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Ehmann’s reagent is a mixture of Ehrlich reagent and Salkowski reagent in a ratio of 1:3 (v/v). It is used to detect indole intermediates during the thin layer chromatography. This reagent is greatly helpful to detect the present of different types of indole intermediate. However, standard indole-compounds must be run along with the sample to compare the retention factor and to determine the presence of intermediates in the sample.
To prepare Ehmann’s reagent, you need to prepare two individual reagents; 1) Ehrlich’s reagent and 2) Salkowski reagent. Ehrlich’s reagent is a 2 % solution of p-dimethylaminobenzaldehyde prepared in 100 ml of a mixture of 95 % ethanol and concentrated HCl in a 1:1 ratio (v/v).
Weigh 2 g of p-dimethylaminobenzaldehyde and dissolve it completely in 50 ml of 95 % ethanol and then add 50 ml of concentrated HCl and mix it well. Label this solution as Ehrlich’s reagent. To prepare Salkowski reagent see my post on how to prepare Salkowski reagent.
Now, take 50 ml of Ehrlich’s reagent and add 150 ml of Salkowski reagent and mix it well and store it in a brown bottle and label it as an Ehmann’s reagent. You can use it to spray over the TLC plates to visualize spots of different indole intermediates along with standards.
During the analysis, indole intermediates, such as indole-3-acetic acid, indole-3-acetamide, indole-3-acetonitrile, tryptamine, tryptophol, and indole-3-pyruvate will be used as standards. These indole standards will be run in the TLC along with the sample to be tested for the presence of indole intermediates.
After running thin layer chromatography, TLC plate will be dried and then sprayed with Ehmann’s reagent. After spraying with the reagent, allow the TLC plate to get dry and wash the TLC plate with water for two times to remove unreacted Ehmann’s reagent. After washing with water, wrap the TLC plate with tissue paper to soak the water and allow it to dry. After drying, hear the plate to 90 ºC until all the spots are visible.