Modified Method: Digestion and distillation of total cyanide by MICRO DIST and FIA
Appendix A / 4.5.2: Modified Method Compilation
[30 November 2000], Revision 2.1
Digestion and distillation of total cyanide in drinking and wastewaters
using MICRO DIST and determination of cyanide by flow injection
analysis
Lachat Instruments QuikChem Method 10-204-00-1-X
1.0
SCOPE AND APPLICATION
1.1
This method is for the determination of total cyanide which is the sum of strong acid dissociable
(SAD), weak acid dissociable (WAD), and free cyanide, in samples of drinking water and
domestic and industrial wastewaters. The method is for use in EPA’s data gathering and
monitoring programs associated with the Clean Water Act, Resource Conservation and Recovery
Act, Comprehensive Environmental Response, Compensation and Liability Act, and Safe
Drinking Water Act.
1.2
The applicable range is 0.005 (minimum level, ML) to 0.500 mg CN-/L. Higher concentrations
can be determined by dilution of the original sample or by reducing the volume of the sample
loop. In a single laboratory, the method detection limit (MDL) is 0.001 mg CN-/L. The MDL
obtained from a pooled interlaboratory study, is 0.002 mg CN-/L. Sample throughput is 80
injections / h, or 45 s / sample.
1.3
Modifications to the distillation procedure beyond those expressly stated in this method are
strictly prohibited. The individual laboratory is permitted to make modifications to the
colorimetric determinative procedure described in this method provided that all quality control
requirements specified in Sections 9 and 17.5 of the method are met. The laboratory must fully
document any such changes for review, as specified in Section 9.
2.0
SUMMARY OF METHOD
By means of a passive miniature distillation device, MICRO DIST, the cyanide in the samples is released
by digesting and acidifying cyanide complexes, converting them to hydrocyanic acid (HCN). The cyanide
ion is trapped in a 1.0 M sodium hydroxide absorbing solution which is diluted to 0.25 M solution during
the distillation. By means of flow injection analysis, the 0.25 M NaOH distillate is converted to cyanogen
chloride by reaction with chloramine-T, pyridine and barbituric acid to give a red-colored complex. The
Modified Method: Digestion and distillation of total cyanide by MICRO DIST and FIA
absorbance of this complex is measured at 570 nm by measuring the peak area resulting from the sample.
The peak area is proportional to the concentration of the cyanide in the sample.
The determinative step of this method is described here only so that a complete method is provided. Any
approved continuous-flow method may be used for the determination of cyanide in the 0.25 M NaOH
MICRO DIST distillates.
NOTE: Other approved distillation procedures may use different concentrations of NaOH in the absorber
or trapping solution. Because the determinative method’s sensitivity is pH dependent, it is important to
ensure that the pH of the absorber solution from any distillation procedure is adjusted to match the pH of
calibration standards specified in the approved determinative method being used.
USEPA Reference Method “Methods for the Chemical Analysis of Water and Wastes”, EPA-600/4-79020, Revised March 1983 and 1979, Method 335.2 specifies that absorbance be measured at 578 nm
using a batch spectrophotometer. USEPA Reference Method “Methods for the Determination of
Inorganic Substances in Environmental Samples”, EPA-600/R-93-100, August 1993, Method 335.4
specifies that absorbance be measured at 570 nm. The latter wavelength is specified in this Modified
Method because Method 335.4 uses an automated continuous flow-through detector similar to that used in
the following flow injection method.
3.0
DEFINITIONS
3.1
CALIBRATION BLANK (CB) -- A volume of reagent water in the same matrix as the
calibration standards, but without the analyte.
3.2
CALIBRATION STANDARD (CAL) -- A solution prepared from the primary dilution standard
solution or stock standard solutions. The CAL solutions are used to calibrate the instrument
response with respect to analyte concentration.
3.3
INSTRUMENT PERFORMANCE CHECK SOLUTION (IPC) -- A solution of one or more
method analytes used to evaluate the performance of the instrument system with respect to a
defined set of criteria.
3.4
LABORATORY SPIKED BLANK (LSB) or LABORATORY FORTIFIED BLANK (LFB) -- an
aliquot of reagent water or other blank matrices to which known quantities of the method analytes
are added in the laboratory. The LSB or LFB is analyzed exactly like a sample, and its purpose is
to determine whether the methodology is in control and whether the laboratory is capable of
making accurate and precise measurements. The LSB or LFB can be used as the INITIAL
PRECISION AND RECOVERY (IPR) and ONGOING PRECISION AND RECOVERY (OPR)
samples as needed.
3.5
LABORATORY REAGENT BLANK (LRB) -- An aliquot of reagent water or other blank
matrices that is digested exactly as a sample including exposure to all glassware, equipment, and
reagents that are used with other samples. The LRB is used to determine if method analytes or
other interferences are present in the laboratory environment, the reagents, or the apparatus.
3.6
LINEAR CALIBRATION RANGE (LCR) -- The concentration range over which the instrument
response is linear.
3.7
MATRIX SPIKE/MATRIX SPIKE DUPLICATE (MS/MSD) -- An aliquot of an environmental
sample to which known quantities of the method analytes are added in the laboratory (MS) – the
fortified sample can then be split to provide a duplicate (MSD). The MS and MSD are analyzed
exactly like a sample, and their purpose is to determine whether the sample matrix contributes
bias or imprecision to the analytical results. The background concentrations of the analytes in the
sample matrix must be determined in a separate aliquot and the measured values or the MS and
MSD corrected for background concentrations.
Modified Method: Digestion and distillation of total cyanide by MICRO DIST and FIA
3.8
MATERIAL SAFETY DATA SHEET (MSDS) -- Written information provided by vendors
concerning a chemical's toxicity, health hazards, physical properties, fire, and reactivity data
including storage, spill, and handling precautions.
3.9
METHOD DETECTION LIMIT (MDL) -- The minimum concentration of an analyte that can be
identified, measured and reported with 99% confidence that the analyte concentration is greater
than zero.
3.10
QUALITY CONTROL SAMPLE (QCS)/ONGOING PRECISION AND RECOVERY SAMPLE
(OPR) -- A solution of method analytes of known concentrations that is used to spike an aliquot
of LRB. The QCS is obtained from a source external to the laboratory and different from the
source of calibration standards. The OPR sample may be prepared from the standard source or
external source (if the latter, the OPR and QCS are equivalent).
4.0
INTERFERENCES
4.1
Most non-volatile interferences are eliminated or minimized by the distillation procedure. Some
of the known interferences are aldehydes, nitrate-nitrite, and oxidizing agents, such as chlorine,
thiosulfate, and sulfide. Multiple interferences may require the analysis of a series of matrix
spikes and duplicates (MS/MSDs) to verify the suitability of the chosen treatment.
4.2
4.3
Thiocyanate will interfere if present.
Sulfides adversely affect the procedure by producing hydrogen sulfide during distillation. If a
drop of the sample on lead acetate test paper indicates the presence of sulfide, treat 25 mL more
than the stabilized sample (pH ≥ 12) than that required for the cyanide determination with
powdered cadmium carbonate. Yellow cadmium sulfide precipitates if the sample contains
sulfide. Repeat this operation until a drop of the treated sample solution does not darken the lead
acetate test paper. Filter the solution through a dry filter paper into a dry beaker, and from the
filtrate, measure the sample to be used for analysis. Avoid a large excess of cadmium and a long
contact time in order to minimize a loss by complexation or occlusion of cyanide on the
precipitated material.
NOTE: If samples contain particulate that would be removed upon filtration, the samples must be
filtered prior to treatment with cadmium carbonate. The collected particulate must be saved, and
the filtrate then treated using the sulfide removal procedure described above. The collected
particulate and treated filtrate must be recombined, homogenized, and included in the total
cyanide distillation.
4.4
Studies have shown that sulfide concentrations of up to to 10 mg S2-/L in the distillate can be
tolerated. However, when is it expected that hydrogen sulfide will be generated from the distilled
sample during the distillation, every effort should be made to analyze distillates within 2 hours
from the start of distillation.
4.5
If exceptionally high concentrations of sulfide (>>10 mg S2-/L) are generated by the
decomposition of sulfur-containing compounds during distillation, they could significantly bias
results, violating the QC criteria in Section 9 of this method. Should this situation occur, a
MICRO DIST tube containg the lead cation, as described in other approved distillation methods,
should be used.
When using a sulfide removal procedure, make sure to initially analyze QC samples to ensure
criteria are being met with the modified procedure.
4.6
High results may be obtained for samples that contain nitrate and/or nitrite. During the distillation
nitrate and nitrite will form nitrous acid that will react with some organic compounds to form
Modified Method: Digestion and distillation of total cyanide by MICRO DIST and FIA
oximes. These oximes will decompose under test conditions to generate HCN. The interference of
nitrate and nitrite is eliminated by pretreatment with sulfamic acid.
4.7
Oxidizing agents, such as residual chlorine, decompose most of the cyanides. Test a drop of the
sample with potassium iodide (KI)-starch paper at time of collection; a blue color indicates the
need for treatment. Add ascorbic acid, a few crystals (about 0.6 g each) at a time, until a drop of
sample produces no color in the indicator paper; then add an additional 0.6 g of ascorbic acid for
each liter of sample volume. Sodium arsenite has also been employed to remove oxidizing agents.
4.8
Method interferences may be caused by contaminants in the reagent water, reagents, and sample
processing apparatus that bias response.
5.0
SAFETY
5.1
The toxicity or carcinogenicity of each reagent used in this method has not been fully established.
Each chemical should be regarded as a potential health hazard and exposure should be as low as
reasonably achievable. Cautions are included for known extremely hazardous materials.
5.2
Each laboratory is responsible for maintaining a current awareness file of OSHA regulations
regarding the safe handling of the chemicals specified in this method. A reference file of Material
Safety Data Sheets (MSDS) should be made available to all personnel involved in the chemical
analysis. The preparation of a formal safety plan is also advisable.
5.3
All waste materials should be disposed of in a responsible manner, in accordance with federal,
state, territory, local, and tribal regulations.
5.4
The following chemicals have the potential to be highly toxic or hazardous, for detailed
explanations consult the MSDS.
5.4.1
5.4.2
5.4.3
5.4.4
Cyanide
Hydrochloric acid
Pyridine
Sulfuric acid
6.0
EQUIPMENT AND SUPPLIES
6.1
6.2
Balance -- analytical, with a 0.0001 g resolution
6.3
Flow injection analysis equipment designed to deliver and react sample and reagents in the
required order and ratios.
Glassware -- Class A volumetric flasks and pipettes or polyethylene containers as required.
Samples may be stored in plastic or glass.
6.3.1
6.3.2
6.3.3
6.3.4
6.3.5
6.4
Autosampler
Multichannel proportioning pump
Reaction unit or manifold
Colorimetric detector
Data system
Special Apparatus
6.4.1 In-line Heating Unit
Modified Method: Digestion and distillation of total cyanide by MICRO DIST and FIA
6.4.2 MICRO DIST Block and Cyanide-1 tubes. The MICRO DIST block is available from
Zellweger Analytics, Lachat Instrument Division, Milwaukee, WI. as Lachat part number
A17100. The Cyanide-1 tubes are available as Lachat part number A17001.
6.4.3 Filter paper, No. 2
7.0
REAGENTS AND STANDARDS
7.1
Preparation of Reagents
Use reagent water, distilled or deionized, free of the analyte of interest, ASTM-Type II or
equivalent, for all solutions.
Degassing with helium:
To prevent bubble formation, degas all solutions except the standards with helium. Use He at 140 kPa
(20 lb/in2) through a helium degassing tube. Bubble He vigorously through the solution for one minute.
Reagent 1.
Carrier, 0.25 M Sodium Hydroxide
In a 1 L plastic container dissolve 10.0 g NaOH [CASRN-1310-73-2] in 1.00 L or 1.00 kg water.
Reagent 2.
Phosphate Buffer, 0.71 M
By Volume: In a 1 L volumetric flask, dissolve 97 g potassium phosphate, monobasic,
anhydrous, (KH2PO4) [CASRN-7778-77-0] in approximately 800 mL water. Dilute to the mark
and invert to mix. Prepare fresh monthly.
By Weight: To a 1 L tared container add 97 g potassium phosphate, monobasic, anhydrous,
(KH2PO4) and 975 g water. Stir or shake until dissolved. Prepare fresh monthly.
Reagent 3.
Chloramine-T
Dissolve 2.0 g chloramine-T hydrate [CASRN-127-65-1] in 500 mL or 500 g water. Prepare
fresh daily.
Reagent 4.
Pyridine-Barbituric Acid Reagent
By Volume: In the fume hood, place 15.0 g barbituric acid [CASRN-67-52-7]in a 1 L beaker and
add 100 mL water, rinsing down the sides of the beaker to wet the barbituric acid. Add 75 mL
pyridine (C5H5N) [CASRN-110-86-1] while stirring and mix until the barbituric acid dissolves.
Add 15 mL concentrated hydrochloric acid (12 M HCl) [CASRN-7647-01-0] and mix. Transfer
to a 1 L volumetric flask, dilute to the mark, and invert to mix. Prepare fresh weekly.
By Weight: In the fume hood, place 15.0 g barbituric acid in a tared 1 L container and add 100 g
water, rinsing down the sides of the beaker to wet the barbituric acid. Add 73 g pyridine (C5H5N)
with stirring and mix until the barbituric acid dissolves. Add 18 g concentrated hydrochloric acid
(12 M HCl), then add an additional 825 g water and mix. Prepare fresh weekly.
Reagent 5.
MICRO DIST Distillation Releasing Agent
This will make 200 mL of 7.11 M sulfuric acid / 0.79 M magnesium chloride. It is best to do this
in the hood as HCl fumes will be released.
Place a 500 mL beaker on a top-loading balance with a 400 g capacity or greater. Tare the
balance. Into the tared 500 mL beaker place 110.8 g water. Then add and dissolve completely
32.2 g magnesium chloride hexahydrate (MgCl2.6H2O) [CASRN-7786-30-3] in this water.
Use 99% A.C.S. reagent as any trace cyanide in this reagent will contribute to a distillation blank.
Slowly add 139 g concentrated sulfuric acid in increments of 40 g at a time, swirling and
allowing to cool. HCl fumes will be released. Use the purest grade you can find as any trace
cyanide in this reagent will contribute to a distillation blank.
CAUTION!
Modified Method: Digestion and distillation of total cyanide by MICRO DIST and FIA
When adding sulfuric acid to water, the solution will become very hot. NEVER ADD WATER TO
CONCENTRATED ACID!
Transfer the solution to the MICRO DIST automatic pipet container. Place the assembled and
calibrated pipet cap on loosely and allow the solution to cool to room temperature in the hood.
When the solution is at room temperature, screw the cap on tightly. Prime the pipet and the
solution is ready to use.
7.2
Preparation of Standards
Pre-calibration with non-distilled standards (see Section 7.2 – STANDARDS NOT TO BE
DISTILLED WITH SAMPLES) is recommended to test instrument operation. However, final
calibration must be performed with distilled standards (See Section 7.2 – STANDARDS TO BE
DISTILLED WITH SAMPLES).
Standard 1.
Stock Standard 1250 mg CN-/L
In a 1 L volumetric flask dissolve 2.0 g potassium hydroxide (KOH) [CASRN-1310-58-3] in
approximately 500 mL water. Add 3.129 g potassium cyanide (KCN) [CASRN-151-50-8] .
CAUTION: KCN IS HIGHLY TOXIC. AVOID INHALATION OF DUST OR CONTACT
WITH THE SOLID OR SOLUTIONS. Dilute to the mark with water and invert three times.
Prepare fresh weekly.
STANDARDS TO BE DISTILLED WITH SAMPLES:
Standard 2.
Stock Standard 12.5 mg CN-/L in water
In a 100 mL volumetric flask add 1.00 mL Standard 1 (1250 mg CN-/L). Dilute to the mark
with water and invert to mix. Prepare fresh weekly.
Table 1 These standards will be distilled
Working Standards (Prepare Daily)
Concentration mg CN-/L
A
0.500
B
0.100
C
0.05
D
0.01
E
0.005
F
0.00
10.0
2.0
1.0
---
---
---
---
---
---
5.0
2.5
---
Weight (g) of stock standard 2
diluted to final weight (~250 g)
divided by factor below with water
10.0
2.0
1.00
---
---
---
Weight (g) of standard A diluted to
final weight (~250 g) divided by
factor below with water
---
---
---
5.0
2.5
---
Division Factor
Divide exact weight of the standard
by this factor to give final weight
0.04
0.008
0.004
0.02
0.01
---
By Volume
Volume (mL) of stock standard 2
diluted to 250 mL with water
Volume(mL) of standard A diluted to
250 mL with DI water
By Weight
STANDARDS NOT TO BE DISTILLED WITH SAMPLES:
Blank in Distillation Matrix (0.25 M NaOH)
By Volume: In a 2 L volumetric flask containing approximately 1200 mL water, add 20 g
sodium hydroxide (NaOH). Dilute to the mark and invert to mix. Prepare the blank in a large
single batch versus several smaller batches as slight changes in the 0.25 M diluent matrix can
Modified Method: Digestion and distillation of total cyanide by MICRO DIST and FIA
adversely affect the determined concentrations. The non distilled standards, blank, and carrier
should all be prepared from this single diluent.
By Weight: To a tared 2 L container, add 2 kg water and 20 g sodium hydroxide (NaOH).
Invert to mix. Prepare the blank in a large single batch versus several smaller batches as slight
changes in the 0.25 M diluent matrix can adversely affect the determined concentrations. The
non distilled standards, blank, and carrier should all be prepared from this single diluent.
Standard 2.
Stock Standard 12.5 mg CN-/L in Distillation Matrix
In a 100 mL volumetric flask add 1 mL of Standard 1 (1250 mg CN-/L). Dilute to the mark
with 0.25 M NaOH and invert to mix. Prepare fresh weekly.
These standards will not be distilled
Working Standards (Prepare Daily)
Concentration mg CN-/L
A
0.500
B
0.100
C
0.05
D
0.01
E
0.005
F
0.00
Volume (mL) of standard 2 diluted
to 250 mL with 0.25 M NaOH
10.0
2.0
1.0
---
---
---
Volume(mL) of standard A diluted
to 250 mL with 0.25 M NaOH
---
---
---
5.0
2.5
---
Weight (g) of standard 2 diluted to
final weight (~250 g) divide by
factor below with 0.25 M NaOH
10.0
2.0
1.0
---
---
---
Weight (g) of standard A diluted to
final weight (250 g) divide by factor
below with 0.25 M NaOH
---
---
---
5.0
2.5
---
Division Factor
Divide exact weight of the standard
by this factor to give final weight
0.04
0.008
0.004
0.02
0.01
---
By Volume
By Weight
Modified Method: Digestion and distillation of total cyanide by MICRO DIST and FIA
8.0
SAMPLE COLLECTION, PRESERVATION AND STORAGE
The following procedure is consistent with 40 CFR 136.3(c) Table II. When this method is used for EPA
compliance monitoring, preservation techniques and holding times may not be altered by the laboratory.
8.1
Samples may be collected in polyethylene or glass bottles. All bottles must be thoroughly cleaned
and rinsed with reagent water. Volume collected should be sufficient to insure a representative
sample, allow for replicate analysis (if required), and minimize waste disposal.
8.2
Sulfides (S2-) may adversely affect the procedure by producing hydrogen sulfide during
distillation. Sulfide also rapidly converts CN- to SCN-, especially at high pH, and the sample must
be analyzed within 24 h of collection unless the sample is treated as follows.
8.2.1 If a drop of the sample on lead acetate test paper indicates the presence of sulfide, treat 25
mL more of the stabilized sample (pH ≥ 12) than that required for the cyanide
determination with powdered cadmium carbonate. Yellow cadmium sulfide precipitates if
the sample contains sulfide. Repeat this operation until a drop of the treated sample
solution does not darken the lead acetate test paper. Filter the solution through a dry filter
paper into a dry beaker, and from the filtrate, measure the sample to be used for analysis.
Avoid a large excess of cadmium and a long contact time in order to minimize a loss by
complexation or occlusion of cyanide on the precipitated material. The resulting filtrate
must have a concentration of 10 mg S2-/L or less.
NOTE: If samples contain particulate that would be removed upon filtration, the samples
must be filtered prior to treatment with cadmium carbonate. The collected particulate
must be saved, and the filtrate then treated using the sulfide removal procedure described
above. The collected particulate and treated filtrate must be recombined, homogenized,
and included in the total cyanide distillation.
8.3
Samples must be preserved with sodium hydroxide at a pH ≥ 12 and cooled to 4oC at the time of
collection. Samples should be analyzed as soon as possible after collection. If storage is required,
preserved samples should be maintained at 4oC and may be held for up to 14 days.
8.4
Oxidizing agents, such as residual chlorine, decompose most of the cyanides. Test a drop of the
sample with potassium iodide (KI)-starch paper at time of collection; a blue color indicates the
need for treatment. Add ascorbic acid, a few crystals (about 0.6 g each) at a time, until a drop of
sample produces no color in the indicator paper. Sodium arsenite has also been employed to
remove oxidizing agents.
9.0
Quality Control
9.1
Each laboratory that uses this method is required to operate a formal quality assurance program.
The minimum requirements of this program consist of an initial demonstration of laboratory
capability, ongoing analyses of standards and blanks as a test of continued performance, and
analyses of matrix spike (MS) and matrix spike duplicate (MSD) samples to assess accuracy and
precision. Laboratory performance is compared to established performance criteria to determine
if the results of analyses meet the performance characteristics of the method.
9.1.1
The analyst shall make an initial demonstration of the ability to generate acceptable
accuracy and precision with this method. This ability is established as described in
Section 9.2. The laboratory must maintain performance records that define the quality of
the data that are generated. See Section 17.5.
Modified Method: Digestion and distillation of total cyanide by MICRO DIST and FIA
9.1.2
9.1.1.1
Each time a modification is made to the method, the analyst is required
to repeat the procedure in Section 9.2. If the change will affect the
detection limit of the method, the laboratory is required to demonstrate
that the MDL (40 CFR Part 136, Appendix B) is lower than the MDL for
that analyte in this method, or one-third the regulatory compliance level,
whichever is higher. If the change will affect calibration, the analyst
must recalibrate the equipment according to Section 10.
9.1.1.2
Alternative determinative techniques and changes that degrade method
performance are not allowed. If an analytical technique other than the
techniques specified in this method is used, that technique must have
specificity equal to or better than the specificity of the techniques in this
method for the analytes of interest.
9.1.1.3
The laboratory is required to maintain records of modifications made to
this method. These records include the following, at a minimum:
9.1.1.3.1
The names, titles, addresses and telephone numbers of the
analyst(s) who performed the analyses and modification, and of
the quality control officer who witnessed and will verify the
analyses and modification.
9.1.1.3.2
A listing of pollutant measured (total cyanide)
9.1.1.3.3
A narrative stating reason(s) for the modification.
9.1.1.3.4
Results from all quality control (QC) tests comparing the modified
method to this method, including:
(a) Calibration (Section 10)
(b) Calibration verification (Section 9.3.3 and 9.7)
(c) Initial precision and recovery (Section 9.2.2)
(d) Analysis of blanks (Section 9.3.4)
(e) Accuracy assessment (Section 9.3 and 9.4)
(f) Ongoing precision and recovery (Section 9.6)
9.1.1.3.5
Data that will allow an independent reviewer to validate each
determination by tracing the instrument output (weight or other
signal) to the final result. This data will include:
(a) Sample numbers and other identifiers.
(b) Extraction dates.
(c) Analysis dates and times.
(d) Analysis sequence/run chronology.
(e) Sample weight or volume.
(f) Extract volume.
(g) Make and model of analytical balance and weights traceable to
NIST.
(h) Copies of logbooks, printer tapes and other recordings of raw
data.
(i) Data system outputs, and other data to link the raw data to the
results reported.
Analyses of matrix spike and matrix spike duplicate samples are required to demonstrate
method accuracy and precision and to monitor matrix interferences (interferences caused
Modified Method: Digestion and distillation of total cyanide by MICRO DIST and FIA
by the sample matrix). The procedure and QC criteria for spiking are described in Section
9.4.
9.2
9.1.3
Analyses of laboratory blanks are required to demonstrate freedom from contamination.
The procedure and criteria for analysis of a blank are described in Section 9.3.4.
9.1.4
The laboratory shall, on an ongoing basis, demonstrate through calibration verification
and analysis of the ongoing precision and recovery sample that the analysis system is in
control. These procedures are described in Sections 9.6 and 9.7, respectively.
9.1.5
The laboratory should maintain records to define the quality of data that is generated.
Development of accuracy statements is described in Sections 9.3.2.
Initial demonstration of laboratory performance.
9.2.1
Method Detection Limit (MDL)— To determine MDL values, take seven replicate
aliquots of the fortified reagent water and process through the entire analytical method.
The fortifying concentration should be 1 to 5 times the MDL (see 40 CFR Part 136,
Appendix B for more information). Perform all calculations defined in the method and
report the concentration values in the appropriate units. Calculate the MDL as follows:
MDL = tS
where t is the Student's t value for a 99% confidence level and 7-1 degrees of freedom; t
= 3.14 for seven replicates, and S is the standard deviation of the replicates.
MDLs should be determined every 6 months, when a new operator begins work, or
whenever there is a significant change in the background or instrument response.
9.2.2
Initial precision and recovery (IPR)—To establish the ability to generate acceptable
precision and accuracy, the analyst shall perform the following operations:
9.2.2.1
Measure four samples of prepared at 0.2 mg-CN/L according to the
procedure beginning in Section 11.
9.2.2.2
Using the results of the set of four analyses, compute the average percent
recovery (x) and the standard deviation of the percent recovery (s) for
cyanide. Use the following equation for calculation of the standard
deviation of the percent recovery:
Equation 1
s =
Σx2 – (Σx)2
n
n–1
where:
n = Number of samples
x = Concentration in each sample
Modified Method: Digestion and distillation of total cyanide by MICRO DIST and FIA
9.2.2.3
9.3
Compare (s) and (x) with the corresponding limits for initial precision
and recovery in Table 2. If (s) and (x) meet the acceptance criteria,
system performance is acceptable and analysis of samples may begin. If,
however, (s) exceeds the precision limit or (x) falls outside the range for
recovery, system performance is unacceptable. In this event, correct the
problem and repeat the test.
Assessing laboratory performance:
9.3.1
Ongoing Precision and Recovery Standard (OPR). The laboratory must analyze at least
one LFB spiked at a level of 0.2 mg-CN/L with each batch of samples. Calculate
accuracy as percent recovery (Sect. 9.4.3). If the recovery of any analyte falls outside the
required control limits [see Section 17.5] the method is judged out of control, and the source
of the problem must be identified and resolved before continuing analyses.
9.3.2
When sufficient internal performance data become available (usually a minimum of 2030 analyses), optional control limits can be developed from the percent mean recovery (x)
and the standard deviation (S) of the mean recovery. These data can be used to establish
the upper and lower control limits as follows:
UPPER CONTROL LIMIT = x+3S
LOWER CONTROL LIMIT = x-3S
The optional control limits must be equal to or better than the required control
limits specified in Section 17.5. After each five to 10 new recovery
measurements, new control limits can be calculated using only the most recent
20-30 data points. Also, the standard deviation (S) data should be used to
establish an ongoing precision statement for the level of concentrations included in
the LFB. These data must be kept on file and be available for review.
9.3.3
Instrument Performance Check Solution (IPC). For all determinations, the laboratory
must analyze the IPC (a midrange check standard) and a calibration blank immediately
following daily calibration, after every 10th sample (or more frequently, if required), and
at the end of the sample run. Analysis of the IPC solution and calibration blank
immediately following calibration must verify that the instrument is still within ± 10
percent. If the calibration cannot be verified with the specified limits, reanalyze the IPC
solution. If the second analysis of the IPC solution confirms calibration to be outside the
limits, sample analysis must be discontinued and the cause determined. In the case of drift,
the instrument should be recalibrated. All samples following the last acceptable IPC
solution must be reanalyzed. The analysis data of the calibration blank and IPC solution
must be kept on file with the sample analyses data.
9.3.4
Laboratory reagent blanks (LRB)—laboratory reagent water blanks are analyzed to
demonstrate freedom from contamination.
9.3.4.1
Measure a laboratory reagent water blank initially (i.e., with the tests in
Section 9.2) and with each analytical batch. The blank must be subject
to the same procedural steps as a sample.
9.3.4.2
If material is detected in the blank at a concentration greater than the
Minimum Level (Section 1.2), analysis of samples is stopped until the
source of contamination is eliminated and a blank shows no evidence of
contamination. All samples must be associated with an uncontaminated
Modified Method: Digestion and distillation of total cyanide by MICRO DIST and FIA
method blank before the results may be reported for regulatory
compliance purposes.
9.4
Matrix spikes/Matrix Spike Duplicates (MS/MSD)—The laboratory must spike, in duplicate, a
minimum of 10 percent of all samples (one sample in each batch of 10 samples) from a given
sampling site or, if for compliance monitoring, from a given discharge. The two sample aliquots
shall be spiked with a cyanide standards
9.4.1
The concentration of the spike in the sample shall be determined as follows:
9.4.1.1
For compliance monitoring, the concentration of cyanide in the sample
being checked against a regulatory concentration limit, and the spiking
level shall be at that limit or one to five times higher than the background
concentration of the sample (determined in Section 9.3.1), whichever
concentration is higher.
9.4.1.2
If the concentration of cyanide in a sample is not being checked against a
limit, the spike shall be at the concentration of the precision and recovery
standard (Section 9.6) or at one to five times higher than the background
concentration, whichever concentration is higher.
9.4.2
Analyze one sample aliquot out of each set of 10 samples from each site or discharge
according to the procedure beginning in Section 11 to determine the background
concentration (B) of cyanide.
9.4.3
Calculate the percent recovery (P) of cyanide in each aliquot using the following
equation:
Equation 2
P =
100 (A – B)
T
where:
A = Measured concentration of analyte after spiking
B = Measured background concentration of cyanide
T = True concentration of the spike
9.4.4
Compare the percent recovery of cyanide with the corresponding QC acceptance criteria
in Section 17.5.
9.4.4.1
If the results of the spike fail the acceptance criteria, and the recovery of
the QC standard in the ongoing precision and recovery test (Section 9.6)
for the analytical batch is within the acceptance criteria in Section 17.5,
an interference is present. In this case, the result may not be reported for
regulatory compliance purposes and the analyst must assess the potential
cause for the interference. If the interference is attributable to sampling,
the site or discharge should be resampled. If the interference is
attributable to a method deficiency, the analyst must modify the method,
repeat the tests required in Section 9.1.2, and repeat the analysis of the
sample and the MS/MSD.
Modified Method: Digestion and distillation of total cyanide by MICRO DIST and FIA
9.4.4.2
9.4.5
If the results of both the spike and the ongoing precision and recovery
test fail the acceptance criteria, the analytical system is judged to be out
of control, and the problem shall be identified and corrected, and the
sample reanalyzed.
Compute the relative percent difference (RPD) between the two results (not between the
two recoveries) using the following equation:
Equation 3
D1 – D2
RPD
=
x 100
(D1 + D2)/2
where:
D1 = concentration of cyanide in the sample
D2 = concentration of cyanide in the second (duplicate) sample
9.4.6
The relative percent difference for duplicates shall meet the acceptance criteria in Section
17.5. If the criteria are not met, the analytical system is judged to be out of control, and
the problem must be immediately identified and corrected, and the analytical batch
reanalyzed.
As part of the QC program for the laboratory, method precision and accuracy for samples
should be assessed and records should be maintained. After the analysis of five spiked
samples in which the recovery passes the test in Section 9.3.4, compute the average
percent recovery (Pa) and the standard deviation of the percent recovery (sp). Express the
accuracy assessment as a percent recovery interval from Pa – 2 sp to Pa + 2 sp. For
example, if Pa = 90 percent and sp = 10 percent for five analyses of cyanide, the accuracy
interval is expressed as 70 – 110 percent. Update the accuracy assessment on a regular
basis (e.g., after each five to 10 new accuracy measurements).
9.5
Depending upon specific program requirements, field replicates and field spikes of the analytes of
interest into samples may be required to assess the precision and accuracy of the sampling and
sample transporting techniques.
9.6
Ongoing precision and recovery (OPR)—To demonstrate that the analysis system is in control,
and acceptable precision and accuracy is being maintained with each analytical batch, the analyst
shall perform the following operations:
9.6.1
Prepare a precision and recovery standard with each analytical batch according to the
procedure beginning in Section 11.
9.6.2
Compare the concentration with the limits for ongoing precision and recovery (OPR) in
Section 17.5. If the concentration is in the range specified, the process is in control and
analysis of blanks and samples may proceed. If, however, the concentration is not in the
specified range, the analytical process is not in control. In this event, correct the
problem, retest the analytical batch, and repeat the ongoing precision and recovery test.
Modified Method: Digestion and distillation of total cyanide by MICRO DIST and FIA
The laboratory should add results that pass the specification in Section 9.6.2 to IPR and
previous OPR data and update QC charts to form a graphic representation of continued
laboratory performance. The laboratory should also develop a statement of laboratory
data quality for each analyte by calculating the average percent recovery and the standard
deviation of the percent recovery (sr). Express the accuracy as a recovery interval from R
– 2sr to R + 2sr. For example, if R = 95 percent and sr = 5 percent, the accuracy is 85
percent to 105 percent.
9.6.3
It is suggested that the laboratory obtain a quality control sample (QCS) from a source
different from the source for the cyanide standard used routinely in this method, and that
the QCS be used for verification of the concentrations of cyanide.
9.7
The standards used for initial precision and recovery (Section 9.2.2), matrix spikes (Section 9.4),
and ongoing precision and recovery (Section 9.6) should be identical so that the most precise
results will be obtained.
9.8
Depending upon specific program requirements, field replicates and field spikes of the analytes of
interest into samples may be required to assess the precision and accuracy of the sampling and
sample transporting techniques.
10.0 CALIBRATION AND STANDARDIZATION
10.1
Prepare a series of at least 3 standards, covering the desired range, and a blank by diluting
suitable volumes of standard solution.
10.2
10.3
Calibrate the instrument, as described in section 11.
10.4
After the calibration has established, it must be verified by the analysis of a suitable quality
control sample (QCS). If measurements exceed +/-10% of the established QCS value, the
analysis should be terminated and the instrument recalibrated. The new calibration must be
verified before continuing analysis. Periodic reanalysis of the QCS is recommended as a
continuing calibration check.
Prepare standard curve by plotting instrument response against concentration values. A
calibration curve may be fitted to the calibration solution concentration/response data using the
computer. Acceptance or control limits should be established using the difference between the
measured value of the calibration solution and the "true value" concentration.
11.0 PROCEDURE
11.1
Sample Pretreatment
11.1.1 Sample Preparation at collection or prior to distillation by MICRO DIST
11.1.1.1
When this method is used for EPA compliance monitoring, preservation
techniques and holding times may not be altered by the laboratory
without approval of a variance by EPA.
Modified Method: Digestion and distillation of total cyanide by MICRO DIST and FIA
11.1.1.2
11.1.1.3
11.1.1.4
11.1.1.5
Sulfides may adversely affect the procedure by producing hydrogen
sulfide during distillation. If a drop of the sample on lead acetate test
paper indicates the presence of sulfide, treat 25 mL more of the stabilized
sample (pH ≥ 12) than that required for the cyanide determination with
powdered cadmium carbonate. Yellow cadmium sulfide precipitates if
the sample contains sulfide. Repeat this operation until a drop of the
treated sample solution does not darken the lead acetate test paper. Filter
the solution through a dry filter paper into a dry beaker, and from the
filtrate, measure the sample to be used for analysis. Avoid a large excess
of cadmium and a long contact time in order to minimize a loss by
complexation or occlusion of cyanide on the precipitated material.
NOTE: If samples contain particulate that would be removed upon
filtration, the samples must be filtered prior to treatment with cadmium
carbonate. The collected particulate must be saved, and the filtrate then
treated using the sulfide removal procedure above. The collected
particulate and treated filtrate must be recombined, homogenized, and
included in the total cyanide distillation.
High results may be obtained for samples that contain nitrate and/or
nitrite. During the distillation nitrate and nitrite will form nitrous acid
that will react with some organic compounds to form oximes. These
oximes will decompose under test conditions to generate HCN. The
interference of nitrate and nitrite is eliminated by pretreatment with
sulfamic acid.
Oxidizing agents, such as residual chlorine, decompose most of the
cyanides. Test a drop of the sample with potassium iodide (KI)-starch
paper at time of collection; a blue color indicates the need for treatment.
Add ascorbic acid, a few crystals (about 0.6 g each) at a time, until a
drop of sample produces no color in the indicator paper. Sodium arsenite
has also been employed to remove oxidizing agents.
If the sample volume originally collected is much greater than the 6.0 mL
sample distilled, and the original sample contains suspended solids,
homogenize the sample (after preservation with NaOH to pH > 12) so
that the 6.0 mL sample distilled contains the same concentration of
suspended solids as in the original sample.
11.1.2 Sample and Standard Distillation by MICRO DIST (MICRO DIST Method
Cyanide-1)
A summary of the procedure for the digestion and distillation of total cyanide is given below. The
user should read and be familiar with the MICRO DIST Reference and Methods Manual supplied
with the MICRO DIST system. The figure references in the following procedure refer to figures
in this manual.
In the following procedure, D and M refer to the marks on the ends of the collector tube. D means
‘distillation’, or ‘discarded’ end and M means ‘measuring’ end as shown below:
Modified Method: Digestion and distillation of total cyanide by MICRO DIST and FIA
Procedure:
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
Set the MICRO DIST block temperature controller to 120°C. Allow the block to warm up. This
will take about 40 min.
With the M end up, put as many collector tubes as you have samples into the collector tube rack.
Put as many sample tubes into the sample tube rack as you have samples to distill; up to 21 for
one block. Place 6.0 mL of sample or standard into each sample tube with an automatic pipet . In
this method, the standards is distilled with the samples.
In order to both release the free cyanide as HCN(g) and to digest complexed cyanides during the
digestion/distillation, first prime the re-pipettor several times into a waste container. Then, add
0.75 mL of 7.11 M sulfuric acid / 0.79 M magnesium chloride solution to the sample tube using
the automatic pipet.
Immediately push the D end of a Cyanide-1 collector tube over the open end of each sample tube
to start the seal.
Place the assembly in the press, putting the sample tube through the hole in the white base.
Before pressing the user should grip the collector tube firmly at the breakaway point to keep the
tube from shifting during the pressing procedure.
The pressing motion should be a smooth constant pressure which is just enough to slide the
sample tube inside the collector tube. A jerky, forced motion may cause added strain to the tube
and could potentially crack it. Press down on the handle until the stop ring on the sample tube
hits the D end of the collector tube.
Put on the heat-resistant gloves. Push the sample tube and D end of each tube all the way into the
preheated block so that the collector tube stop ring touches the block. Placing 21 tubes should
take less than one minute.
Set the timer for 30 min.
When 30 min is up, put on the heat-resistant gloves. Remove the first tube from the block and
immediately pull off its sample tube using a downward, twisting motion as opposed to a
sideways motion. You must pull off the sample tube within 4 s of removing it from the block or
suck-back of the sample will occur. Drop the sample tube and the hot solution left in it into a
waste bucket reserved for this purpose.
Modified Method: Digestion and distillation of total cyanide by MICRO DIST and FIA
11.
12.
13.
14.
15.
16.
17.
11.2
Invert each collector tube and place it into the collector tube rack, now with the D end up. It
should take less than two minutes to pull and separate all 21 tubes.
Allow tubes to cool for at least 10 min.
For each collector tube, hold the tube horizontally and rinse its walls with the distillate in order to
homogenize it. Slowly roll the distillate around in the tube to gather all droplets clinging to the
tube walls into the bulk of the distillate. Then, slowly return the collector tube to an upright
position so that the D end is up. Stubborn drops will often fall into the M end when the tube is
flicked with your finger.
With the D end still up, break the collector tube in half by pulling the D end hard towards
yourself to break it, then twisting and tearing off the D end. Discard the D end.
In the remaining M end of the collector tube, dilute to the 6.0 mL mark with reagent water. This
results in the original sample volume, but now in 0.25 M NaOH.
Push the second cap onto the open end of the tube (Figure 11) as far as it will go. Shake the tube
with a gentle whipping motion to mix in diluent water (Figure 12). Do not invert the sample.
With the M end down, place the tube into the collector tube rack (Figure 5).
Determine the solution in the tube with the flow injection method described in this method or
other approved continuous flow method. Using the following flow injection analysis method,
there is enough distillate for two 25 s sample periods. Samples should be determined as quickly
after analysis as possible. If a batch of samples is being analyzed (so that immediate analysis
every sample is not possible), seal both ends of the tube with Parafilm until analysis can be
performed.
FLOW INJECTION ANALYZER START-UP PROCEDURE
11.2.1 Prepare reagent and standards as described in section 7.
11.2.2 Set up manifold as shown in section 17.
11.2.3 Input data system parameters as in section 17.
11.2.4 Pump reagents and allow the system to equilibrate. Allow 15 minutes for heating unit to
warm up to 60°C.
11.2.5 Place samples and/or standards in the autosampler. Input the information required by the
data system, such as concentration, replicates and QC scheme.
11.2.6 Calibrate the instrument by injecting the standards.
11.3
System Notes
11.3.1 If sample concentrations are greater than the high standard, the distilled sample should be
diluted with 0.25 M sodium hydroxide (NaOH) diluent. When an automated diluter is
used, 0.25 M NaOH should be used as the diluent. Do not dilute distilled samples or
standards with water as this will cause a large positive matrix effect.
11.3.2 For analyzing distilled samples, prepare a standard curve by plotting the peak area of
distilled standards against concentration values. Compute concentrations by comparing
sample peak area with the standard curve.
12.0 DATA ANALYSIS AND CALCULATIONS
12.1
The data system will then prepare a calibration curve by plotting instrument response against
standard concentration. Sample concentration is calculated from the linear regression equation:
Modified Method: Digestion and distillation of total cyanide by MICRO DIST and FIA
Concentration = (Slope) Instrument Response + Intercept.
12.2
Report only those values that fall between the lowest and the highest calibration standards.
Samples exceeding the highest standard should be diluted and reanalyzed.
12.3
Report results in mg /L.
13.0 METHOD PERFORMANCE
13.1
Typical method performance data are presented as support data in section 17.
14.0 POLLUTION PREVENTION
14.1
Pollution prevention encompasses any technique that reduces or eliminates the quantity or
toxicity of waste at the point of generation. Numerous opportunities for pollution prevention
exist in laboratory operation. Whenever feasible, laboratory personnel should use pollution
prevention techniques to address their waste generation. When wastes cannot be feasibly reduced
at the source, the USEPA recommends recycling as the next best option.
14.2
The quantity of chemicals purchased should be based on expected usage during its shelf life and
disposal cost of unused material. Actual reagent preparation volumes should reflect anticipated
usage and reagent stability.
14.3
All waste materials should be disposed of in a responsible manner, in accordance with federal,
state, territory, local, and tribal regulations.
14.4
For information about pollution prevention that may be applicable to laboratories and research
institutions, consult "Less is Better: Laboratory Chemical Management for Waste Reduction,"
available from the American Chemical Society's Department of Government Regulations and
Science Policy, 115 16th Street N. W., Washington D. C. 20036, (202) 872-4477.
15.0 WASTE MANAGEMENT
15.1
The Environmental Protection Agency (USEPA) requires that laboratory waste management
practice be conducted consistent with all applicable rules and regulations. Excess reagents,
samples and method process wastes should be characterized and disposed of in an acceptable
manner. The agency urges laboratories to protect the air, water, and land by minimizing and
controlling all releases from hoods, and bench operations, complying with the letter and spirit of
any waster discharge permit and regulations, and by complying with all solid and hazardous
waste regulations, and by complying with all solid and hazardous waste regulations, particularly
the hazardous waste identification rules and land disposal restrictions. For further information on
waste management consult the "Waste Management Manual for Laboratory Personnel", available
from the American Chemical Society at the address listed in Sect. 14.3.
16.0 REFERENCES
16.1
U.S. Environmental Protection Agency, Methods for Chemical Analysis of Water and Wastes,
EPA-600/4-79-020, Revised March 1983, Method 335.4
16.2
Guideline and Format for EMSL-Cincinnati Methods. EPA-600/8-83-020, August 1983.
Modified Method: Digestion and distillation of total cyanide by MICRO DIST and FIA
16.3
Standard Methods For the Examination of Water and Wastewater, 20th ed., AWWA/WEF/APHA
(1999), Method 4500-CN--N, p4-50.
17.0 TABLE, DIAGRAMS, FLOWCHARTS, AND VALIDATION DATA
17.1
Flow Injection Analysis Manifold diagram
CARRIER is 0.25 M sodium hydroxide solution (Reagent 1).
All manifold tubing is 0.8 mm (0.030 in) i.d. This is 5.2 uL/cm.
4.5 is 70 cm of tubing on a 4.5 cm coil support
APPARATUS: An injection valve, a 10 mm path length flow cell , and a colorimetric detector module is
shows 650 cm of tubing wrapped around the heater block at the specified
required. The
temperature.
Note 1: 2 meter back pressure loop, 0.52 mm i.d.
17.2
Typical parameters for Lachat Instruments QuikChem AE Flow Injection
Analyzer
Sample throughput: 80 samples/h, 45 s/sample
Pump speed: 35
Cycle Period: 45 s
Inject to Start of Peak Period: 42 s
Modified Method: Digestion and distillation of total cyanide by MICRO DIST and FIA
17.3
Typical parameters parameters for Lachat Instruments QC 8000 Flow
Injection Analyzer
Sample throughput: 80 samples/h, 45 s/sample
Pump speed: 35
Cycle Period: 45 s
Analyte Data:
Concentration Units: mg CN-/L
Peak Base Width: 39 s
% Width Tolerance: 100 %
Threshold: 25000
Inject to Peak Start: 42 s
Chemistry: Direct
Calibration Data:
Levels
Concentration mg/L
1
2
3
4
5
6
0.500
0.100
0.050
0.010
0.005
0.000
Calibration Fit Type: 1st Order Polynomial
Calibration Rep. Handling: Average
Weighting Method: 1/X
Sampler Timing:
Min. Probe in Wash Period: 14 s
Probe in Sample Period: 20 s
Valve Timing:
Load Time: 0.0 s
Load Period: 20 s
Inject Period: 25 s
Modified Method: Digestion and distillation of total cyanide by MICRO DIST and FIA
17.4
Typical data for Lachat Instruments QC 8000 Flow Injection Analyzer
Calibration Graph and Statistics
Level
Area
mg CN-/L
Determined
Rep 1
Rep 2
Replica
STD
Replica
%RSD
% residual
1
31230856
0.5
0.503
3120856
31241620
6214.6
0.0
-0.6
2
615006
0.1
0.099
6150060
6158613
4937.8
0.1
1.0
3
295031
0.05
0.0475
2950321
2972790
12972.8
0.4
5.0
4
636723
0.01
0.0103
636723
659544
13175.8
2.0
-2.5
5
333965
0.005
0.0054
333965
356110
12784.4
3.7
-7.5
6
0
0
0
0
0.0
0.0
0.0
---
Method Detection Limit using Distilled Standards
Modified Method: Digestion and distillation of total cyanide by MICRO DIST and FIA
17.5
Method Performance
The method is required to perform according to the following QC Acceptance Criteria:
Acceptance Criterion
Section
NPDES
Limit (%)
NPDWR
Limit (%)
32
65-129
26
66-118
62-132
35
63-121
29
62-132
63-121
9.2.2
Initial precision and recovery
Precision (s)
Recovery (X)
Matrix spike/matrix spike
duplicate
Recovery
RPD
9.2.2.2
9.2.2.2
9.3
9.3.4
9.3.5
9.6
Ongoing precision and recovery
Recovery
9.2.3