Standard Operating Procedure
Cyanogen Bromide
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☐ Process
☒Hazardous Chemical
☐ Hazardous Class
Purpose
Cyanogen bromide is an inorganic compound with the formula CNBr. This colorless crystalline compound,
similar in appearance to sugar, is highly soluble in water and organic solvents. CNBr is used in organic
synthesis, parasiticide, fumigating compositions, rat exterminants, cyaniding reagent in gold extraction
process. For selective peptide cleavage, e.g. methionine, and for use in protein immobilisation
procedures. Cyanogen bromide is often used to immobilize proteins by coupling them to reagents such as
agarose for affinity chromatography. Cyanogen bromide must be kept dry at all times. Exposure to
moisture results in formation of HCN gas which is highly toxic.
Physical & Chemical Properties/Definition of Chemical Group
CAS#: 506-68-3
Class: Acutely toxic chemical/Corrosive
Molecular Formula: CNBr
Form (physical state): Crystalline powder
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Color: Colorless
Boiling point: 61 - 62 °C (142 - 144 °F) - lit.
Potential Hazards/Toxicity
Target Organs: Central nervous system, eyes, thyroid, blood.
Potential Health Effects:
Eye: Causes eye burns, also vapors or mists may be extremely irritating. Cyanide can be absorbed
through the eyes causing the symptoms described for inhalation.
Skin: May be fatal if absorbed through the skin. Contact with skin causes irritation and possible burns,
especially if the skin is wet or moist. Causes symptoms similar to those of inhalation. Skin absorption may
cause unconsciousness. Concentrated HCN vapor may also be absorbed through the skin.
Ingestion: May be fatal if swallowed. Causes gastrointestinal tract burns. May cause effects similar to
those for inhalation exposure. May cause tissue anoxia, characterized by weakness, headache,
dizziness, confusion, cyanosis (bluish skin due to deficient oxygenation of the blood), weak and irregular
heart beat, collapse, unconsciousness, convulsions, coma and death. Contains cyanide. Human fatalities
have been reported from acute poisoning. Large doses of cyanide may result in sudden loss of
consciousness and prompt death; small doses will prolong the above symptoms 1 to 2 hours.
Inhalation: May be fatal if inhaled. Causes respiratory tract irritation. Inhalation may result in symptoms
similar to cyanide poisoning which include tachypnea, hyperpnea (abnormally rapid or deep breathing),
and dyspnea (labored breathing) followed rapidly by respiratory depression. Pulmonary edema may
occur. Early symptoms include weakness, headache, giddiness, dizziness, confusion, anxiety, nausea
and vomiting. In severe cases breathing is rapid and deep then becomes slow and gasping; an irregular
heartbeat and tightness in the chest may be experienced.
Chronic: There is some evidence that human exposure to the material may result in developmental
toxicity. This evidence is based on animal studies where effects have been observed in the absence of
marked maternal toxicity, or at around the same dose levels as other toxic effects but which are not
secondary non-specific consequences of the other toxic effects.
Long term exposure to high dust concentrations may cause changes in lung function i.e.,
pneumoconiosis; caused by particles less than 0.5 micron penetrating and remaining in the lung. Prime
symptom is breathlessness; lung shadows show on X-ray.
Chronic intoxication with ionic bromides, historically, has resulted from medical use of bromides but not
from environmental or occupational exposure; depression, hallucinosis, and schizophreniform psychosis
can be seen in the absence of other signs of intoxication. Bromides may also induce sedation, irritability,
agitation, delirium, memory loss, confusion, disorientation, forgetfulness (aphasias), dysarthria,
weakness, fatigue, vertigo, stupor, coma, decreased appetite, nausea and vomiting, diarrhoea,
hallucinations, an acne like rash on the face, legs and trunk, known as bronchoderma (seen in 25-30% of
case involving bromide ion), and a profuse discharge from the nostrils (coryza). Ataxia and generalised
hyperreflexia have also been observed. Correlation of neurologic symptoms with blood levels of bromide
is inexact. The use of substances such as brompheniramine, as antihistamines, largely reflect current day
usage of bromides; ionic bromides have been largely withdrawn from therapeutic use due to their toxicity.
Several cases of foetal abnormalities have been described in mothers who took large doses of bromides
during pregnancy.
Chronic exposure to cyanides and certain nitriles may result in interference to iodine uptake by thyroid
gland and its consequent enlargement. This occurs following metabolic conversion of the cyanide moiety
to thiocyanate. Thyroid insufficiency may also occur as a result of metabolic conversion of cyanides to the
corresponding thiocyanate. Exposure to small amounts of cyanide compounds over long periods are
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reported to cause loss of appetite, headache, weakness, nausea, dizziness, abdominal pain, changes in
taste and smell, muscle cramps, weight loss, flushing of the face, persistent runny nose and irritation of
the upper respiratory tract and eyes. These symptoms are not specific to cyanide exposure and therefore
the existence of a chronic cyanide toxicity remains speculative. Repeated minor contact with cyanides
produce a characteristic rash with itching, papules (small, superficial raised spots on the skin) and
possible sensitization. Concerns have been expressed that low-level, long term exposures may result in
damage to the nerves of the eye.
Personal Protective Equipment (PPE)
Respirator Protection
A respiratory protection program that meets OSHA's 29 CFR 1910.134 and ANSI Z88.2 requirements or
European Standard EN 149 must be followed whenever workplace conditions warrant respirator use. A
full-face particle respirator type N100(US) or type P3 (EN 143) respirator cartridges as a backup to
engineering controls is recommended. If the respirator is the sole means of protection, use a full-face
supplied air respirator recommended.
Respirators should be used only under any of the following circumstances:
 As a last line of defense (i.e., after engineering and administrative controls have been
exhausted).
 When Permissible Exposure Limit (PEL) has exceeded or when there is a possibility that PEL will
be exceeded.
 Regulations require the use of a respirator.
 An employer requires the use of a respirator.
 There is potential for harmful exposure due to an atmospheric contaminant (in the absence of
PEL)
 As PPE in the event of a chemical spill clean-up process
Lab personnel intending to use/wear a respirator mask must be trained and fit-tested by EH&S. This is a
regulatory requirement. (https://www.ehs.ucla.edu/ep/ih/resp)
Hand Protection
Wear Natural Rubber, Neoprene, Butyl, PVC or Viton gloves. Any of these glove types are suitable for
handling cyanogen bromide. Be sure that you take into account that the gloves are resistant to whatever
solvent you are dissolving the cyanogen bromide in. Double-gloving is recommended. Inspect gloves
frequently for tears and other breakdown.
NOTE: Consult with your preferred glove manufacturer to ensure that the gloves you plan on using are
compatible with Cyanogen Bromide.
Refer to glove selection chart from the links below:
http://www.ansellpro.com/download/Ansell_8thEditionChemicalResistanceGuide.pdf
OR
http://www.allsafetyproducts.com/glove-selection-chart-chemical-breakthrough-ratings.html
OR
http://www.showabestglove.com/site/default.aspx
OR
http://www.mapaglove.com/
Eye Protection
Wear chemical splash goggles.
Skin and Body Protection
Lab coats should be worn. These laboratory coats must be appropriately sized for the individual and be
buttoned to their full length. Laboratory coat sleeves must be of a sufficient length to prevent skin
exposure while wearing gloves. Full length pants and close-toed shoes must be worn at all times by all
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individuals that are occupying the laboratory area. The area of skin between the shoe and ankle should
not be exposed.
Hygiene Measures
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Engineering Controls
Local exhaust ventilation is required where solids are handled as powders or crystals; even when
particulates are relatively large, a certain proportion will be powdered by mutual friction. Exhaust
ventilation should be designed to prevent accumulation and recirculation of particulates in the workplace.
If in spite of local exhaust an adverse concentration of the substance in air could occur, respiratory
protection should be considered. Such protection might consist of:
(a): particle dust respirators, if necessary, combined with an absorption cartridge;
(b): filter respirators with absorption cartridge or canister of the right type;
(c): fresh-air hoods or masks
Powder handling equipment such as dust collectors, dryers and mills may require additional protection
measures such as explosion venting.
First Aid Procedures
IMPORTANT: ESTABLISH A FIRST AID PLAN BEFORE WORKING WITH CYANIDES. ANTIDOTES
SHOULD BE AVAILABLE ON SITE.
If inhaled
SPEED IS ESSENTIAL, OBTAIN MEDICAL AID IMMEDIATELY. POISON material. If inhaled, get
medical aid immediately. Remove victim to fresh air. If not breathing, give artificial respiration. If breathing
is difficult, give oxygen.
In case of skin contact
POISON material. In case of contact, get medical aid immediately. Immediately flush eyes or skin with
plenty of water for at least 15 minutes while removing contaminated clothing and shoes. Wash clothing
before reuse. Destroy contaminated shoes.
In case of eye contact
Treat patient as for inhalation. In case of contact, immediately flush eyes with plenty of water for at least
15 minutes. Get medical aid immediately.
If swallowed
Get medical aid immediately. SPEED IS ESSENTIAL. A DOCTOR MUST BE NOTIFIED AT ONCE.
POISON material. If swallowed, get medical aid immediately. Only induce vomiting if directed to do so by
medical personnel. Never give anything by mouth to an unconscious person.
Notes to Physician: Prompt action is essential in all cases of contact. Exposure should be treated as a
cyanide poisoning.
Signs symptoms of acute cyanide poisoning reflect cellular hypoxia and are often non-specific. A
bradycardia, hypertensive and tachypneic patient suggests poisoning especially if CNS and
cardiovascular depression subsequently occurs. Immediate attention should be directed towards assisted
ventilation, administration of 100% oxygen, insertion of intravenous lines and institution of cardiac
monitoring. Obtain an arterial blood gas immediately and correct any severe metabolic acidosis (pH
below 7.15).
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Mildly symptomatic patients generally require supportive care alone. Nitrites should not be given
indiscriminately - in all cases of moderate to severe poisoning, they should be given in conjunction with
thiosulfate. As a temporizing measure supply amyl nitrite perles ( 0.2ml inhaled 30 seconds every minute)
until intravenous lines for sodium nitrite are established. 10 ml of a 3% solution is administered over 4
minutes to produce 20% methemoglobin in adults. Follow directly with 50 ml of 25% sodium thiosulfate, at
the same rate, IV. If symptoms reappear or persist within 1/2-1 hour, repeat nitrite and thiosulfate at 50%
of initial dose. As the mode of action involves the metabolic conversion of the thiosulfate to thiocyanate,
renal failure may enhance thiocyanate toxicity. Methylene blue is not an antidote. [Ellenhorn and
Barceloux: Medical Toxicology]
If amyl nitrite intervention is employed then Medical Treatment Kits should contain the following:
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One box containing one dozen amyl nitrite ampoules
Two sterile ampoules of sodium nitrite solution (10 mL of a 3% solution in each)
Two sterile ampoules of sodium thiosulfate solution (50 mL of a 25% solution in each)
One 10 mL sterile syringe. One 50 mL sterile syringe. Two sterile intravenous needles. One
tourniquet.
One dozen gauze pads.
Latex gloves
A "Biohazard" bag for disposal of bloody/contaminated equipment.
A set of cyanide instructions on first aid and medical treatment.
- Notes on the use of amyl nitrite:
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AN is highly volatile and flammable - do not smoke or use around a source of ignition.
If treating patient in a windy or draughty area provide some shelter or protection (shirt, wall, drum,
cupped hand etc.) to prevent amyl nitrite vapor from being blown away. Keep ampoule upwind
from the nose, the objective is to get amyl nitrite into the patient's lungs.
Rescuers should avoid AN inhalation to avoid becoming dizzy and losing competence.
Lay the patient down. Since AN dilates blood vessels and lowers blood pressure, lying down will
help keep patient conscious.
DO NOT overuse - excessive use might put the patient into shock. Experience at DuPont plants
has not shown any serious after-effects from treatment with amyl nitrite.
Antidote: For cyanide poisoning, administer cyanide antidote kit (contains amyl nitrite, sodium nitrite and
sodium thiosulfate).
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Major medical treatment procedures may vary e.g. US (FDA method as recommended by
DuPont) uses amyl nitrite as a methemoglobin generator, followed by treatment with sodium
nitrite and then sodium thiosulfate.
MODES OF ACTION: Amyl nitrite (AN) reacts with hemoglobin (HB) to form about 5% methemoglobin
(MHB). Sodium nitrite (NaNO2) reacts with hemoglobin to form approximately 20-30% methemoglobin.
Methemoglobin attracts cyanide ions (CN) from tissue and binds with them to become
cyanmethemoglobin (CNMHB). Sodium thiosulfate (Na2S2O3) converts cyanmethemoglobin to
thiocyanate (HSCN) which is excreted by the kidneys. i.e. AN + HB = MHB NaNO 2 + HB = MHB CN +
MHB = CNMHB Na2S2O3 + CNMHB + O2 = HSCN
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The administration of the antidote salts is intravenous in normal saline, Ringers lactate or other
available IV fluid.
Special Handling and Storage Requirements
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Precautions for safe handling
Avoid contact with skin and eyes. Avoid formation of dust and aerosols. Provide appropriate exhaust ventilation at places where dust is
formed.
Conditions for safe storage
Keep container tightly closed in a dry and well-ventilated place. Never allow product to get in contact with water during storage. Do not store
near acids. Recommended storage temperature: 2 - 8 °C Light sensitive. Moisture sensitive. Hydrolyses readily
Spill and Accident Procedure
Chemical Spill Dial 911 and x59797
Spill – Help contaminated or injured persons. Evacuate the spill area. Avoid breathing vapors utilizing a
self-contained breathing apparatus. Eliminate sources of ignition if the chemical is flammable. If
possible, confine the spill to a small area using a spill kit or absorbent material. Vacuum or sweep up
material and place into a suitable disposal container. Avoid runoff into storm sewers and ditches which
lead to waterways. Clean up spills immediately, observing precautions in the Protective Equipment
section. Avoid generating dusty conditions. Decontaminate trace cyanide in the spill area with a strong
sodium or calcium hypochlorite solution and flush waste to a holding area for potassium removal. Provide
ventilation. Prevent spreading of vapors through sewers, ventilation systems and confined areas.
Evacuate unnecessary personnel. Keep others from entering contaminated area (e.g., use caution tape,
barriers, etc.)
Small (<1 L) – If you have training, you may assist in the clean-up effort. Use appropriate personal
protective equipment and clean-up material for chemical spilled. Double bag spill waste in clear plastic
bags, label and take to the next chemical waste pick-up.
Large (>1 L) – Dial 911 (or 310-825-1491 from cell phone) and EH&S at x59797 for assistance.
Chemical Spill on Body or Clothes – Remove clothing and rinse body thoroughly in emergency shower
for at least 15 minutes. Seek medical attention. Notify supervisor and EH&S at x59797 immediately.
Chemical Splash Into Eyes – Immediately rinse eyeball and inner surface of eyelid with water from the
emergency eyewash station for 15 minutes by forcibly holding the eye open. Seek medical attention.
Notify supervisor and EH&S at x59797 immediately.
Medical Emergency Dial 911 or x52111
Life Threatening Emergency, After Hours, Weekends And Holidays – Dial 911 (or 310-825-1491 from
cell phone) or contact the Ronald Reagan UCLA Medical Center (emergency room) directly at x52111
(located at 757 Westwood Plaza, enter from Gayley Avenue). Note: All serious injuries must be reported
to EH&S at x59797 within 8 hours.
Non-Life Threatening Emergency – Go to the Occupational Health Facility (OHF), x56771, CHS room
67-120 (This is on the 6th floor, 7th corridor, room 120. Enter through the School of Dentistry on Tiverton
Drive and proceed to the “O” elevator to the 6th floor.)Hours: M - F, 7:30 a.m. to 4:30 p.m. At all other
times report to Ronald Regan UCLA Medical Center (emergency room) at x52111. Note: All serious
injuries must be reported to EH&S at x59797 within 8 hours.
Needle stick/puncture exposure (as applicable to chemical handling procedure) – Wash the affected
area with antiseptic soap and warm water for 15 minutes. For mucous membrane exposure, flush the
affected area for 15 minutes using an eyewash station. Page the needle stick nurse by dialing 231 from a
campus phone, enter 93333 when prompted and then enter your extension. Hours: M – F, 8:00 a.m. to
4:00 p.m. At all other times report to Ronald Regan UCLA Medical Center (emergency room) at x52111.
Note: All needle stick/puncture exposures must be reported to EH&S at x59797 within 8 hours.
Decontamination/Waste Disposal Procedure
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1. Cyanogen bromide and all cyanogen bromide waste shall be disposed of into waste containers
specifically designated for cyanogen bromide. Examples of cyanogen bromide waste material
include gloves, pipette tips, paper towels that have been contaminated with cyanogen bromide.
2. Once the waste container is full, dispose of according the UCLA EH&S hazardous waste
guidelines.
General hazardous waste disposal guidelines:
Label Waste
 Affix an on-line hazardous waste tag on all waste containers using the WASTe Online Tag
Program https://ehs.ucop.edu/waste as soon as the first drop of waste is added to the container
Store Waste
 Store hazardous waste in closed containers, in secondary containment and in a designated
location
 Double-bag dry waste using transparent bags
https://www.ehs.ucla.edu/hazwaste/management/containers
 Waste must be under the control of the person generating & disposing of it
Dispose of Waste
 Dispose of regularly generated chemical waste within 90 days
 Call EH&S at x61887 for questions
 Empty Containers
o Dispose as hazardous waste if it once held extremely hazardous waste (irrespective of
the container size) https://www.ehs.ucla.edu/hazwaste/types/extremely-hazardous
o Consult waste pick-up schedule
https://www.ehs.ucla.edu/hazwaste/management/pick-ups
Prepare for transport to pick-up location
 Check on-line waste tag
 Write date of pick-up on the waste tag
 Use secondary containment
Safety Data Sheet (SDS) Location
Online SDS can be accessed at http://msds.ehs.ucla.edu.
Protocol/Procedure
Quantities covered by this SOP:
0-25 mg for a cleavage reaction
Conditions covered by this SOP:
2°C - 26°C
Cyanogen bromide can result in toxic exposure if inhaled or if it comes into contact with skin or mucous
membranes. Always wear appropriate PPE and work with in a fume hood making sure to maintain dry
conditions at all times. Cyanogen bromide is commonly used in peptide cleavage at methionine bonds
and it is also used in purifying matrixes.
Cleavage at Met-X Bonds by Cyanogen Bromide
Reduction
1. Dissolve the protein in 0.2 M ammonium bicarbonate to a final concentration of 10-20 mg/ml.
2. Add β-mercaptoethanol to between 1% and 5% (v/v).
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3. Blow nitrogen over the solution to displace oxygen, cap the tube, and incubate overnight at room
temperature.
4. Dry the sample. Warming the sample will help evaporate all of the ammonium bicarbonate.
Cyanogen Bromide Cleavage
1. Dissolve the dried sample (10-20 mg/ml) in 70% formic acid ( an example would be 200 ul of 70%
formic acid for 4 mg protein sample)
2. Add solid cyanogen bromide (2 mg of reagent per mg of protein) [an example would be 8 mg
cyanogen bromide] directly to the protein solution. For very small amounts of protein, add one
small crystal of cyanogen bromide. Cyanogen bromide is usually present at 20- to 100-fold
excess with respect to methionyl residues, which is equivalent to approximately equal amounts of
protein and cyanogen bromide.
3. Cap the tube and incubate the reaction for 18-20 hours at room temperature in the dark.
4. Terminate the reaction by diluting the reaction mixture with ~5 volumes of water and removing the
excess reagents by lyophilization.
5. Separate the products by liquid chromatography or gel electrophoresis. Alternatively, store the
samples at -20ºC for later analysis.
6. To remove all traces of cyanogen bromide, decontaminate equipment (e.g., spatulas, tubes) by
immersion in sodium hypochlorite (bleach) solution for a few minutes until bubbling stops.
NOTE
Any deviation from this SOP requires approval from PI.
Documentation of Training (signature of all users is required)
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Prior to conducting any work with Cyanogen Bromide, designated personnel must provide training to
his/her laboratory personnel specific to the hazards involved in working with this substance, work
area decontamination, and emergency procedures.
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The Principal Investigator must provide his/her laboratory personnel with a copy of this SOP and a
copy of the SDS provided by the manufacturer.
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The Principal Investigator must ensure that his/her laboratory personnel have attended appropriate
laboratory safety training or refresher training within the last one year.
Principal Investigator SOP Approval
Print name __________________________Signature___________________________
Approval Date:
I have read and understand the content of this SOP:
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