TITLE: Hydrogen cyanide determination in cassava leaves 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 prepare and analyse cassava leaf samples for their total hydrogen cyanide content by the picrate paper method. Cyanide in leaves of crops other than cassava can also be prepared and analysed using this protocol. The picrate paper method for leaf cyanide determination relies on endogenous enzymes to breakdown the cyanogenic glucosides (linamarine) in fresh cassava leaf samples. In this protocol cassava leaves are crushed to bring together the cell contents of cassava leaves, including the enzyme linamarase and the cyanogenic glucosides. Hydrogen cyanide is liberated in the process and it reacts with picrate papers that have a colour pigment which darkens with released cyanide. The variations in the darkening of picrate papers is then used to measure the amount of hydrogen cyanide released from fresh cassava leaf samples. </div></div><div class = "text-block"><div class = "justify" style = "text-align:left"></div></div><div class = "text-block"><span>This protocol has been already well outlined by Dr J.H. Bradbury and his team, so please refer to their document (see the attached document below). A kit for cyanide analysis in cassava roots called PROTOCOL E: DETERMINATION OF TOTAL CYANIDE IN LEAVES, has also been designed by them. This protocol describes how the Protocol E kit can be used to determine cyanide in cassava leaves. This protocol is not a replacement of the original protocol. Some of the contents of this protocol have however been directly copied from the original protocol; they are indicated in </span><span style = "font-weight:bold;">bold text</span><span>. This protocol however contains additional insights from things encountered during the use of the kit; this information is in </span><span style = "font-weight:bold;">normal text</span><span>. In addition to this, this protocol includes insights that Dr Bradbury shared to help troubleshoot a few unforeseen issues; these insights are in </span><span style = "font-weight:bold;font-style:italic;">italics</span><span style = "font-weight:bold;font-style:italic;">.</span></div><div class = "text-block"><div class = "justify" style = "text-align:left"></div></div><div class = "text-block"><div class = "justify" style = "text-align:left"></div></div><div class = "text-block"><div class = "justify" style = "text-align:left"><a style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;"> </span></a></div></div></div>

[STEPS]
?. [Setting up your work area]
A bench or table will be needed as a working surface. Place the mortar, pestle, scissors, paper napkins and various contents of the Protocol E kit on the work table. The mortar and pestle should have been thoroughly cleaned before the start of the analysis and so should have been the scissors. It would be convenient for you to work in or near a room with a refrigerator. In this way you will be able to keep the leaves and picrate papers cool while you work. Leaf samples should be kept refrigerated until right before preparing them for cyanide analysis. A wash area is needed for washing up the mortar and pestle after use. The mortar and pestle should be washed to avoid sample contamination with each sample ground. If the room you are working in has a tap and sink this would be ideal for washing up apparatus. A basin and water in a bucket or container could alternatively be used if the room has no washing facility. Place a small brush at the sink for scrubbing the mortar and pestle. The napkins should be used for drying the mortar and pestle and for wiping the scissors clean. Throw them after each use.
?. [Bibliography]
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.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.
?. [As leaf contents will crushed and mixed steps 2 to 6 should be carried out very quickly to minimise losses in hydrogen cyanide from the samples.]
Leaves are chopped up with scissors and the small pieces immediately ground up in a mortar and pestle. 
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							Notes:If the leaves still have petioles, remove the petioles from all leaves as it is the cyanide content in the leaf blade that is being measured. However, if you are interested in the cyanide content of petioles, its best you carry out the procedure on petioles alone. It is important that you first check the quantity of leaves in your sample before you begin cutting them up into smaller pieces. This is because, large quantities of cassava leaves need to be sub-sampled to reduce their amount before cyanide analysis. The mortar should never be filled with leaves when grinding, as a large quantity of leaves cannot be effectively and quickly ground together. That is why sub-sampling is important. The sampled leaves could also be large in size and would hence also give more than the needed amount of leaf material for cyanide analysis. Sub-sampling allows you to take a smaller but representative sample of the collected leaves. To carry out sub-sampling, you will have to cut the leaves into three parts i.e. the inner, middle and outer leaf parts as shown in Fig 1.   Put the inner leaf parts together and cut them up into smaller pieces (1 cm x 1 cm or smaller) using a scissors. Do the same for the middle and outer leaf parts. Thoroughly mix together each pile of leaf part pieces and pick a small amount of cut leaves from each pile. Place the selected cut leaf part pieces in the mortar. Remember to place an amount that can be quickly and effectively ground to give a small quantity of ground leaf sample. Leaf grinding should be done very quickly to avoid losses in hydrogen cyanide.If leaves are few and small in size they can straight away be cut up and ground without sub-sampling.Put the inner leaf parts together and cut them up into smaller pieces (1 cm x 1 cm or smaller) using a scissors. Do the same for the middle and outer leaf parts. Thoroughly mix together each pile of leaf part pieces and pick a small amount of cut leaves from each pile. Place the selected cut leaf part pieces in the mortar. Remember to place an amount that can be quickly and effectively ground to give a small quantity of ground leaf sample. Leaf grinding should be done very quickly to avoid losses in hydrogen cyanide.If leaves are few and small in size they can straight away be cut up and ground without sub-sampling.
?. [As leaf contents will crushed and mixed steps 2 to 6 should be carried out very quickly to minimise losses in hydrogen cyanide from the samples.]
Immediately weigh out 100 mg of leaves using an analytical balance in the laboratory. If in the field, use a small portable plastic balance which may be supplied in the kit if needed, which has a 100 mg weight glued inside one spoon. 
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							Notes: The small portable plastic balance supplied with the kit is a useful tool for weighing the needed amount of cassava leaf samples, while working in the field. 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 high accuracy. The less time ground leaves are left exposed to air the less the introduction of errors through 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 small weights like 100 mg, so the small portable balance supplied with the kit will still have to be used.
?. [As leaf contents will crushed and mixed steps 2 to 6 should be carried out very quickly to minimise losses in hydrogen cyanide from the samples.]
Immediately place a round filter paper disc loaded with buffer at pH 6 in a flat bottomed plastic bottle (supplied in the kit), add the ground-up leaves and add 1 mL of water. Notes:
?. [As leaf contents will crushed and mixed steps 2 to 6 should be carried out very quickly to minimise losses in hydrogen cyanide from the samples.]
Immediately add a yellow picrate paper attached to a plastic strip. The picrate paper must not touch the liquid in the bottle. Immediately close the bottle with a screw capped lid. Notes:
?. [As leaf contents will crushed and mixed steps 2 to 6 should be carried out very quickly to minimise losses in hydrogen cyanide from the samples.]
Prepare another sample as above but with no leaves, to serve as a blank.
?. [As leaf contents will crushed and mixed steps 2 to 6 should be carried out very quickly to minimise losses in hydrogen cyanide from the samples.]
As a control (or standard) to check on the method, place a filter paper disc loaded with buffer and linamarase in a bottle, add a linamarin paper (see below), 0.5 mL water and a yellow picrate paper. Immediately close the bottle with a screw cap lid.
?. [As leaf contents will crushed and mixed steps 2 to 6 should be carried out very quickly to minimise losses in hydrogen cyanide from the samples.]
Allow the bottles to stand for 16-24 hour at room temperature (20-35oC). Notes: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 at any time.You can take note of the hourly changes in room temperatures during incubation. On cooler days you could decide to leave the picrate papers longer i.e. for the full 24 hours, to allow a fuller reaction time.
?. [As leaf contents will crushed and mixed steps 2 to 6 should be carried out very quickly to minimise losses in hydrogen cyanide from the samples.]
Open the bottles and match the colour of the paper against the colour chart supplied. Notes:Once you open the bottles, carefully remove the picrate papers, still on the plastic strip, and place them on their plastic caps or on A4 sized 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 them to incubate other samples. Using the A4 paper for drying however allows you to continue with other samples.To easily remember the label identifying each reacted picrate paper that has been left to dry on the A4 paper, write the label on the A4 paper next to the drying picrate paper. Once dry you should label the picrate papers, by placing their identity marks on the plastic sheet (Fig 5).
?. [As leaf contents will crushed and mixed steps 2 to 6 should be carried out very quickly to minimise losses in hydrogen cyanide from the samples.]
From the colour chart, read off the amount of total cyanide in ppm in the leaves. Also check that the blank corresponds to zero and the control gives the expected value. Notes: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 however 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. However, do not 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 plastic. Gladwrap can be used for this purpose. You can alternatively place the picrate papers in tiny plastic bags (2 cm x 10 cm). Remember to group together individually wrapped reacted picrate papers from the same field. 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 in the deep freezer once you have arrived at your destination.Storing reacted picrate papers in this manner is also necessary when you have to wait to take the spectrophotometer readings. Although picrate papers can stay stable indefinitely in the deep freezer try to carry out the spectrophotometer reading within 1 – 2 days.
?. [USING THE SPECTROPHOTOMETER TO DETERMINE LEAF HCN LEVELS FROM THE PICRATE COLOUR CHANGES]
Carefully remove the plastic backing sheet from the picrate paper.
?. [USING THE SPECTROPHOTOMETER TO DETERMINE LEAF HCN LEVELS FROM THE PICRATE COLOUR CHANGES]
Place the picrate paper in a test tube and add 5.0 ml of water measured accurately with a pipette. Notes: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 is also easier when beakers are used. However, getting several beakers of this size may be difficult thus any lab ware that can be used as a beaker can be alternatively used.Be careful not to spill out any of the solution while swirling the contents.
?. [USING THE SPECTROPHOTOMETER TO DETERMINE LEAF HCN LEVELS FROM THE PICRATE COLOUR CHANGES]
Leave the test tube at room temperature for about 30 min with occasional gentle stirring. Notes:
?. [USING THE SPECTROPHOTOMETER TO DETERMINE LEAF HCN LEVELS FROM THE PICRATE COLOUR CHANGES]
Measure the absorbance at 510 nm of the picrate solution from 13 against the blank from 6.Notes:
?. [USING THE SPECTROPHOTOMETER TO DETERMINE LEAF HCN LEVELS FROM THE PICRATE COLOUR CHANGES]
The total cyanide content in ppm is calculated by the Equation 1  
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							Total cyanide content (ppm) fresh weight = 396 x absorbance ….. Equation 1
?. [USING THE SPECTROPHOTOMETER TO DETERMINE LEAF HCN LEVELS FROM THE PICRATE COLOUR CHANGES]
The cyanide content obtained for the same sample of leaf, from both the colour chart and spectrophotometer, should be about the same. Also check that the standard value agrees using both methods.
?. [USING THE SPECTROPHOTOMETER TO DETERMINE LEAF HCN LEVELS FROM THE PICRATE COLOUR CHANGES]
Carefully remove the plastic backing sheet (it may be washed and used again) from the picrate paper.