Sample Requirements

Samples of biomolecules or biofluids submitted to the NMR facility or staff should be safe and nonhazardous.

Task: NMR Sample Preparation

Equipment Used: Centrifuge, pH meter, NMR spectrometers, fumehood, fridges, freezers, tube cleaner

Location: Rooms G14, G16, G23 and G24 in the Henry Wellcome Building for Biomolecular NMR Spectroscopy, University of Birmingham

Sample requirements table
 Hazards  Risk  (‘high’, ‘medium’ or ‘low’):
Radiation: danger to people and equipment due to inappropriate submission of radioactive samples. Low risk: All Users must understand and agree that no samples are allowed that require any form of radiological monitoring
Chemical: danger to people and equipment due to submission of carcinogenic, corrosive, flammable or toxic samples. Medium: Use of hazardous chemicals is discouraged. Users must follow all Control of Substances Hazardous to Health (COSHH) regulations. Hazardous chemicals brought into the building must be reported and approved by HWB•NMR staff, and labelled and stored in a safe manner at all times. Excluded Chemical Weapons Convention Scheduled Chemicals are listed in Appendix 2. COSHH Assessment NMR002 ‘Protein NMR Sample Preparation’ on the School of Cancer Sciences COSHH database covers a number of chemicals commonly used in biomolecular NMR. Copies are available from S. Rhodes.
Biological: danger to people due to submission of biological hazards such as toxins, infectious agents, viruses, pathogenic bacteria. Low: Use of any biological hazard is discouraged and must follow all COSHH regulations. Some of the disallowed biological agents are listed in Appendix 1.  All samples must be labelled and stored in a safe manner at all times
Ethical: samples submitted may be or human or animal origins, requiring consent. Low: No testing of human or animal subjects shall be conducted on the premises.  Ethical approvals for human or animal tissues or biofluids must be obtained from the approproate ethical review body and submitted to HWB•NMR staff prior to initiation of NMR analysis.
Physical: Inappropriate use of manual centrifuge can cause sample breakage and debris, and involves spinning an exposed rotor. The insertion of samples into NMR magnets requires climbing access platform near the magnets. Low: Use of equipment in the laboratory and NMR chambers requires appropriate user induction and appropriate access systems.
Financial (e.g. damage to NMR systems): NMR sample tubes can break and release their contents onto and damage probes. Probes can be damaged by insertion of hazardous samples, stripping the probe tuning rods by forced overturning, or use of excessive RF pulse power or probe temperatures.  These probes can cost over £100000, and full insurance cover is not affordable. Medium: NMR samples can only be inserted and experiments initiated by fully trained users and NMR staff who understand the risks and accept their responsibilities.  Users are not permitted to modify the NMR hardware or control software or attempt new pulse sequences unless these actions have been authorized by the NMR staff.

Safety Pre-Requisites:

  1. Ensure compliance with COSHH regulations
  2. Submit completed form for NMR time or sample analysis indicating all hazards.  Provide MSDS datasheets for any hazardous chemicals.
  3. All Users must obtain approval from the Operations Manager before initiating NMR experiments, including the completion of an orientation for the safe use of the NMR systems.

Key Points:

  1. In case of any accident, concern or question regarding these protocols please notify the Preparers (see above)
  2. It is the user’s responsibility to be familiar with and comply with these procedures.
  3. Negligence or non-compliance can result in barring from future use of HWB-NMR resources.
  4. Disputes will be handled by the Health and Safety Committee.
  5. Approved chemicals (e.g. 2H2O) and glassware (e.g. NMR tubes) may be stocked in the HWB•NMR sample preparation laboratory,  and are available upon request from Sue Rhodes.

Considerations for Protein NMR Sample Preparation

  1. Molecular weight: Larger molecules have longer correlation times, leading to faster relaxation and increased line-widths.  NMR is best suited to characterizing folded proteins in the 5-40 kDa range, larger proteins and complexes require exponentially more time and expense.  Tags used for affinity purification (e.g. glutathione S transferase or thioredoxin) should be cleaved and removed unless they are small (under 10 residues, e.g. His-tags).
  2. Concentration: Protein concentrations should ideally be between 0.5 and 1.5 mM for structural analysis (1mM = 10mg/ml for a 10kDa protein), noting that doubling the concentration requires approximately a quarter of the acquisition time to obtain a given signal to noise ratio, but increases sample viscosity. Proteins should be purified to >90% homogeneity, i.e. a single clean band on a SDS-PAGE gel or, even better, a single peak on a size exclusion chromatography column.  The sequence and exact size of the construct should be verified by DNA or protein sequencing and mass spectrometry, respectively. Ideally the monomeric (or oligomeric state) should be demonstrated by size exclusion chromatography, dynamic light scattering, analytical ultracentrifugation or other analytical measure.
  3. Stability: The sample may be in the NMR spectrometer for many hours or days, with temperatures for data collection ranging from 4 - 40ºC.  The standard operating temperature is 25ºC. Highly concentrated proteins tend to aggregate and precipitate, yielding poor NMR spectra.
  4. Buffer: Ideally 20 mM, although 0-50 mM is common. Phosphate buffer is an economical non-protonated buffer, and there also are a variety of perdeuterated buffers (d-Tris, d-HEPES) available that do not obscure protein NMR signals.
  5. pH: Usually acidic pH is required (typically 5-7) because many NMR experiments require observation of exchangeable amide protons which are difficult to observe at higher pH.  Protein structure and interactions can be pH sensitive, so the final sample pH should be verified.
  6. Ionic Strength: Salt (e.g. KCl) can increase protein solubility. However, high ionic strength (>150mM) demands longer 90º pulses, increases sample heating, reduces signal to noise (especially using cryogenic probes) and makes it more difficult to tune the probe. 
  7. Paramagnetics: Paramagnetic metals such as Cu(II), Mn(II), Cr(III), Fe(III) and Co(II) lead to NMR line broadening, and should typically be avoided.
  8. Volume: Each NMR sample should consist of a final volume of 550 L, including all additives, for a standard tube (e.g. Wilmad 535).
  9. Additives: 2H2O is added to 10% (or 5% for high value samples) for locking on the NMR signal frequencies, unless a 100% 2H2O solution is being used to clearly observe nonexchangeable protons. Sodium azide (usually 1-3mM) is added to prevent microbial contamination. A reducing agent such as DTT (<1mM or higher concentration - 30mM - if perdeuterated DTT) can be added to prevent protein oxidation.  The internal standard 2,2-dimethylsilapentane-5-sulfonic acid (DSS) can be added (typically 50 M) to reference the chemical shifts. If sample gets proteolysed, protease inhibitors can be added at low concentrations (<50M)
  10. Sample Tubes: Wilmad 535 NMR tubes (7 inch) are standard for protein NMR. New NMR tubes are not 'analytically clean' when delivered, but usually have organic or inorganic residues. Ensure tubes are clean (a rinse with water or buffer is advisable) and not chipped or warped by excessive heat. Shigemi tubes can be used for low volume samples, but require special care to eliminate bubbles.  Tubes should be capped and the cap parafilmed to avoid evaporative loss. 
  11. Isotope labelling: Although proteins can initially be assessed for suitability for structural analysis in unlabelled forms, detailed studies require labelling with 15N for small (~50-100 residues), 15N and 13C for medium (~100-150 residues), and 15N, 13C and 2H for large (>~150 residues) proteins.

Procedure – Operational Notes

  1. Use of concentrators: Proteins are often exchanged into their final solution conditions using concentrators with molecular weight cut off filters. The filters are stored with glycerol, which must be removed by at least three washes or spins of the concentrator with deionized water.
  2. Washing NMR tubes: NMR tube washers or Solvent Jet Cleaners can be purchased from GPE Limited, Sigma Aldrich and Wilmad, an economical 9 tube washer unit can be built by the School of Chemistry glassblowing shop, and will be provided by the HWB-NMR wet-lab  and Overduin’s laboratory (School of Cancer Sciences, S313 ). Strong acids such as Nochromix (Godax Laboratories) are available to remove adhered materials and deposits by overnight soaking, followed by washes with water or buffer.
  3. Transport of NMR tubes: Individual NMR tubes can be safely transported in NMR tube racks made by, for example, Kimble-Kontes or Wilmad, graduated cylinders, or inverted and taped 15 mL Falcon tubes.
  4. Short term storage of NMR tubes: use specialized NMR tube racks at the consoles and refrigerator to minimize risks of breakage of the delicate NMR tubes.
  5. Long term storage of NMR tubes: We recommend flash freezing proteins in liquid nitrogen and storage at -80C to minimize risks of oxidation or degradation. A small scale freeze and thaw trial experiment is advised to assess the risk of protein precipitation during sample warm up.

Sources of NMR reagents and consumables:

  1. Spectra Gases  sells isotope labelled reagents for NMR
  2. Cambridge Isotope Laboratories - sells isotope labelled reagents for NMR
  3. Isotec -  (a division of Sigma/Aldrich) sells isotope labelled reagents for NMR
  4. Silantes GmbH - sells isotope labelled reagents for NMR
  5. CDN ISOTOPES - sells isotope labelled reagents for NMR
  6. Medical Isotopes, Inc. - sells isotope labelled reagents for NMR
  7. Wilmad Glass Company: The standard NMR tube is Wilmad product 5mm 535-PP 7, although a less expensive tube (528-PP 7) can also be used for routine use. 
  8. Shigemi, Inc: For low volume samples use 5 mm tubes from Shigemi, Inc (412-444-3011). Ensure that tubes are matched to the appropriate solvent.
  9. GPE Limited http://www.gpelimited.co.uk/  sells glassware for NMR
  10. Godax Laboratories sells Nochromix for cleaning tubes

Articles which may provide useful information:

  1. Chemical shift standards: DS Wishart et al. (1995) 1H, 13C and 15N chemical shift referencing in biomolecular NMR, J Biomol NMR 6, 135-140.
  2. NMR Buffers: CH Schein (1990) Solubility as a function of protein structure and solvent components, Biotechnology 8, 308-316
  3. J Freund and HR Kalbitzer (1995) Physiological buffers for NMR spectroscopy, J Biomol NMR 5, 321-322.
  4. Three Methods in Enzymology volumes (176, 177 and 239) are dedicated to biomolecular NMR. See Norman Oppenheimer's article "Sample Preparation" pp 78-89, Vol 176 for useful hints on preparing NMR samples.
  5. Protein solubility: AP Golovanov et al. A simple method for improving protein solubility and long-term stability. J Am Chem Soc. 2004 126:8933-9.

Excluded Infectious Agents, Bacteria, Viruses and Toxins:

Note: The following list of excluded substances is not necessarily exhaustive. Please contact NMR staff if you are unsure about a particular substance.

ANTI-TERRORISM, CRIME AND SECURITY ACT 2001

Part 7 of the Anti-Terrorism, Crime and Security Act 2001 is concerned with the security of dangerous substances that may be targeted or used by terrorists. These substances are listed in Schedule 5 of the Act. The Schedule was amended in 2007. The current list is shown below and includes viruses, rickettsiae, fungi, bacteria and toxins. The toxins are also included in HAZDAT.

The provisions set out in Part 7 (and Schedules 5 and 6) place an obligation on managers of laboratories and other premises holding stocks of specified disease-causing micro-organisms and toxins to notify their holdings, and to comply with any reasonable security requirements which the police may impose.

It also requires managers of laboratories and other premises, on request, to furnish the police with details of people with access to the dangerous substances held there. The Secretary of State is given power to direct that a named individual must not be allowed access to such disease strains or the premises in which they are held.

Reporting etc, is co-ordinated through the University Health and Safety Unit.

VIRUSES

  1. Chikungunya virus
  2. Congo-crimean haemorrhagic fever virus
  3. Dengue fever virus
  4. Dobrava/Belgrade virus
  5. Eastern equine encephalitis virus
  6. Ebola virus
  7. Everglades virus
  8. Getah virus
  9. Guanarito virus
  10. Hantaan virus
  11. Hendra virus (Equine morbillivirus)
  12. Herpes simiae (B virus)
  13. Influenza viruses (pandemic strains)
  14. Japanese encephalitis virus
  15. Junin virus
  16. Kyasanur Forest virus
  17. Lassa fever virus
  18. Louping ill virus
  19. Lymphocytic choriomeningitis virus
  20. Machupo virus
  21. Marburg virus
  22. Mayaro virus
  23. Middleburg virus
  24. Mobala virus
  25. Monkey pox virus
  26. Mucambo virus
  27. Murray Valley encephalitis virus
  28. Nipah virus
  29. Ndumu virus
  30. Omsk haemorrhagic fever virus
  31. Polio virus
  32. Powassan virus
  33. Rabies virus
  34. Rift Valley fever virus
  35. Rocio virus
  36. Sabia virus
  37. Sagiyama virus
  38. Sin Nombre virus
  39. St Louis encephalitis virus
  40. Tick-borne encephalitis virus (Russian Spring-Summer encephalitis virus)
  41. Variola virus
  42. Venezuelan equine encephalitis virus
  43. West Nile fever virus.
  44. Western equine encephalitis virus
  45. Yellow fever virus

RICKETTSIAE

  1. Coxiella burnetii
  2. Rickettsia prowazeki
  3. Rickettsia rickettsii
  4. Rickettsia typhi (mooseri).

BACTERIA

  1. Bacillus anthracis
  2. Brucella abortus
  3. Brucella canis
  4. Brucella melitensis
  5. Brucella suis
  6. Burkholderia mallei (Pseudomonas mallei)
  7. Burkholderia pseudomallei (Pseudomonas pseudomallei)
  8. Chlamydophila psittaci
  9. Clostridium botulinum
  10. Clostridium perfringens
  11. Enterohaemorrhagic Escherichia coli, serotype 0157 and verotoxin producing strains
  12. Francisella tularensis
  13. Mycobacterium tuberculosis
  14. Salmonella paratyphi A, B, C
  15. Salmonella typhi
  16. Shigella boydii
  17. Salmonella paratyphi (Multiple-drug resistant)
  18. Shigella dysenteriae
  19. Shigella flexneri.Vibrio cholerae
  20. Yersinia pestis

FUNGI

  1. Cladophialophora bantiana
  2. Cryptococcus neoformans."

TOXINS (TOXIN, CAS NUMBER)

  1. Abrin Modeccin toxin, Abrin, 1393-62-0
  2. Botulinum toxins, Botulin D, 93384-46-4
  3. Botulinum toxin A, 93384-43-1,
  4. Botulinum toxin B
  5. Botulinum toxin F
  6. Clostridium botulinum toxin
  7. Clostridium perfringens toxins
  8. Clostridium botulinum neurotoxin
  9. Clostridium perfringens, epsilon toxin
  10. Clostridium perfringens, type A enterotoxin
  11. Conotoxin, Conotoxin, 123210-68-4
  12. Modeccin, Modeccin, 65988-88-7
  13. Ricin, Ricin, 9009-86-3
  14. Saxitoxin, Saxitoxin, 35523-89-8
  15. Shiga and Shiga-like toxins, Shiga toxin
  16. DNA (Escherichia coli strain KY-019 clone pKTN1054 Shiga-like toxin SLT-II subunit A gene plus Shiga-like toxin SLT-II subunit B gene), 53834-56-1
  17. DNA (Escherichia coli strain KY-019 clone pKTN1054 Shiga-like toxin SLT-II subunit A gene), 153834-58-3
  18. DNA (Escherichia coli strain KY-019 clone pKTN1054 Shiga-like toxin SLT-II subunit B gene), 153834-60-7
  19. DNA (Escherichia coli strain TK-051 clone pKTN1050 Shiga-like toxin SLT-II subunit A gene plus Shiga-like toxin SLT-II subunit B gene), 153834-57-2
  20. DNA (Escherichia coli strain TK-051 clone pKTN1050 Shiga-like toxin SLT-II subunit A gene), 153834-59-4
  21. Verotoxin 1 (Shiga shigella B subunit), 620190-09-2
  22. Staphylococcal enterotoxins, Staphylococcal enterotoxin A (Staphylococcus aureus gene SEA), 915245-87-3
  23. Staphylococcal enterotoxin B (Staphylococcus aureus aureus strain COL gene seb), 811333-16-1
  24. Staphylococcal enterotoxin C-bovine (Staphylococcus aureus host cattle gene sec-bov), 349587-80-0
  25. Staphylococcal enterotoxin E (Staphylococcus aureus) 197981-85-4
  26. Tetrodotoxin, Tetrodotoxin, 4368-28-9
  27. Viscum Album Lectin 1 (Viscumin), Viscum Album Lectin 1, 83590-17-4
  28. Volkensin toxin, Volkensin toxin, 91933-11-8

Any reference to a micro-organism or toxin includes:

  • any genetic material containing any nucleic acid sequence
    • associated with the pathogenicity of the micro-organism or
    • for the coding of the toxin; and
  • any genetically modified organism containing any such sequence.
    • Any reference to a toxin includes subunits of the toxin.
  1. Abrin
  2. Aflatoxins
  3. African horsesickness virus
  4. African swine fever
  5. Akabane virus
  6. Avian influenza (highly pathogenic)
  7. Bacillus anthracis
  8. Bluetongue virus (exotic)
  9. Botulinum toxins
  10. Bovine spongiform encephalopathy agent
  11. Brucella abortus,
  12. Brucella melitensis
  13. Brucella suis
  14. Burkholderia (Pseudomonas) mallei
  15. Burkholderia (Pseudomonas) pseudomallei
  16. Camel pox virus
  17. Classical swine fever
  18. Clostridium botulinum
  19. Clostridium perfringens epsilon toxin
  20. Coccidioides immitis
  21. Conotoxins
  22. Cowdria ruminantium (heartwater)
  23. Coxiella burnetii
  24. Crimean-Congo haemorraghic fever virus
  25. Diacetoxyscerpinol
  26. Eastern equine encephalitis virus
  27. Ebola viruses
  28. Equine morbillivirus (Hendra virus)
  29. Foot-and-mouth disease virus
  30. Francisella tularensis
  31. Goat pox virus
  32. Japanese encephalitis virus
  33. Lassa fever virus
  34. Liberobacter africanus
  35. Liberobacter asiaticus
  36. Lumpy skin disease virus
  37. Malignant catarrhal fever
  38. Marburg virus
  39. Menangle virus
  40. Mycoplasma capricolum /M. F38/M. mycoides capri (contagious caprine
  41. Mycoplasma mycoides mycoides (contagious bovine pleuropneumonia)
  42. Newcastle disease virus (exotic)
  43. Nipah virus
  44. Peronosclerospora philippinensis
  45. Peste des petits ruminants
  46. Phakopsora pachyrhizi
  47. pleuropneumonia)
  48. Plum pox potyvirus
  49. Prion
  50. Ralstonia solanacearum Race 3
  51. Ricin
  52. Rickettsia prowazekii
  53. Rickettsia rickettsii
  54. Rift Valley fever virus
  55. Rinderpest virus
  56. Saxitoxin
  57. Sclerophthora rayssiae var. zeae
  58. Sheep pox
  59. Shigatoxin
  60. South American haemorraghic fever viruses
  61. Staphylococcal enterotoxins
  62. Swine vesicular disease virus
  63. Synchytrium endobioticum
  64. T-2 toxin
  65. Tetrodotoxin
  66. Tick-borne encephalitis complex viruses
  67. Variola major virus (smallpox)
  68. Venezuelan equine encephalitis virus
  69. Vesicular stomatitis (exotic)
  70. Viruses causing hantavirus pulmonary syndrome
  71. Xanthomonas oryzae pv. oryzicola
  72. Xylella fastidiosa (citrus variegated chlorosis strain)
  73. Yellow fever virus
  74. Yersinia pestis

Appendix 2: Excluded chemicals:

  • Chemical Weapons Convention Scheduled Chemicals.
  • The following Schedules list toxic chemicals and their precursors.
  • (Whenever reference is made to groups of dialkylated chemicals, followed by a list of alkyl groups in arentheses, all chemicals possible by all possible combinations of alkyl groups listed in the parentheses are considered as listed in the respective Schedule as long as they are not explicitly exempted.)

Schedule 1

A. Toxic chemicals: CAS registry number

  1. (1) O-Alkyl (<C10, incl. cycloalkyl) alkyl(Me, Et, n-Pr or i-Pr)-phosphonofluoridates, e.g. Sarin: O-Isopropyl methylphosphonofluoridate 107-44-8, and Soman: O-Pinacolyl methylphosphonofluoridate 96-64-0
  2. O-Alkyl (<C10, incl. cycloalkyl) N,N-dialkyl (Me, Et, n-Pr or i-Pr)
  3. phosphoramidocyanidates, e.g. Tabun: O-Ethyl N,N-dimethylphosphoramidocyanidate 77-81-6
  4. O-Alkyl (H or <C10, incl. cycloalkyl) S-2-dialkyl(Me, Et, n-Pr or i-Pr)-aminoethyl alkyl (Me, Et, n-Pr or i-Pr) phosphonothiolates and corresponding alkylated or protonated salts, e.g. VX: O-Ethyl S-2-diisopropylaminoethyl methyl phosphonothiolate 50782-69-9
  5. Sulfur mustards: 2-Chloroethylchloromethylsulfide 2625-76-5
  6. Mustard gas: Bis(2-chloroethyl)sulfide 505-60-2
  7. Bis(2-chloroethylthio)methane 63869-13-6; Sesquimustard: 1,2-Bis(2-chloroethylthio)ethane 3563-36-8; 1,3-Bis(2-chloroethylthio)-n-propane 63905-10-2; 1,4-Bis(2-chloroethylthio)-n-butane 142868-93-7; 1,5-Bis(2-chloroethylthio)-n-pentane 142868-94-8; Bis(2-chloroethylthiomethyl)ether 63918-90-1; O-Mustard: Bis(2-chloroethylthioethyl)ether 63918-89-8
  8. Lewisites: Lewisite 1: 2-Chlorovinyldichloroarsine 541-25-3; Lewisite 2: Bis(2-chlorovinyl)chloroarsine 40334-69-8; Lewisite 3: Tris(2-chlorovinyl)arsine 40334-70-1
  9. Nitrogen mustards: HN1: Bis(2-chloroethyl)ethylamine 538-07-8; HN2: Bis(2-chloroethyl)methylamine 51-75-2; HN3: Tris(2-chloroethyl)amine 555-77-1
  10. Saxitoxin 35523-89-8
  11. Ricin 9009-86-3

B. Precursors:

  1. Alkyl (Me, Et, n-Pr or i-Pr) phosphonyldifluorides, e.g. DF: Methylphosphonyldifluoride 676-99-3
  2. O-Alkyl (H or <C10, incl. cycloalkyl) O-2-dialkyl (Me, Et, n-Pr or i-Pr)-aminoethyl alkyl (Me, Et, n-Pr or i-Pr) phosphonites and corresponding alkylated or protonated salts, e.g. QL: O-Ethyl O-2-diisopropylaminoethylmethylphosphonite 57856-11-8
  3. Chlorosarin: O-Isopropyl methylphosphonochloridate 1445-76-7
  4. Chlorosoman: O-Pinacolyl methylphosphonochloridate 7040-57-5

Schedule 2

A. Toxic chemicals: CAS registry number

  1. Amiton: O,O-Diethyl S-[2-(diethylamino)ethyl]phosphorothiolate and corresponding alkylated or protonated salts 78-53-5
  2. PFIB: 1,1,3,3,3-Pentafluoro-2-(trifluoromethyl)-1-propene 382-21-8
  3. BZ:3-Quinuclidinyl benzilate (*) 6581-06-2

B. Precursors:

  1. Chemicals, except for those listed in Schedule 1, containing a phosphorus atom to which is bonded one methyl, ethyl or propyl (normal or iso) group but not further carbon atoms, e.g. Methylphosphonyl dichloride 676-97-1, Dimethyl methylphosphonate 756-79-6, Exemption: Fonofos: O-Ethyl S-phenylethylphosphonothiolothionate 944-22-9
  2. N,N-Dialkyl (Me, Et, n-Pr or i-Pr) phosphoramidic dihalides
  3. Dialkyl (Me, Et, n-Pr or i-Pr) N,N-dialkyl (Me, Et, n-Pr or i-Pr)-phosphoramidates
  4. Arsenic trichloride 7784-34-1
  5. 2,2-Diphenyl-2-hydroxyacetic acid 76-93-7
  6. Quinuclidin-3-ol 1619-34-7
  7. N,N-Dialkyl (Me, Et, n-Pr or i-Pr) aminoethyl-2-chlorides and corresponding protonated salts
  8. N,N-Dialkyl (Me, Et, n-Pr or i-Pr) aminoethane-2-ols and corresponding protonated salts, Exemptions: N,N-Dimethylaminoethanol and corresponding protonated salts 108-01-0 and N,N-Diethylaminoethanol and corresponding protonated salts 100-37-8
  9. N,N-Dialkyl (Me, Et, n-Pr or i-Pr) aminoethane-2-thiols and corresponding protonated salts
  10. Thiodiglycol: Bis(2-hydroxyethyl)sulfide 111-48-8
  11. Pinacolyl alcohol:3,3-Dimethylbutan-2-ol 464-07-3

Schedule 3

A. Toxic chemicals: CAS registry number

  1. Phosgene: Carbonyl dichloride 75-44-5
  2. Cyanogen chloride 506-77-4
  3. Hydrogen cyanide 74-90-8
  4. Chloropicrin: Trichloronitromethane 76-06-2

B. Precursors:

  1. Phosphorus oxychloride 10025-87-3
  2. Phosphorus trichloride 7719-12-2
  3. Phosphorus pentachloride 10026-13-8
  4. Trimethyl phosphite 121-45-9
  5. Triethyl phosphite 122-52-1
  6. Dimethyl phosphite 868-85-9
  7. Diethyl phosphite 762-04-9
  8. Sulfur monochloride 10025-67-9
  9. Sulfur dichloride 10545-99-0
  10. Thionyl chloride 7719-09-7
  11. Ethyldiethanolamine 139-87-7
  12. Methyldiethanolamine 105-59-9
  13. Triethanolamine * Please just inform us if you intend to use TEA 102-71-6
 

 

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The health and safety of our users and staff is of paramount importance. Please let the staff know if your sample poses any significant risks.