EPA-Method-3352
METHOD #: 335.2Approved for NPDES (Technical Revision 1980) TITLE:Cyanide, Total (Titrimetric; Spectrophotometric) ANALYTE:CN Cyanide
INSTRUMENTATION:Spectrophotometer
STORET No.00720
1.0Scope and Application
1.1This method is applicable to the determination of cyanide in drinking, surface
and saline waters, domestic and industrial wastes.
1.2The titration procedure using silver nitrate with
p-dimethylamino-benzal-rhodanine indicator is used for measuring
concentrations of cyanide exceeding 1 mg/L (0.25 mg/250 mL of absorbing
liquid).
1.3The colorimetric procedure is used for concentrations below 1 mg/L of cyanide
and is sensitive to about 0.02 mg/L.
2.0Summary of Method
2.1The cyanide as hydrocyanic acid (HCN) is released from cyanide complexes by
means of, a reflux-distillation operation and absorbed in a scrubber containing
sodium hydroxide solution. The cyanide ion in the absorbing solution is then
determined by volumetric titration or colorimetrically.
2.2In the colorimetric measurement the cyanide is converted to cyanogen
chloride, CNCl, by reaction with chloramine-T at a pH less than 8 without
hydrolyzing to the cyanate. After the reaction is complete, color is formed on
the addition of pyridine-pyrazolone or pyridine-barbituric acid reagent. The
absorbance is read at 620 nm when using pyridine-pyrazolone or 578 nm for
pyridine-barbituric acid. To obtain colors of comparable intensity, it is essential
to have the same salt content in both the sample and the standards.
2.3The titrimetric measurement uses standard solution of silver nitrate to titrate
cyanide in the presence of a silver sensitive indicator.
3.0Definitions
3.1Cyanide is defined as cyanide ion and complex cyanides converted to
hydrocyanic acid (HCN) by reaction in a reflux system of a mineral acid in the
presence of magnesium ion.
4.0Sample Handling and Preservation
4.1The sample should be collected in plastic or glass bottles of 1 liter or larger
size. All bottles must be thoroughly cleansed and thoroughly rinsed to remove
soluble material from containers.
4.2Oxidizing agents such as chlorine decompose most of the cyanides. Test a drop
of the sample with potassium iodide-starch test paper (KI-starch paper); a blue
color indicates the need for treatment. Add ascorbic acid, a few crystals at a
time, until a drop of sample produces no color on the indicator paper. Then
add an additional 0.06 g of ascorbic acid for each liter of sample volume.
4.3Samples must be preserved with 2 mL of 10 N sodium hydroxide per liter of
sample (pH 2 > or = 12) at the time of collection.
4.4Samples should be analyzed as rapidly as possible after collection. If storage is
required, the samples should be stored in a refrigerator or in an ice chest filled
with water and ice to maintain temperature at 4°C.
5.0Interferences
5.1Interferences are eliminated or reduced by using the distillation procedure
described in Procedure 8.1, 8.2 and 8.3.
5.2Sulfides adversely affect the colorimetric and titration procedures. Samples that
contain hydrogen sulfide, metal sulfides or other compounds that may produce
hydrogen sulfide during the distillation should be distilled by the optional
procedure described in Procedure 8.2. The apparatus for this procedure is
shown in Figure 3.
5.3Fatty acids will distill and form soaps under the alkaline titration conditions,
making the end point almost impossible to detect.
5.3.1Acidify the sample with acetic acid (1 + 9) to pH
6.0 to
7.0.
Caution: This operation must be performed in the hood and the sample left
there until it can be made alkaline again after the extraction has been
performed.
5.3.2Extract with iso-octane, hexane, or chloroform (preference in order
named) with a solvent volume equal to 20% of the sample volume. One
extraction is usually adequate to reduce the fatty acids below the
interference level. Avoid multiple extractions or a long contact time at
low pH in order to keep the loss of HCN at a minimum. When the
extraction is completed, immediately raise the pH of the sample to
above 12 with NaOH solution.
5.4High results may be obtained for samples that contain nitrate and/or nitrite.
During the distillation nitrate and nitrite will form nitrous acid which will
react with some organic compounds to form oximes. These compounds formed
will decompose under test conditions to generate HCN. The interference of
nitrate and nitrite is eliminated by pretreatment with sulfamic acid.
6.0Apparatus
6.1Reflux distillation apparatus such as shown in Figure 1 or Figure 2. The boiling
flask should be of 1 liter size with inlet tube and provision for condenser. The
gas absorber may be a Fisher-Milligan scrubber.
6.2Microburet, 5.0 mL (for titration).
6.3Spectrophotometer suitable for measurements at 578 nm or 620 nm with a 1.0
cm cell or larger.
6.4Reflux distillation apparatus for sulfide removal as shown in Figure 3. The
boiling flask same as 6. 1. The sulfide scrubber may be a Wheaton Bubber
#709682 with 29/42 joints, size 100 mL. The air inlet tube should not be fritted.
The cyanide absorption vessel should be the same as the sulfide scrubber. The
air inlet tube should be fritted.
6.5Flow meter, such as Lab Crest with stainless steel float (Fisher 11-164-50).
7.0Reagents
7.1Sodium hydroxide solution, 1.25N: Dissolve 50 g of NaOH in distilledwater,
and dilute to 1 liter with distilled water.
7.2Lead acetate: Dissolve 30 g of Pb (C H O)3H O in 950 mL of distilled water.
2322
Adjust the pH to 4.5 with acetic acid. Dilute to 1 liter.
7.5Sulfuric acid; 18N: Slowly add 500 mL of concentrated H SO to 500 mL of
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distilled water.
7.6Sodium dihydrogenphosphate, 1 M: Dissolve 138 g of NaH2PO4*H2O in 1 liter
of distilled water. Refrigerate this solution.
7.7Stock cyanide solution: Dissolve 2.51 g of KCN and 2 g KOH in 900 mL of
distilled water. Standardize ù with 0.0192 N AgNO3. Dilute to appropriate
concentration so that 1 mL = 1 mg CN.
7.8Standard cyanide solution, intermediate: Dilute 100.0 mL of stock (1 mL = 1
mg CN) to 1000 mL with distilled water (1 mL = 100.0 ug).
7.9Working standard cyanide solution: Prepare fresh daily by diluting 100.0 mL
of intermediate cyanide solution to 1000 mL with distilled water and store in a
glass stoppered bottle. 1 mL = 10.0 ug CN.
7.10Standard silver nitrate solution, 0.0192 N: Prepare by crushing approximately 5
g AgNO3 crystals and drying to constant weight at 40°C. Weigh out 3.2647 g
of dried AgNO, dissolve in distilled water, and dilute to 1000 mL (1 mL = 1
3
mg CN).
7.11Rhodanine indicator: Dissolve 20 mg of p-dimethyl-amino-benzalrhodanine in
100 mL of acetone.
7.12Chloramine T solution: Dissolve 1.0 g of white, water soluble Chloramine T in
100 mL of distilled water and refrigerate until ready to use. Prepare fresh
daily.
7.13Color Reagent--One of the following may be used:
7.13.1Pyridine-Barbituric Acid Reagent: Place 15 g of barbituric acid in a 250
mL volumetric flask and add just enough distilled water to wash the
sides of the flask and wet the barbituric acid. Add 75 mL of pyridine
and mix. Add 15 mL of conc. HCl, mix, and cool to room temperature.
Dilute to 250 mL with distilled water and mix. This reagent is stable for
approximately six months if stored in a cool, dark place.
7.13.2Pyridine-pyrazolone solution:
7.13.2.13-Methyl-1-phenyl-2-pyrazolin-5-one reagent, saturated
solution: Add 0.25 g of 3-methyl-1-phenyl-2-pyrazolin-5-one
to 50 mL of distilled water, heat to 60°C with stirring. Cool
to room temperature.
7.13.2.23,3'Dimethyl-1, 1'-diphenyl-[4,4'-bi-2 pyrazoline] -5,5'dione
(bispyrazolone): Dissolve 0.01 g of bispyrazolone in 10 mL of
pyridine.
7.13.2.3Pour solution (7.13.2.1) through non-acid-washed filter
paper. Collect the filtrate. Through the same filter paper
pour solution (7.13.2.2) collecting the filtrate in the same
container as filtrate from (7.13.2.1). Mix until the filtrates are
homogeneous. The mixed reagent develops a pink color but
this does not affect the color production with cyanide if used
within 24 hours of preparation.
7.14Magnesium chloride solution: Weight 510 g of MgCl6H O into a 1000 mL
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flask, dissolve and dilute to 1 liter with distilled water.
7.15Sulfamic acid.
8.0Procedure
8.1For samples without sulfide.
8.1.1Place 500 mL of sample, or an aliquot diluted to 500 mL in the 1 liter
boiling flask. Pipet 50 mL of sodium hydroxide (7.1) into the absorbing
tube. If the . apparatus in Figure 1 is used, add distilled water until the
spiral is covered. Connect the boiling flask, condenser, absorber and
trap in the train. (Figure 1 or 2).
8.1.2Start a slow stream of air entering the boiling flask by adjusting the
vacuum source. Adjust the vacuum so that approximately two bubbles
of air per second enters the boiling flask through the air inlet tube.
Proceed to 8.4.
8.2For samples that contain sulfide.
8.2.1Place 500 mL of sample, or an aliquot diluted to 500 mL in the 1 liter
boiling flask. Pipet 50 mL of sodium hydroxide (7.1 ) to the absorbing
tube. Add 25 mL of lead acetate (7.2) to the sulfide scrubber.Connect
the boiling flask, condenser, scrubber and absorber in the train. (Figure
3) The flow meter is connected to the outlet tube of the cyanide
absorber.
8.2.2Start a stream of air entering the boiling flask by adjusting the vacuum
source. Adjust the vacuum so that approximately 1.5 liters per minute
enters the boiling flask through the air inlet tube. The bubble rate may
not remain constant while heat is being applied to the flask. It may be
necessary to readjust the air rate occasionally. Proceed to 8.4.
8.3If samples contain NO, and or NO add 2 g of sulfamic acid solution (7.15)
32
after the air rate is set through the air inlet tube. Mix for 3 minutes prior to
addition of H SO.
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8.4Slowly add 50 mL 18N sulfuric acid (7.5) through the air inlet tube. Rinse the
tube with distilled water and allow the airflow to mix the flask contents for 3
min. Pour 20 mL of magnesium chloride (7.14) into the air inlet and wash
down with a stream of water.
8.5Heat the solution to boiling. Reflux for one hour. Turn off heat and continue
the airflow for at least 15 minutes. After cooling,the boiling flask, disconnect
absorber and close off the vacuum source.
8.6Drain the solution from the absorber into a 250 mL volumetric flask. Wash the
absorber with distilled water and add the washings to the flask. Dilute to the
mark with distilled water.
8.7Withdraw 50 mL or less of the solution from the flask and transfer to a 100 mL
volumetric flask. If less than 50 mL is taken, dilute to 50 mL with 0.25N
sodium hydroxide solution (7.4). Add 15.0 mL of sodium phosphate solution
(7.6) and mix.
8.7.1Pyridine-barbituric acid method: Add 2 mL of chloramine T (7.12) and
mix. See Note 1. After 1 to 2 minutes, add 5 mL of pyridine-barbituric
acid solution (7.13.1) and mix. Dilute to mark with distilled water and
mix again. Allow 8 minutes for color development then read
absorbance at 578 nm in a 1 cm cell within 15 minutes.
8.7.2Pyridine-pyrazolene method: Add 0.5 mL of chloramine T (7.12) and
mix. See Note 1 and 2. After 1 to 2 minutes add 5 mL of
pyridine-pyrazolone solution (7.13.1) and mix. Dilute to mark with
distilled water and mix again. After 40 minutes read absorbance at 620
nm in a 1 cm cell.
NOTE 1:Some distillates may contain compounds that have a chlorine
demand. One minute after the addition of chloramine T, test
for residual chlorine with KI-starch paper. If the test is
negative, add an additional 0.5 mL of chlorine T. After one
minute, recheck the sample.
NOTE 2:More than 05. mL of chloramine T will prevent the color
from developing with pyridine-pyrazolone.
8.8Standard curve for samples without sulfide.
8.8.1Prepare a series of standards by pipeting suitable volumes of standard
solution (7.9) into 250 mL volumetric flasks. To each standard add 50
mL of 1.25 N sodium hydroxide and dilute to 250 mL with distilled
water. Prepare as follows:
ML of Working Standard Solution Conc. g CN
(1 mL = 10 g CN)per 250 mL
0BLANK
1.010
2.020
5.050
10.0100
15.0150
20.0200
8.8.2It is not imperative that all standards be distilled in the same manner as
the samples. It is recommended that at least two standards (a high and
low) be distilled and compared to similar values on the curve to insure
that the distillation technique is reliable. If distilled standards do not
agree within ±10% of the undistilled standards the analyst should find
the cause of the apparent error before proceeding.
8.8.3Prepare a standard curve by plotting absorbance of standard vs.
cyanide concentrations.
8.8.4To check the efficiency of the sample distillation, add an increment of
cyanide from either the intermediate standard (7.8) or the working
standard (7.9) to 500 mL of sample to insure a level of 20 g/L.
Proceed with the analysis as in Procedure (8.1.1).
8.9Standard curve for samples with sulfide.
8.9.1It is imperative that all standards be distilled in the same manner as the
samples. Standards distilled by this method will give a linear curve, but
as the concentration increases, the recovery decreases. It is
recommended that at least 3 standards be distilled.
8.9.2Prepare a standard curve by plotting absorbance of standard vs.
cyanide concentrations.
8.10Titrimetric method.
8.10.1If the sample contains more than 1 mg/L of CN, transfer the distillate
or a suitable aliquot diluted to 250ml, to a 500 mL Erlenmeyer flask.
Add lO-12 drops of the benzalrhodanine indicator.
8.10.2Titrate with standard silver nitrate to the first change in color from
yellow to brownish-pink. Titrate a distilled water blank using the same
amount of sodium hydroxide and indicator as in the sample.
8.10.3The analyst should familiarize himself with the end point of the
titration and the amount of indicator to be used before actually titrating
the samples.
9.0Calculation
9.1If the colorimetric procedure is used, calculate the cyanide, in g/L, in the
original sample as follows:
where:
A = g CN read from standard curve
B = mL of original sample for distillation
C = mL taken for colorimetric analysis
9.2Using the titrimetric procedure, calculate concentration of CN as follows:
where:
A = volume of AgNO for titration of sample.
3
B = volume of AgNO for titration of blank.
3
10.0Precision and Accuracy
10.1In a single laboratory (EMSL), using mixed industrial and domestic waste
samples at concentrations of 0.06, 0.13, 0.28 and 0.62 mg/L CN, the standard
deviations were ±0.005, ±0.007, ±0.031 and ±0.094, respectively.
10.2In a single laboratory (EMSL), using mixed industrial and domestic waste
samples at concentrations of 0.28 and 0.62 mg/L CN, recoveries were 85% and
102%, respectively.
Bibliography
1.Bark, L. S., and Higson, H. G. "Investigation of Reagents for the Colorimetric
Determination of Small Amounts of Cyanide", Talanta, 2:471-479 ( 1964).
2.Elly, C. T. "Recovery of Cyanides by Modified Serfass Distillation". Journal Water
Pollution Control Federation 40:848-856 (1968).
3.Annual Book of ASTM Standards, Part 31, "Water", Standard D2036-75, Method A, p
503 (1976).
4.Standard Methods for the Examination of Water and Wastewater, 14th Edition, p 367
and 370, Method 41 3B and D (1975).
5.Egekeze, J. O., and Oehne, F. W., "Direct Potentiometric Determination of Cyanide in
Biological Materials," J. Analytical Toxicology, Vol. 3, p. 119, May/June 1979.
6.Casey, J. P., Bright, J. W., and Helms, B. D., "Nitrosation Interference in Distillation
Testsfor Cyanide," Gulf Coast Waste Disposal Authority, Houston, Texas.