Title or Type of Procedure: Generation of an aqueous solution of hydrogen cyanide (HCN) for
synthetic purposes (pH-adjustment approach)
P.I.: Romas Kazlauskas
Lab Location: 174 Gortner Laboratories
Original Issue Date: November 2012-12-01
Revision Date: none
Prepared by: Jan von Langermann
Approval Signature: not required
This procedure yields an ~250 mM aqueous solution of HCN mainly for synthetic purposes. For
experiments that require a known HCN-concentration, measure the HCN concentration using
Andexer et al. (2006). (Another option is to distill HCN, but this is not recommended for this laboratory due to its high toxicity and explosive nature in combination with air.)
!! READ REVIEW ON HCN TOXICITY: WOLFSIE & SHAFFER, 1959. PERFORM ALL
EXPERIMENTS IN A WELL-VENTILATED FUME HOOD!! (CHECK HOOD FUNCTION
WITH A TISSUE TEST.) IF AVAILABLE, USE AN ELECTROCHEMICAL HYDROGEN
CYANIDE-DETECTOR! TELL OTHERS IN THE LAB ABOUT YOUR EXPERIMENT. HYDROGEN CYANIDE (HCN) IS HIGHLY TOXIC AND SHOULD NOT BE INHALED. FURTHERMORE, HCN WITHIN AQUEOUS SOLUTIONS CAN PENETRATE SKIN SO USE
GLOVES, LAB COAT AND GOOGLES!
The lethal dose of HCN is ~1 mg/kg absorbed for humans. This procedure generates 135 mg of
HCN. For a 70 kg human, absorption of approximately half of this amount of HCN is lethal, so
follow all safety procedures carefully.
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I. Required materials and glassware
1. 50-mL two-neck-round-bottom flask
2. pH-electrode with an internal temperature probe
3. glass stoppers to ensure no leakage of HCN-solutions
4. ice for cooling
5. 245 mg NaCN or 325 mg KCN (5 mmol)
6. 1 M HCl-solution (approximately 5 mL needed)
7. aqueous buffer that will later form the reaction media for the desired reaction
8. a HCN-waste container to store excess HCN-solutions
9. 30 wt% H2O2 for the oxidation of HCN-waste solutions
10. aqueous sodium hypochlorite solution (bleach) for the oxidation of small hydrogen cyanide
spills.
15. cyanide test strips (from U-Store: ‘CYANIDE TEST STRIP, EMD-10044-1)
16. cyanide antidote (May not be available due to restriction, contact the lab safety officer for
more information + a doctor visit may also be required to learn how to use the kit. Currently the
‘Cyanide Antidote Kit’ is the most widely used kit. It is also known as the Lilly Kit, Pasadena Kit
or the Taylor Kit.
II. Hydrogen cyanide-formation
This procedure generates ~20 mL of 250 mM hydrogen cyanide and 250 mM NaCl or KCl in
aqueous buffer.
The weighted amount of NaCN (or KCN) is transferred into the two-neck-round-bottom flask and
dissolved in 20 mL buffer. From this point on the two-neck-round-bottom flask is cooled externally with ice to ensure internal temperatures below 10 °C. Higher temperatures may allow outgassing of HCN (bp = 25 °C). Subsequently, the pH-electrode is added to the two-neck-roundbottom flask while ensuring that the tip of the electrode is covered by the aqueous solution. The
position of the electrode within the neck of the flask (ground glass) can be fixed with Parafilm,
which also seals partly this opening. A dropping funnel is added to close the experimental set-up.
After an internal temperature of <5°C is reached the 1 M HCl-solution (approximately 5 mL) is
slowly dropwise to lower the pH of the solution to <7 (pKa of HCN = 9.2). The pH will decrease
slowly while the cyanide is being protonated; once an excess of acid is added, the pH will deop
rapidly. Herein NaCl (or KCl) is formed. The temperature of the aqueous phase should NOT exceed 10°C!! The resulting solution is transferred to a separate vial and all other required substrates and enzymes are added to start the reaction.
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II Disposal of HCN-waste-solutions
All HCN-containing aqueous waste solutions are combined are combined within a high beaker
(Do NOT fill above 50%) and adjusted to pH 10-11 (pH paper) in well-ventilated fume hood.
The mixture is stirred and hydrogen peroxide (30 wt%) is slowly added. Gas formation may occur (mainly NH3), which shows the ongoing oxidation reaction. Continue adding hydrogen peroxide until no more gas is formed. The reaction is highly exothermic and needs to cool down afterwards. Then the pH is adjusted to 8-9, which will results in additional gas formation. Again,
the solution is allowed to cool, the final cyanide concentration tested with a cyanide test stripe,
and poured down the drain as nonhazardous aqueous waste.
Hazard identification and risk of exposure to the hazards:
Hydrogen cyanide is a very strong poison, explosive within an air/hydrogen cyanide mixture and
should be handled very carefully.
Broken flasks: Do not handle broken glassware directly. Remove it using a brush and dustpan,
tongs or forceps.
Exposure Controls Specific to Above Risk of Exposure
Use only small amount of cyanide to prevent intoxication in the first place. Wear gloves, a lab
coat and a suitable eye protection! Cool all relevant solutions and glassware to prevent the generation of gaseous hydrogen cyanide.
Fume hood: Execute all relevant procedure steps within a well-ventilated fume hood. Store all
reactants and waste solutions within the fume hood. Inform your colleagues about the HCNexperiments and label all flasks.
Biological Waste Disposal Procedures – not required
Spill Response Procedures:
Small hydrogen cyanide spill: small quantities of hydrogen cyanide-solutions are wiped away
with a paper towel or tissue and left in the fume hood. Hydrogen cyanide will slowly evaporate
from the towel and the towel can be disposed after a week without any further treatment.
Sodium hypochlorite solution (bleach) can be used to oxidize small spills directly.
Large hydrogen cyanide spill: With large quantities of hydrogen cyanide-spill the risk of intoxication arises significantly and in this case of large amounts of free HCN the laboratory should be
evacuated immediately. If possible open the windows! Warn all persons in laboratories near and
above you (HCN rises as it is lighter than air). The room may only be entered with a working gas
mask, which is equipped with an effective HCN-filter until the HCN-concentration is low enough
(e.g. 10 ppm, which can be tolerated without any health-risks for 8h/day).
Accident Response Procedures
If spills result in a hazard exposure (e.g. face or eye splash, cut or puncture with sharps,
contact with non-intact skin):
 Wash exposed skin with soap and water, flush eyes for 15 min.
 Call 911 and seek medical attention. If symptoms of hydrogen cyanide poisoning appear
medical attention needs to very, very fast due to fast internal suffocation of the patient! Mention the possible risk of hydrogen cyanide intoxication to all relevant persons!
 If available, use a cyanide antidote. Unfortunately most antidotes require an intravenous (IV)
injection, which can only be used by trained personal.
 DO NOT perform mouth-to-mouth/nose ventilation due to the risk of secondary intoxication.
 For urgent care employees you may also go to HealthPartners Occupational and Environmental Medicine (M/F day time or Urgent Care after hours), or UMMC-Fairview Hospital
(24hrs). You may seek medical attention at the closest available medical facility or your own
healthcare provider.
(Follow-up must be done by HealthPartners Occupational and Environmental Medicine.)
 Report the incident to your supervisor, fill out the appropriate documentation. These documents are important to collect workers compensation should any complications from the hazardous exposure arise.
- Employee First Report of Injury
- Supervisor Incident Investigation Report
References
J. H. Wolfsie, C. B. Shaffer (1959) Hydrogen cyanide – hazards, toxicology, prevention and
management of poisoning, J. Occup. Med. 281-8.
J. Andexer, J. K. Guterl, M. Pohl, T. Eggert (2006) A high-throughput screening assay for hydroxynitrile lyase activity, Chem. Commun. 4201-3.