3b-scientific Experiment Set - On the Trail of Flavour Enhancers User Manual download pdf (Page 6)

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V. Experiment

Sample Preparation

Weigh 3 g of a sample and put it into an Erlenmeyer flask. Mix it with 5 ml of extraction solution and finely

ground it in a mortar. If there is not a large enough mortar available, the sample may also be processed

portion by portion. Filter the extract through a paper filter. The filtrate shall be used for the thin-layer

chromatography.

If you want to carry out the thin-layer chromatography later, you can store the filtrate frozen at -18 °C for

several weeks.

Thin-Layer Chromatography

Note: Please do not touch the chromatography paper with your fingers. Please wear powder-free gloves

use tweezers and touch only the edge of the paper when moving it. The ruler that you need for later expe-

rimental steps should be absolutely clean!

First, draw a thin horizontal line with a pencil about 1.5 cm from the bottom of the chromatography paper

(see Fig 1). This is the starting line for the samples and the glutamine acid standard. Below this starting

line, at spaces of 8 mm, mark the numbers 1,2,3 etc. from left to right where the traces will run. Part of the

above mentioned glutamic acid standard (1 mg/ ml) shall be diluted 1:3 and 1:5 diluted with H

O, so that

then 3 standards with the concentrations 1mg/ml, 0.33 mg/ml and 0.2 mg/ml are available. Fill the chro-

matography chamber with the solvent mixture to about 7 mm and seal it with the lid. For the saturation of

the chamber, it should be incubated for at least 15 min.

Using capillary tubes, apply both the samples and the standards (10 µl each) on the starting line at equally

spaced intervals (above the numbers). For each sample and each standard a new capillary tube must be

used.

For application, place the chromatography paper horizontally on the table, then open the chromatography

chamber, and place the chromatography paper loaded with samples and standards with the starting line

pointing downwards into the chamber. Close it again with the lid.

Now the solvent mixture rises upward in the chromatography paper. When the solvent has risen about 7cm

(measured from the starting line), remove the paper from the chamber and put it on the table to dry. The

drying process can be accelerated using a hair dryer on a low setting or in an oven. The chromatogram now

ready to be stained, however if you prefer it is possible to store the chromatogram in the refrigerator pro-

tected from light for a few days.

Staining of Amino Acids

Note: The bowl has to be absolutely clean (no fingerprints etc.) as any protein contamination would be

stained, too!

Pour some ninhydrin staining solution into a suitable bowl (glass or plastic). Then pull the chromatography

paper very briefly (1 sec.) through the staining solution and let the paper dry

in the air or an oven at about 105°C. Now the chromatogram is ready for analysis.

The stained chromatogram can be stored at room temperature in the dark. Sticking it in a workbook may

best be done with scotch tape.

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3b-scientific Experiment Set - On the Trail of Flavour Enhancers User Manual Page 6