Kovac's reagent - difficulty making

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Jeremy Carson

Apr 20, 1997, 3:00:00 AM4/20/97

For detecting indole I use Kovac's reagent which I have made for many
years without difficulty until recently. In fact I and my colleagues
seem to have totally lost the plot now when making this reagent.

The formula I use is as follows:

p-dimethylaminobenzaldehyde 5g
iso-amyl alcohol (3-methyl butan-1ol) 75ml
conc. HCl 25ml

I dissolve the aldehyde in the amyl alcohol by warming in a water bath
at 50C. Once cooled to RT, I add the acid. Normally I end up with a
pale straw colour reagent, but recently all I get is a dark amber/red
coloured fluid which is totally useless for detecting indole.

I have read that such problems may be due to mixed alcohols in the
iso-amyl alcohol (eg. 2-methyl butan-1ol). I have tried using an
analytical grade iso-amyl alcohol but with the same undesirable
result. I have tried analytical grade p-dimethylaminobezaldehyde and
HCl but with no success either.

My colleagues have tried to make the reagent as well, I thinking that
I was making some procedural error. They have not had any success

Does anyone have any suggestions as to what is going on and most
importantly what to do to make this rather essential reagent?

The obvious answer is to buy the reagent ready made but that is a
defeat I'm not prepared to accept quite yet!

Thanks in advance

Jeremy Carson
Department of Primary Industry
Tasmania, Australia


Apr 20, 1997, 3:00:00 AM4/20/97

I had the same problem the first time I made it. THe problem was that the
glassware that I was using had not been cleaned properly. I washed everything
with Sparkleen, rinsed well with distilled water, and had no problems getting
that pale straw-coloured reagent afterwards. Also, contamination of any of
the ingredients could be another possibility.

I hope this helps.



Sep 30, 2015, 6:37:11 PM9/30/15
I have a different end result. My Kovac's reagent has more of a green coloration instead of the straw yellow that people mention. My guess is that one of the ingredients I used has going bad. When I started this position 6 years ago none of the chemicals had any dates written on them to denote when they were purchased or opened.

I tested out my green Kovac's reagent and it's showing the correct result based on using control organisms. I've had to start ordering all new chemicals due to the fact that the ones I currently have are long expired.

Robert Corbett
Department of Biology
Lamar University
Beaumont, TX
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