (2 drops) KOH-CH
3
OH solution to the small
test tube
containing the sample, and crush the sample with a glass rod, then place it 6 to 8h. Add 0.15ml (2 to 3 drops) 1mol/L HCl and 0.1ml (2 drops) of petroleum ether.
(ii) dot on the side of the paper with a pencil gently draw a straight line, every 1.5cm with a pencil gently tap a mark, number. Soak the layering paper in a solution of shira oil and remove the cool dry (make the number up). At each number of the laminate paper, a small hole is pierced with a needle, and then 0.01 ml of liquid is removed from the sample with a capillary tube, which drips into the hole by number (after each sampling, wash the needle of the microscope with petroleum ether).
(iii) Expand the solvent system and conditions to fix a good point of the analysis paper on the layering frame (so that the number is down), into a layer tank containing acetic acid solution, acetic acid solution to soak the paper but the liquid surface is lower than the point sample is appropriate. Until the spread layerer rises 1cm from the top of the filter paper, remove the layering paper and cool it dry.
(iv) color: the layering paper immersed in copper acetate solution, so that the production of fatty acid copper salts. Rinse slightly with water, then put in a clean porcelain dish and rinse with running water about 0.5h. Remove the paper carefully, dry it in a 40C oven after absorbing most of the moisture with a dry towel or filter paper. The layering paper is passed by in the erythione solution, and the action of the fatty acid copper salt immediately shows blue-gray spots.
. The results
a diagram of the location of the five unsaturated fatty acids
chromatography
on the inverse paper chromatography.
: The order of unsaturated fatty acids in rapeseed in the inverse paper laminate map is bottom-up: mustard acid, peanut oleic acid, oleic acid, flaxic acid.