Why Did My Kastle-Meyer Test Yield a False Negative?

[Ellyse]

How to prepare the Kastle-Meyer reagent?
I had search from google but there got a lot of way.
Is that use phenolphthalein only? or need to add others substance into phenolphthalein such as zinc powder?
I very blur with this, and I do not know whether if i use the pure phenolphthalein is work for the experiment.

 

[chemisttree]

Phenothalein is reduced with zinc dust to produce phenothalin (note the difference in spelling). The latter produces a light yellow anion at high pH. Something like NaOH or NaCO3 is added to the reagent mix so that the anion is always present. When hemoglobin is present with peroxide, the phenothelin is oxidized back to bright pink phenothalein.

 

[Ellyse]

So how long time we need to reflux the phenothalin? Did you tried before?

 

[chemisttree]

I would think that you would add the phenolthalein to a basic solution of ethanol and reflux it over zinc dust until the pink color is completely discharged to the basic form of phenolthalin. The endpoint of the reaction is self-indicating.

 

[Ellyse]

Ok .. I have tried the experiment juz now.
first, I reflux the phenolphthalein
and then .. i prepare 3 solutions which are
1. Ethanol
2. 2cm³ of phenolpthalin, 10cm³ of distilled water and 2cm³ of ethanol (mixed)
3. 3% Hydrogen peroxide
The experiment is work at first (give a positive result). However, after 1 hour I redo the test again by using the same phenolpthalin, I got the negative result which are after I dropped the second solution, it shows the pink colour. Why this will happen? Is that the phenolpthalin been oxidized? How to keep the phenolpthalin well? is that decant it into a brown bottle(with a small amount of zinc powder) and store it in refrigenarator?

** The blood that I use is only animal blood in the whole experiment

 

[chemisttree]

Ok .. I have tried the experiment juz now.
first, I reflux the phenolphthalein
and then .. i prepare 3 solutions which are
1. Ethanol
2. 2cm³ of phenolpthalin, 10cm³ of distilled water and 2cm³ of ethanol (mixed)
3. 3% Hydrogen peroxide
The experiment is work at first (give a positive result). However, after 1 hour I redo the test again by using the same phenolpthalin, I got the negative result which are after I dropped the second solution, it shows the pink colour. Why this will happen? Is that the phenolpthalin been oxidized? How to keep the phenolpthalin well? is that decant it into a brown bottle(with a small amount of zinc powder) and store it in refrigenarator?

Yes, it can oxidize in air to form a pink anion and give you a false positive. Don't ever put something you intend to reduce into a closed container unless you are sure it can handle the H2 pressure it will generate.

Try sparging with argon and storing cold under an argon atmosphere. I would sparge it with argon, attach the lid (a little dexterity required here), tape it shut and store it in a secondary jar (small mayo jar) that was sparged with argon and fitted with a tight, taped cap. Make sure that all of your solutions are sparged with argon before use and during storage. Oxygen can get into those as well and give you a false positive.

 

[Ellyse]

besides of argon, zinc powder can also prevent oxidized rite?

 

[minerva]

If the phenolpthalin has been oxidised in the storage bottle into phenolpthalein, you should see the pink color of the phenolpthalein in the reagent, before you add any other chemical or material to it.

Storing the phenolpthalin reagent in a dark glass bottle in the refrigerator with a little excess zinc powder in the bottom of the bottle should probably be OK if you don't have access to argon, yes.

 

[Ellyse]

Minerva, so, in your opinion, why the experiment gave me a negative result (refer to post #5)?

 

[~christina~]

Ok .. I have tried the experiment juz now.
first, I reflux the phenolphthalein
and then .. i prepare 3 solutions which are
1. Ethanol
2. 2cm³ of phenolpthalin, 10cm³ of distilled water and 2cm³ of ethanol (mixed)
3. 3% Hydrogen peroxide
The experiment is work at first (give a positive result). However, after 1 hour I redo the test again by using the same phenolpthalin, I got the negative result which are after I dropped the second solution, it shows the pink colour. Why this will happen? Is that the phenolpthalin been oxidized? How to keep the phenolpthalin well? is that decant it into a brown bottle(with a small amount of zinc powder) and store it in refrigenarator?

** The blood that I use is only animal blood in the whole experiment

I'm not sure I'm understanding you. The test procedures should be 1. Add the KM reagent (Kastle-Meyer) 2. add substance to be tested (nothing should happen here unless false +) 3. add 3% H2O2 (see color change) It's important to note that you would need to run proper controls. (positive/negative/unknown)

The solution in my lab was stored in a brown bottle in the fridge with some solid zinc at the bottom.

Minerva, so, in your opinion, why the experiment gave me a negative result (refer to post #5)?

As for the negative result, is this with a known blood sample? The reason I ask is that false negatives are possible with this test. Situations which could cause false -'s include the presence of strong reducing agents, presence of substances that cause pH shifts from the optimal, and conditions that destroy the hemoglobin catalyst. Thus controls are necessary to determine this. Of course, the reagent preparation could be the problem here if what I just described is not the case. If I may ask, what is your experiment exactly?

There are other reagents that can also be used to test for the possible presence of blood (i.e. Leucomalachite Green(LMG) and Luminol). The thing is that the color tests don't confirm the presence of blood since other substances can give false +'s (plant peroxidase/strong oxidants etc.) These when combined with crystal tests confirm the presence of blood.