HYDROGEN CYANIDE ESTIMATION IN SORGHUM Scope: HCN Estimation in Sorghum

Scope: This is an analytical method to estimate the HCN in Sorghum plants: leaves, stems and but no roots in the respective labs.

Responsibility: The scope of analysis is responsible to the personals who are working in the respective departments.

Required Apparatus: Visible Spectrophotometer; vortex mixer; Cuvettes 10mm cells 1pair.

Required Glass ware:

General use: 2 no’s of 500mL Volumetric standard flasks;2no’s of 250mL volumetric standard flasks;2no’s of 100mL volumetric standard flasks; 1 no capacity 5g glass Petridis; pipettes: 25mL-1no, 10mL-1no, 2mL-1no, 1mL – 1no; wash bottle volume 500mL -1no; tissue rolls-2no’s; Brush to clean the tubes-1no; bulb suckers for pipette-2no’s; funnel,60deg.angle,long stem-1no; polyethylene tin with cap capacity 10liters-1no;

Test use: Nessler tubes-60no’s; Bark corks-70no’s; whatman filter paper No.1 -1Box; sharp edge Blades-2no’s; apron-2no’s; nose mosks-2no’s; eye specticals-2no’s; surgical gaggles-4pairs; scale-1no and H.B pencil-1no. Forceps-1no; dettol soap-1no.

Chemicals & Reagents:

Potassium cyanide grade AR; Picric acid grade AR; Sodium Carbonate grade AR, Hydrochloric acid grade AR, Chloroform grade GR; required distilled water in liters:5-10 approx.

Make: Preferable Merck / qualigens or fisher scientific

Reagents preparation:

Alkaline Picric acid:

Weigh about 12.5 gm of anhydrous Sodium carbonate (AR) in 500mL volumetric flask and transfer into 100mL distilled water previously taken, dissolve completely and into it add 2.5 gm of picric acid slowly to the same flask and stir to dissolve, prevent precipitation and make up to the mark.

Note: Picric acid must never be allowed to dry out, especially on metal or concrete surfaces. It is highly sensitive to heat, shock, or friction, because of explosive nature it is among the most hazardous substance found in the laboratory.

Before go for analysis one should know how to use this kind of chemical & better to have more awareness before use. So, go for its MSDS to know about necessary precautions: Picric acid is

available in the market as slurry form (powder in water).This is to be dried and used. The below given drying method suggested by the chemical supplier (Fisher Scientifics).

Drying Procedure: Switch on oven by setting it to 100 deg.C. Take picric acid slurry into Petridis about 3gms & close it with lid, then Keep it in oven, dry it for 3 hours and later, bring down to room temperature. Now, it is ready for use the analysis. Do not keep any other glassware whose rinsed with any solvents.

2. Preparation of bark cork with paper strips:

Take whatman No.1 cut them in size of 1 cm width, 10 cm long like as paper strip, dip these strips into alkaline picric acid solution, allow to dry and arrange them in-between the two half’s of bark cork.

3. Preparation of Hydrochloric acid (3M):

Transfer approximately 63mL of Conc. Hydrochloric acid (AR grade) into 100 mL distilled water previously taken of 250mL volumetric flask, mix it slowly, if flask is in hot allow it room-temperature and make up to the mark.

PRECAUTION INFORMATION ABOUT KCN:

* Very toxic by inhalation, ingestion and through skin contact. Inhalation, ingestion or skin contact may

be fatal. Note low LD50s below

* Personal protection Safety glasses, gloves, good ventilation. Only use if no suitable alternative

chemical is available.

*Do not work on your own. Keep a cyanide poisoning kit available at all times, and ensure that fellow workers know how to use it. Do not release into the environment.

Safety phrases (The meaning of any safety phrases which appear in this section is given here.) S1 S2 S7 S9 S13 S16 S28 S29 S45.

4.Preparation of KCN Standard Stock Solution:

(a)Weigh accurately 0.241 gms of KCN, transfer into 500mL volumetric flask add distilled water dissolve it then make up to the mark and leave it for 2hrs and label it as KCN stock, whose concentration 200mg/L.

(b)Intermediate Standard solution

Transfer 50mL of the above stock into 100 mL volumetric flask using the Pipette & sucker and make up to mark with distilled water. It strength equal to 100mg/L.

(c) Working Standard:

Transfer 50mL of the above stock into 100 mL volumetric flask using the Pipette & sucker and make up to mark with distilled water. It strength equal to 50mg/L.

Preparation of Calibration Standards:

1. Blank 0.5 mL of distilled water = 0 mg/L HCN

2. 0.2mL of 50mg/L = 10 mg as HCN

3. 0.4mL of 50mg/L = 20 mg as HCN

4. 0.6mL of 50mg/L = 30 mg as HCN

5. 0.8mL of 50mg/L = 40 mg as HCN

6. 1.0mL of 50mg/L = 50 mg as HCN

7. 1.2mL of 50mg/L = 60 mg as HCN

8. 1.4mL of 50mg/L = 70 mg as HCN

9. 1.6mL of 50mg/L = 80 mg as HCN

10.1.8mL 0f 50mg/L = 90mg as HCN

After, transferring standard solution in to all Nessler tubes, into it adds 2mL of 3.0 N HCL to each test tube including blank and immediately suspend the filter paper strip by closing with bark corks (which is prepared earlier). These are in yellow color, leave the set up as it is in the laboratory conditions for 24 hrs. Hydrochloric acid will produce Hydrogen Cyanide gas; it is adsorbed by filter paper and turns to pink color. Replace them with newly immersed fresh strips and leave for another 24hrs i.e., total reaction time 48hrs.

Measure optical density of each test tube after 24 hrs and 48hrs.

1st 24 hrs Reading: Take out the filter paper strip from test tube and immerse into another 15ml test tube contains accurate volume of 10 mL distilled water, then mix the strip inside water carefully by vortexing it. Using a test tube vortex mixer until all color of the strip is dissolved in distilled water, 2 or 3 times vortexing is enough, remove filter paper strip from distilled water, measure optical density of the colored distilled water in a spectrophotometer at 515 nm wave length, and record the reading as OD1

2nd 24hrs Reading: After 2nd 24hrs, take out the filter paper strip and put this strip into 10mL distilled water in another set of separate 15mL capacity glass test tubes. Immerse this strip completely in 10mL distilled water using glass rod, mix the strip inside water carefully by vortexing it using a test tube vertex mixer until all color of the strip is dissolved in distilled water. Remove the filter paper strip from distilled water, take optical density of colored distilled water in a spectrophotometer at 515nm, and record the reading as OD2.

Make total optical density of 24hrs (OD1) and 48hrs (OD2) for each subsample tube and use this data to plot a calibration curve in the instrument and save it in the instrument memory for sample analysis. Developed method provides the stability, reproducibility & accuracy.

0 10 20 30 40 50 60 70 80 90 1000

0.2

0.4

0.6

0.8

1

1.2

f(x) = 0.0121652631578947 xR² = 0.998803985980547

HCN Graph -surgham

HCN Concentration

abso

rban

ce

Graph: generated based for analysis of HCN in Sorghum plant

Sample Preparation:

Collect at least three representative plants for each treatment / genotype. Cut root portion and discard. Wash the plant in tap water to remove any insects, dust on the stem or leaves, dry the plants in fold of filter paper, chop each plant (including leaves and stem but not roots) separately into as small as possible. Mix and take a representative 1gm of sample in duplicate from each plant, finally chop the 1gm sample with sharp blade into small pieces as possible and put each selected sample (1gm) in separate thoroughly cleaned and dried 15mL capacity glass Nessler tube, into it add 0.5 mL chloroform and suspend a filter paper strip dipped in alkaline picric acid along with the fixing of bark cork. Keep the set up for 24hrs. After 24 hrs take out filter paper strip and put this strip into another fresh 15mL nessler tube contains 10 mL distilled water, immerse this strip completely inside of distilled water, mix this strip carefully by vortex mixer until all color of the strip is dissolved in the water. Then remove filter paper strip from the distilled water, measure its concentration by recalling the calibration curve, record it as reading(1) and in the chopped test sample remaining in the test tube, again add 0.5 mL chloroform and place fresh filter paper dipped in picric acid with the bark cork, as done before and allow it remain for another 24hrs. Similarly, take done reading by follow the method as prescribed for 1 st 24 hrs and in this way, will have two readings add readings of first 24hrs and 48hrs for each sample to get final concentration of HCN content of each sample in each tube separately using the standard curve.

Disposal details of waste (Solid & Liquid):

This method tells how to treat waste either liquid/ solid waste of their disposals without facing any evil effects & their impact. Collect all waste in a closed container including from the pipette wash to wiping of cuvettes and preserve till all the will completes. After, the analysis estimate approximate volume of the waste/ effluent which was produced of the Cyanide analysis and prepare 10% Sodium hydroxide solution mix this into the waste, mix it thoroughly and dilute the treated waste with equal volume of tap water.dig a pit (approx.size:2 ×2 feet) and discord it in the pit, then separate the filter papers, tissue paper etc; fire them immediately and finally, close the pit by putting mud to back immediately.