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Hydrogen Cyanide Estimation in Sorghum
Hydrogen Cyanide Estimation in Sorghum
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.
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.
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.
Reagents preparation:
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.
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.
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.
* Very toxic by inhalation, ingestion and through skin contact. Inhalation, ingestion
or skin contact may
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.
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.
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.
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.
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 1st 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.