TITLE: Determination of Total Hydrogen Cyanide Levels in Fresh Cassava Roots using the picrate paper method
AUTHORS: Matema Imakumbili

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><div class = "justify" style = "text-align:left">This protocol describes how to analyse total cyanide (HCN) levels in fresh cassava roots using the picrate paper method. The method gives the potential cyanide content of cassava roots on a fresh weight basis. </div></div></div>

[STEPS]
?. Place the small plastic balance on its U-shaped mount so that it swings freely. It has a 100 mg weight glued inside one spoon. 
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							The small plastic balance is a useful tool for weighing the needed amount of cassava root sample. If used correctly it is an accurate tool. Exercise patience when using it to weigh your samples and learn to use it quickly and with precision. Carrying out this step quickly will lessen the time that roots are left exposed to air, which will reduce cyanide losses. If you are working from a lab you can use a 0.0001 g (0.1 mg) digital analytical balance to weigh your sample. Some labs will however not have balances that can accurately weigh weights as small as 100 mg.
?. A 1-2 mm thick cross section of the clean cassava root is cut across the middle of the root. The peel is removed and a sector is cut and its weight adjusted to 100 mg by cutting off small pieces along the straight edge of the sector and weighing on the small balance. 
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							Notes:See diagram from original document for demonstration.This is the step where you will have to use the knives for cutting. The large knife is best for cutting roots in half and for taking a slice of it. The small knife is best for cutting out the sector.Sometimes roots are very large and a sector cut in this manner will weigh more than 100 mg no matter its size. When this is the case cut out small sections along the sector to get an average representation of the HCN level of the sector. See Fig 1.
?. Place a round paper disc containing buffer at pH 6 in a flat-bottomed plastic bottle and place a 100 mg sector of cassava root on top of it. Note. Do not leave these sectors standing in the air for more than one hour before setting up the analysis. 
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?. Add 0.5 ml of clean water using the plastic pipette. 
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?. Immediately add a yellow picrate paper attached to a plastic strip. The picrate paper must not touch the liquid in the bottle. 
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?. IMMEDIATELY close the bottle with a screw capped lid.
?. As a blank, prepare another sample but with no cassava present.
?. Every time you run a set of experiments you should check that the method works OK, using a standard pink linamarin paper from the kit.
?. Place a round paper disc with a black spot containing buffer and enzyme in the bottle. Add a pink standard paper and then 0.5 ml water from a pipette and the picrate paper. IMMEDIATELY close the bottle with the screw capped lid.
?. Allow the bottles to stand for 16-24 hour at room temperature. Notes:
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							This is the incubation step. This step should be carried out in the dark. You could use a cupboard for this. Just ensure that you arrange the picrate bottles in manner that will not block you from easily accessing any of them.You can take note of the hourly changes in temperatures. On cooler days you could decide to leave the picrate papers longer i.e. for the full 24 hours.
?. Open the bottles and match the colour of the picrate papers against the shades of colour of the colour chart supplied. 
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							Once you open the bottles, carefully remove the picrate papers, still attached to the plastic strip, and place them on the plastic cap or on A4 size paper (Figs. 4 and 5). This is done to allow the picrate papers to first dry-up. Picrate papers become a bit moist during incubation and must not be touched or made to brush against anything before they dry (and avoid directly touching them even when they are dry).  Use the plastic strip to hold the picrate paper. The plastic caps are used for drying when you do not have to immediately reuse the plastic bottles. Using the A4 paper for drying allows you to continue with other samples needing the bottles.To easily remember the identification of samples that have been left to dry on the A4 paper, place the 12 picrate papers belonging to the same field on the same A4 paper and arrange them in a manner that will help you to remember their order. You must place the field label on the A4 paper.Once dry you should label the picrate papers, by placing their ID mark on the plastic sheet (Fig. 5).
?. Read off from the colour chart the total cyanide content in ppm in the cassava root. Also check out that the blank is zero and the standard gives a colour of about 50 ppm. 
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							Colour chart readings are often the same as spectrophotometer readings and are usually just as reliable. If you are not sure of how to take the chart readings then consider using the spectrophotometer. It is ultimately better to get readings from a spectrophotometer. When taking colour chart readings, do not remove the picrate papers from the plastic strips if you plan to later get spectrophotometer readings from the same picrate papers. Do not however compare the reacted picrate papers by comparing the side covered by the plastic strip with the colour chart. It is the uncovered reacted picrate paper that must be used to get the colour reading.This is why it is advised to remove the plastic strip from the picrate paper when taking colour chart readings. You might however risk losing the label on the plastic strip if you remove the picrate paper from the plastic strip. If you have to travel to a research lab to use a spectrophotometer, then you will have to prepare the picrate papers for transportation. It is advisable to still take the colour reading in case something accidentally happens to the picrate papers while in transit. To better preserve the picrate papers they will need to be individually wrapped in small plastic bags. Gladwrap can be used for this purpose. Remember to group individually wrapped reacted picrate papers from the same field together. Remember to still keep the picrate papers away from light and do not also expose them to room temperatures for long. The firm opaque storage container and cool box will again be handy. Do the transportation as quickly as possible and immediately place the picrate papers still in the storage container in the deep freezer. Although picrate papers can stay stable indefinitely in the deep freezer try to carry out the spectrophotometer reading within 1 – 2 days. Storing reacted picrate papers in this manner is also necessary when you have to wait to take spectrophotometer readings despite working from a lab with all needed equipment.
?. [USING THE SPECTROPHOTOMETER TO DETERMINE ROOT HCN LEVELS FROM THE PICRATE COLOUR CHANGES (steps 13 - 19)]
Carefully remove the plastic backing sheet (it may be washed and used again) from the picrate paper.
?. [USING THE SPECTROPHOTOMETER TO DETERMINE ROOT HCN LEVELS FROM THE PICRATE COLOUR CHANGES (steps 13 - 19)]
Place the picrate paper in a test tube and add 5.0 ml of water measured accurately with a pipette. 
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							25 - 50 ml beakers can also be used instead of test tubes. Unlike test-tubes they are wider and picrate papers can be easily removed from them. Swirling the contents of the beakers together is also easier in beakers. However, getting several beakers of this size may be difficult thus any lab ware that can be used as beaker can be alternatively used.Be careful not to spill out any of the solution while swirling the contents.
?. [USING THE SPECTROPHOTOMETER TO DETERMINE ROOT HCN LEVELS FROM THE PICRATE COLOUR CHANGES (steps 13 - 19)]
Leave the test tube at room temperature for about 30 min with occasional gentle stirring. 
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?. [USING THE SPECTROPHOTOMETER TO DETERMINE ROOT HCN LEVELS FROM THE PICRATE COLOUR CHANGES (steps 13 - 19)]
Take the blank picrate paper (see 7 above), remove its plastic sheet and place the yellow picrate paper in 5.0 ml of water for about 30 min with occasional gentle stirring.
?. [USING THE SPECTROPHOTOMETER TO DETERMINE ROOT HCN LEVELS FROM THE PICRATE COLOUR CHANGES (steps 13 - 19)]
Measure the absorbance at 510 nm of the picrate solution from 15 against the blank from 16.
?. [USING THE SPECTROPHOTOMETER TO DETERMINE ROOT HCN LEVELS FROM THE PICRATE COLOUR CHANGES (steps 13 - 19)]
The total cyanide content in ppm is calculated by the equation1 
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							Total cyanide content (ppm) = 396 x absorbance ….. Equation 1
?. [USING THE SPECTROPHOTOMETER TO DETERMINE ROOT HCN LEVELS FROM THE PICRATE COLOUR CHANGES (steps 13 - 19)]
The cyanide content obtained for the same sample of root, from both measurements 12 and 18, should be about the same. Also check that the standard value agrees using both methods.
?. [BIBLIOGRAPHY]
Egan SV, Yeoh HH, Bradbury JH. Simple picrate paper kit for determination of the cyanogenic potential of cassava flour. J Sci Food Agric. 1998;76: 39–48. Bradbury MG, Egan SV, Bradbury JH. Picrate paper kits for determination of total cyanogens in cassava roots and all forms of cyanogens in cassava products. J Sci Food Agric. 1999;79: 593–601.