Introduction: Dragondroff Reagent Detection of Alkaloids by Serial Dilution.

About: I am a part-time hobbyists biotechnologist with a photonics background. I also have a YouTube channel too. I work full time now.

Hello, future Instructables People.

I am doing an Instructables on Bismuth Quatraiodide (BiI4) in water that produces a brown to a yellow colored solution and forms a precipitate. BiI4 is Dragondroff's Reagent for Alkaloid tests. I am testing Nicotine chloride, Quinne chloride in HCl-Methanol, Pepperdine from Peppers (Black peppers 5-10 percent), and PE (phenylephrine chloride) extract diluted before from 3000 ppm to 300 ppm.

There are other reagents to specifically test for alkaloids like Mandelin reagent (Ammonia metavanadate in Conc Sulfuric acid) or Mecke reagent (Selenious acid) in Concentrated Sulfuric acid 98 percent. However, these all react with sugars in my tests and are dangerous if skin contact occurs (Toxic plus) for both and extremely corrosive.

The dragondroff reagent is still toxic but not nearly as toxic as these agents.


Methanol with Alkaloids is quite toxic. Exposure to Nicotine, for example, can cause cardiac arrest and death but the patch only contains 114 mg of it and 500-1000 mg is required for a potential Lethal dose usually but wear gloves and safety goggles, etc when handling Alkaloids. PE can speed up the heart rate and lead to seizures if ingested potentially leading to death.

31.45 percent Hydrochloric acid and Iodide with KOH and bismuth are toxic and corrosive. Avoid skin contact. The bismuth tetraiodide stains stuff brown due to free iodide I3-, etc present.

I was being fairly cautious about using these Alkaloids and respect is needed to be safe with them.

Step 1: Theory Behind the Dragendorff Reagent With Alkaloids.

Most of the Alkaloids are set for 300 mg an L expect for Pepperdine which could be up to 600 mg an L which explains the dark color for the test. Here what bismuth tetraiodide does to each alkaloid in acidic conditions pH1 to pH 2.

C10H13N2H + BiI4 >> C10H13N2H-BiI4. (Complex) + H+.


C20H23N2O2H + BiI4 >>> C20H23N2O2H-BiI4 (Complex) + H+.

C9H12NO2H + BiI4 >>> C9H12NO2H-BiI4 + H+.

And Pepperdine which i am not 100 percent sure on.

C17H18NO3H + BiI4 >>> C17H18NO3H-BiI4(Complex) + H+.

Here some links for more info.

Step 2: Setup for Procedures and Material and Data Required.

Here some materials that are required.


Nicotine patches 114 mg.

Black Pepper 7.7 grams.

Tonic water with Quinine.

Phenylephdrine chloride 1 percent nose decogesent.

Methanol 99.99 percent Pure.

100 percent pure Bismuth Oxide.

Hydrochloric acid 31.45 percent.

Potassium hydroxide 95 percent.

Citric acid.


100 ml graduated flask.

250 ml graduated flask.

1000-4000 RPM, 0-60 minute Centrifgue (20 ml times 6).

Centrifgue tubes 20 ml times 6.

10 ml glass Pipette.

Disposable 3 ml PE pipettes.

Distillation setup (1 L two-necked flat Boiling flask), Oil bath, Distillation Reflux column.

Boiling chips (glass).

Hot plate (Preferably one with Magnetic stirring).

Support clamps and base.

100 g times 0.01 g Plus or Minus Digital Scale.

500 g times 0.1 g plus or Minus Digital Scale.

Step 3: Making Nicotine Solution, Etc.

Here how to make the nicotine solution. Take a 113-114 mg Nicotine patch put it into a 1 L beaker with 100 ml of 99.99 percent Pure Methanol with 1 ml of 31.45 percent HCl. Place a stir bar over the top of the patch with the methanol and using plastic wrap seal the beaker with plastic and a rubber elastic as well.

Step 4: Quinine HCl From Tonic Water and Hydrochloric Acid 1 Ml.

Here Tonic water 700 ml of it will be boiled down to 200 ml solution with 1 drop of HCl afterwords. The setup took 5 hours of high heat boil but I got a slightly yellow concentrated tonic water 290 ppm (290 mg an L) Quinine chloride solution.

It must be Kept in a dark bottle (glass that can hold at least 200 ml of the sample) to be protected from excessive light.

Step 5: Making Pepperdine OH Extraction.

I am going to describe how I extracted the Black pepper first here.

Grind up 7-8 grams Black pepper with a coffee grinder and weight out that amount. I got 7.7 grams of black pepper so I added 50 ml Methanol and started stirring it on a magnetic stirrer for a few days then I set up for simple distillation -- Reflux.

Add 7.7 grams of the pepper and the methanol to the flask with 50 ml methanol extra** and add 20 grams of boiling glass chips. This is very important and set up an Oil bath with Vegetable Oil. I am using Canola Oil. Without the Oil, there is a risk of the glass cracking and the distillation EXPLODING!

Let this solution of pepper in 100 ml Methanol with black pepper boil and reflux for 7 hrs for 5 days to get as much Pepperdine extracted as possible.

Set it Up for Centrifuging with centrifuge tubes at 2000 RPM for 10 minutes. Before that freeze fracture the bottle in the fridge to remove other impurities and let it get to room temp before using the Polypropylene centrifuge tubes. About 90-95 percent of Impurities will be removed.

2 grams of KOH (Potassium hydroxide) is added to the methanol-pepper extract allowed to stir and some HCl is added but still, the pH is near 13. So 10 ml of a 2 g of citric acid in 250 ml water was added to get the pH near 9.0 instead of 13. Since citric acid a very weak acid it did not allow it to go to the pH 7 or below.

Step 6: Phenylephrine Extract Prep.

A solution of 1 percent PE (Phenylephrine HCl) is added to 100 ml methanol with 1 ml HCl and allowed to stir at high concentrations. Then after 5 days, it is set in the fridge at -10 deg C to cause citric acid, and sodium citrate as well as sugars to crash out in theory.

The solution of PE is now 3000 mg a Liter but we need around 300 mg an L for most samples expect the Pepper extract. It has to be diluted 10 ml sample with 100 ml H20 (Water) to get a starting PE chloride salt of 300 mg an L.

Optional is to centrifuge the sample right after freezing to remove as much sugar, etc from the sample since we are not using Sulfuric acid like Mandelin reagent that would possibly ruin color results this is NOT a big issue here.

Step 7: Making the Dragendorff Reagent.

The dragondroff's Reagent consists of two chemical Iodine with Potassium iodide. (7 g iodide crystals and 10 g Potassium hydroxide in 300 ml water) and 10 g Bismuth oxide in 15 percent to 20 percent Hydrochloric acid.

The samples are when mixed Light brown but age and turn yellow to colorless within 24 hrs so each time Fresh samples of each KI and BiCl3 must be mixed.

The mixture is 15 ml KI and 15 ml BiCl3 in excess HCl. This makes a 30 ml sample. For 20 ml you need a 1:1 ratio of KI and BiCl3*H20 for this to work well.

Both the Bismuth chloride (BiCl3) and Potassium iodide (KI) should be stored in plastic or glass bottles at 4-10 degrees C to prolong the life shelf. If left for 3 months then NEW samples of each must be made!

Step 8: Dragondroff Reactivity With Alkaloids.

To 4 -50 ml tubes measure out 4 ml of the alkaloids and add enough water for 40 ml of total sample. With new pipettes pipette 4 ml of tube 1 into tube 2 and add up to 40 ml water do this until you have 1/10 serial dilution, 1/100 serial dilution, 1/1000 serial dilution and finally 1/10,000 serial dilution.

To each sample add 3 ml of "Freshly made Dragondroff's Reagent" to each tube. For PE it needs to be diluted first by 10 times to get it from 3000 mg an L to 300 mg an L!

For many Alkaloids, the limit of detection is 0.1 mg an L or 0.1 ppm. We will be going way past that to prove the limit of the Bismuth tetraiodide reagent.

Nicotine 114 mg per 100 ml.
so 1140 ppm. However, I assume only 1/3 dissolved due to patch and methanol used. Why? Weighed patch after being evaporated and the amount difference was 33 mg.

1/10 = 37.62 ppm. 1/100 = 3.762 ppm. 1/1000 = 0.3762 ppm. 1/10,000 = 0.0376 ppm.

290 ppm Quinine chloride.

1/10 = 29 ppm. 1/100 = 2.9 ppm. 1/1000 = 0.29 ppm. 1/10,000 = 0.029 ppm.

Phenylephdrine. 3000 mg a L diluted by 1/10 first.

1/10 = 30 ppm. 1/100 = 3 ppm. 1/1000 = 0.3 ppm. 1/10,000 = 0.03 ppm.

I am assuming Pepperdine at 600 ppm due to the difference in color-change, not 300 ppm despite what i say in the pictures!!

600 ppm Pepperdine extract.
1/10 = 60 ppm. 1/100 = 6 ppm. 1/1000 = 0.6 ppm. 1/10,000 = 0.06 ppm.

Step 9: Final Results.

All of the Alkaloids based on Similar concentration expect the Pepperdine (Pepper methanol extract) were the same color. It gives a rough concentration of 290 to 300 mg an L diluted to 1/10,00 max. The limit is 0.1 mg an L for most Alkaloids with this reagent. Shown with the tubes themselves!

It is an easy way if you're careful to test with UV spec with better technology than I have and measure accurate concentration. This is to prove that these chemicals do react. The only chemical Alkaloid that does not react would be Caffeine and maybe Theophylline. They are structured differently thus the BiI4 cannot make a complex with these. Not sure why? The theory is very very complex it has to do with oxygen or hydrogen bonds when the solution is acidified.

A final note all these alkaloids plus the reagent are toxic and must be handled as Labelled toxic waste as well. DO NOT DUMP IT DOWN THE DRAIN.