Greening Forensic Chemistry: Identification of Blood

Katherine Shaw and Duyen Ha Organic Chemistry Fall 2009
University of New Hampshire Manchester ,
In cooperation with Mrs. Ashworth's Chemistry Course at Manchester Memorial High School


Electron Microscope Image of Circulating Blood

Purpose: To green Mrs. Ashworths Identification of Blood lab, in accordance with thetwelve principles of green chemistry

Background: The original lab to be modified consisted of three different tests to identify blood. These were:
1.Luminol Test
2.leuchomalite green test
3.o-tolindine test

Proposal A:
Teichmann Crystallization Test


To replace the o-tolindine test in the experiment with a safer alternative. After research a suitable alternative test was found, the Teichmann Crystalliztion test. In this test the hemoglobin of blood reacts with acetic acid and sodium chloride to form linear and rectangular crystals. This test confirms whether a sample is blood, based on the appearance of Teichmann Crystals. This alternative test encompasses several green chemistry principles. The first being that safer chemicals should be used- this is accomplished by removing o-tolindine (principle 4). The o-tolindine solution is a known carcinogen and also requires the use of a solvent. By replacing the o-tolindine the use of a solvent is no longer necessary. Eliminating the use of a solvent is a second green chemistry principle (principle 5). The Teichmann Crystallization Test for blood differs from the test in the preliminary experiment. It involves a crystallization reaction versus a color change reaction which all three original tests were. This test is not only greener, it also adds variety to the experiment.

The Teichmann Crystallization test for blood was discovered by polish anatomist Ludwig Teichmann in 1853. While attending school in Germany he researched the reactions of various organic compounds in blood, and also discovered the crystals that form in the above experiment. These Teichmann crystals were named after Ludwig Teichmann.

each group or individual will need the following:

1.) microscope slides
2.) NaCl anyhrdrous
3.) sheeps blood
4.) glacial acetic acid
5.) heat source
6.) pipettes
7.) watch glass
8.) microscope


1. Using a pipette drop 3 to 4 drops of sample blood onto a clean microscope slide.
2. Using a plastic pipette place .3 mL of glacial acetic acid on microscope slide. Place acetic acid directly on blood.(Very Important)
3. Weigh .5 g of NaCl and place on sample
4. Place a clean watchglass on heatsource and place slide on top of the watchglass
5. Heat on low heat for 5 to 10 minutes or until sample looks completely dry
6. Take sample off of heat source and allow to cool for 5 minutes
7. Observe sample on microscope; if it is blood you should observer rhombic crystals also known as Teichmann crystals.


Wear appropriate lab clothing, including goggles, lab coat and gloves
Use caution when heating sample
Exposure to Acetic Acid can cause burns and irritation
Exposure to sheep blood can cause an immune response.
See links below for more information:

Acetic Acid MSDS
Sheep Blood Safety Information


Hemoglobin is reacting with acetic acid and sodium chloride to form Teichmann Crystals depicted in red in the diagram below.
Reaction of Teichmann Crystallization Test


Although we could not find a step by step procedure, we were able to devise one after reading various descriptions of the test. After the first trial we had recovered Teichmanns Crystals and decided to run through the experiment several times while manipulating the amounts of blood, reagents and temperature. Through trial and error we found the above procedure to be the most reliable in the recovering of crystals. This method was repeated several times to ensure crystals would develop. Resulting in three successful trials.
The reaction by which Teichmann crystals are formed is by the reaction of the hemoglobin in blood with the glacial acetic acid and sodium chloride. The hemoglobin appears to gain chloride ions and thus transform into a crystal. It should be noted that the whole sample was not crystallized, only sprecific segments were crystallized. These crystals were dark red and orange as well as long and rhombic shaped. Some undissolved salt crystals can be viewed under the microscope but these cubic crystals do not confirm blood- just salt. More ways of identifying blood could be researched to see if there are already greener alternatives available. Also the chemical mechanism of this reaction, is not very well known. After learning about the mechanism it would probably be easier to find a greener alternative.

Proposal B:

If potentially hazardous chemicals cannot be replaced with green alternatives, these tests could be removed from the experiment or reduced.


The original lab consisted of 3 tests for blood. Each of these tests were to be done on 6 dilutions of blood and 7 nonblood samples. This could be reduced by only testing 3 dilutions of blood and 4 non blood samples. This would reduce the testing chemicals needed by 46%. This certainly encompasses green principle 1 which states "it is better to prevent waste". This would allow the original lab protocal to be used and maintain the authenticity of the experiment while reducing waste.

No experimental procedure was run for this lab due to the successful of plan A.

Plan C:

Blood Typing Activity

Objective: To avoid using hazardous materials, a simple blood typing technique can be performed. This experiment is designed to identify the blood type in an ABO system and Rh system from blood samples of a mother, her child, and 3 possible fathers. The idea is to classify who is the child's biological father.


1. Add one drop of anti-a antiserum to each of the five boxes in the 5-compartment plastic tray.
2. Put in one drop of the mother’s blood in the first box with anti-a antiserum, one drop of infant’s blood in the second box with anti-a antiserum. One drop of each of the three man’s blood in the last 3 boxes with anti-a antiserum.
3. Then mix it up well with a plastic rod. If there’s clumps formed, it should look grainy. If there’s no clump formed, it should look even and homogeneous.

4. Repeat the steps with anti-b and anti-d (anti-rh) antisera using a different plastic rod in order to prevent cross-contamination.


Blood sample of a mother, her kid and 3 possible fathers
Blood typing antisera (anti-a, anti-b, anti-d (anti-rh))
5-Compartment plastic test trays
plastic stirring rods

Safety: Wear gloves, lab coat, and goggle.
Biohazard of working with human blood. MSDS data

Proposal C was not performed, due to the success of proposal A.

Collaboration with teacher was not possible.


Reaction of Teichmann Crystallization Test
Electron Microscope Image of Blood

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