Isocyanates, Total (MAP) (5525)

by National Institute for Occupational Safety and Health

ISOCYANATES, TOTAL (MAP) Formula: R-NCO

MW: Appendix C

METHOD: 5525, Issue 1

5525

CAS: Table 1

EVALUATION: PARTIAL PROPERTIES:

OSHA: Table 1 NIOSH: Table 1 ACGIH: Table 1

RTECS: Table 1 Issue 1: 15 March 2003

Table 1

NAMES & SYNONYMS: Table 2 SAMPLING SAMPLER:

FILTER (glass fiber, impregnated); or IMPINGER; or IMPINGER + FILTER (see Table 3)

FLOW RATE: 1 to 2 L/min VOL-MIN: -MAX:

1 L @ 5 ppb 500 L

SHIPMENT:

Cold; impinger solution: vial with PTFE-lined cap; filter: wide-mouth jar with PTFE-lined cap, containing 5.0 mL MAP extraction solution

SAMPLE STABILITY:

REAGENT STABILITY:

Extracted filters: store cold and process as soon as possible; prepared samples: at least 3 months at -10° C (freezer) in the dark

MAP on filters: 6 months at -10° C (freezer) in dark; MAP solutions: irreversible precipitation occurs within a few weeks in 2 mg/mL solutions

BLANKS:

Minimum of 3 field controls, 3 field blanks per set

BULKS:

Necessary for oligomer analysis

MEASUREMENT TECHNIQUE:

HPLC, UV/FLUORESCENCE DETECTION

ANALYTE:

MAP derivatives of isocyanates

SAMPLE PREP:

Impinger samples: solid-phase extraction (SPE); filter samples: acetylate with acetic anhydride

INJECTION VOLUME:

30 µL

MOBILE PHASE: 65:35 (v/v) acetonitrile:triethylammonium phosphate/formate (100 mM in both), pH gradient 6.0 to 1.6, 1.5 mL/min. POST-COLUMN SOLUTION:

65:35 (v/v) acetonitrile- 4.4 N phosphoric acid, 0.7 mL/min.

COLUMN:

Reversed phase InertsilT M C 8, 150 x 4.6 mm, 5-µm, titanium frits in a constant temperature column oven set at 30o C.

DETECTORS:

UV @ 253 nm; fluorescence (xenon lampex: 368 nm, em: 409 nm; or deuterium lampex: 254 nm, em: 409 nm).

CALIBRATION:

MAP derivatives of monomeric isocyanates, with FL or UV for monomer; mandatory use of UV peak area for oligomer.

RANGE:

0.5 nmole NCO per species to 300 nmole total NCO per sample (e.g., 0.04 to 25 µg HDI per sample).

ACCURACY RANGE STUDIED:

Not studied

BIAS:

Not determined

OVERALL PRECISION ( Ö r T ):

Not determined

ESTIMATED LOD: 0.2 nmole NCO per species per sample (0.2 nmole NCO equals 0.017 µg HDI per sample).

ACCURACY:

Not determined

PRECISION ( þ r ):

Filters: HDI 0.05; 2,4-TDI 0.06; MDI 0.06 Impingers: not determined.

APPLICABILITY: The useful air concentration range for this method is approx. 1.4 to 840 :g/m3 NCO total isocyanate group for a 15-L air sample. This is equivalent to 0.4 ppb to 250 ppb for a diisocyanate. The method determines the air concentration of monomeric and oligomeric isocyanates. The applicability of the method depends on the correct choice of sampler for a given environment [1,2]. INTERFERENCES: Any non-isocyanate compound that reacts with MAP. Any compound that elutes at or after the monomer retention time and absorbs at 253 nm may potentially interfere with isocyanate quantification by UV. Interfering compounds that do not fluoresce using 368 nm excitation and 409 nm emission but give an interfering UV signal can be separated by altering the pH gradient. OTHER METHODS: NIOSH Method 5522 [3] is for monomer and total isocyanate. NIOSH Method 5521 [4] is for monomer and aliphatic oligomeric isocyanate. A chapter in the NIOSH Manual of Analytical Methods, a related journal article, and the references therein describe other methods and method selection criteria [1,2].

NIOSH Manual of Analytical Methods (NMAM), Fourth Edition ISOCYANAT ES, TOTA L (MAP): METHO D 5525, Issue 1, dated 15 March 2003 - Page 2 of 17 REAGENTS:

EQUIPMENT:

For Glass-fiber Filter (GFF) Sam pling and An alysis 1. 1-(9-anthracenylmethyl)piperazine (MAP) [5,6] impregnated filters. a. 37-m m : 1.1 mg M AP/filter. b. 25-m m : 0.5 mg M AP /filter. c. 13-m m : 0.13 mg MAP /filter. 2. Extracting solution: 1x10-4 M MAP in acetonitrile (2.76 mg MAP per 100 mL acetonitrile). 3. Acetic an hydride (HP LC grad e).* 4. Aceton itrile (HPLC grad e).*

1. Sam pler: Glass-fiber filter, Type A/E BinderFree, 13- or 37-m m (Gelm an or equivalent), coated with appropriate amount of MAP [5,6] (see Reagents) in an opaque cassette filter holder with cellulose bac kup; or 25-m m glass-fiber filter coated with MAP (see Reagents) in an IOM sampler with stainless steel cassette; or impinger. 2. Personal sampling pump, capable of sam pling at 1 to 2 L/m in with flexible connecting tubing. NO TE : Avoid leaching of plasticizer into solvent during impinger sampling. For impingers, Fluran TM tubing is acceptable, PVC tubing is not. 3. W ide-m outh glass jars (2 0-m L) with PT FE lined cap fo r extra ction a nd tra nsp ort. 4. Insulated shipping container with ice packs. 5. High performance liquid chromatograph (HPLC) with binary solvent and gradient capabilities, PEEK tubing, and post-column addition pump, interfaced with an autoinjection system, em ploying 150x 4.6-m m Ine rtsilTM C8, 5-:m column with titanium frits, in a column oven set to 30 °C (or a minimum of 5 degrees above ambient). Adaption of m eth od to a narrowe r bo re colum n is acc epta ble. 6. UV detector s et to 25 3 nm . 7. Fluorescence (FL) detector with either (a) Xenon source: excitation 368 nm, emission 409 nm; or (b) Deuterium source: excitation 254 nm, emission 409 nm. 8. Volumetric flasks, glass, various sizes. 9. Visiprep TM or equivalent solid phase extraction (SPE) manifold with drying attachment and disposable valve liners. 10. Supelclean TM LC-Si or equivalent SPE tubes (0.5g) with 6-mL reservoir. 11. Disposable 20-mL glass vials with PTFElined caps. 12. Microsyringes. 13. HPLC vials. 14. Pipets, disposable glass. 15. PTF E syringe filters (0.45-µm pore size). 16. Forceps for handling filters. 17. Sealing bands. 18. Top-loading balance , 2 to 3 decimal place. 19. Analytical balance, 5 place.

Fo r Im pinger Sam pling and Analysis 5. Impinger solution: 1x10 -4 M MAP [5,6 ] in butyl benzo ate (2.76 m g per 100 m L butyl benzoate). 6. Butyl benzoate: Purified by running through a silica gel column. 7. Methylene chloride (HP LC grad e).* 8. Acetonitrile (HPL C grade ). 9. Me than ol (HPLC grade ).* 10. 90:10 aceto nitrile-m eth anol. Chromatography 11. Phosp horic acid, 85% (99.9 99% ).* 12. Form ic acid (AC S reage nt gra de).* 13. Acetonitrile (HPL C grade ). 14. Triethylam ine (99.5% ).* 15. Hydroch loric ac id (conce ntrate d).* 16. 100 m M p hos pha te-form ate buffer, pH 6.0 and pH 1.6, and aceton itrile-buffer m obile phase s (APP END IX A). 17. Post-column phosphoric acid (4.4 N) and aceto nitrile-a cid post-colum n addition m obile phase (APPE ND IX B). 18. Monomer standards in acetonitrile: MAPderivatives, 2x10 -8 N to 3x10-5 N (NCO groups per liter). Synthesis of MAPderivatives (APPEN DIX C ). C

See SPECIAL PRECAUTIONS

NIOSH Manual of Analytical Methods (NMAM), Fourth Edition ISOCYANAT ES, TOTA L (MAP): METHO D 5525, Issue 1, dated 15 March 2003 - Page 3 of 17

SPECIAL PRECAUTIONS: Diisocyanates are highly toxic and known respiratory irritants. Follow manufacturer’s guidelines when handling pure products. Follow hazardous material shipping requirements when transporting. Preparation of urea derivatives, samples, and standards should be don e in a h ood to avo id exp osu re to iso cyanate and s olven t vapo rs. T oxicity of MA P is unknow n. Acetic anhydride is a severe irritant; acetonitrile is flamm able and an irritant. Methylene chloride is an irritant and suspected carcinogen. Methanol is toxic and flamm able. Strong acids are toxic and corrosive. Triethylamine is toxic and corrosive.

SAMPLING: NOT E 1: Use sampling conditions listed below for the sampler selected: glass fiber filter (1); impinger (2); impinger followed by glass fiber filter (3). See Table 3 for examples of exposure scenarios and which sampler to choose. Refer to the chapter on isocyanates [1] for more detailed informa tion on how to choos e the appropriate sam pler. NOT E 2: Field controls are made by sampling in an area near the analyte sampling location but where one would not expect to find analyte. These samplers are then treated exactly like the samples from this po int on. NOT E 3: Fie ld blanks are samplers handled exactly th e sam e as sam ples except tha t no air is drawn through them. 1. Filter sampling a. Calibrate each sampling pump with a representative sampler in line. NOTE: W hen sampling aerosol with aerodynamic diameter >20 µm, an IOM sam pler with stainless stee l cassette or other su itable inhalable sam pler shou ld be u sed [1,2]. b. Attach filter cassette to sampling pump with flexible tubing. c. Sam ple at a rate of 1 to 2 L/min (2 L/min for the IOM) for a total sample of at least 1 L. d. Im m ediate ly after sampling, place filter in a wide-mouth jar with a PTFE-lined screw c ap c onta ining 5.0 mL of 1 x 10 -4 M M AP in ace tonitrile. Secure sc rew cap with a s ealing ban d. NOTE 1: If an IOM sam pler is use d, plac e en tire stainless steel cassette and filter in the jar with 10.0 m L of e xtrac ting so lution ins tead of 5.0 m L, rec ap a nd s wirl to we t all surfa ces of the IOM sam pler. NOT E 2: If co llec ting vapo r-o nly s amples, since in-field extraction is not necessary, ship the unextracted samplers with ice packs to the lab for analysis. e. Store samples in a refrigerator and ship the samples with ice packs. f. Obta in a bulk sam ple (at least 3 mL) and a Mate rial Safety Data Sheet (M SD S) for pro ducts used in the process giving rise to expos ure (e.g., for a two-part polyurethan e co ating s ystem , a sam ple of both the isocyanate product and the polyol product). These bulk samples are necessary for oligomer analysis. Ship bulks in a separate container from the air samples. g. Obta in a minimum of three field controls and three fie ld blanks per sam ple set or at least one fie ld control and one field blank for every ten sam ples. T hese controls and blanks are necessary for distinguishing be twee n artifacts a nd iso cyanates . 2. Impinger sampling a. Calibrate each sampling pump with a representative sampler in line. b. Transfer 15.0 mL of 1 x 10 -4 M MAP in purified butyl benzoate to each impinger and connect impinger to sampling pump with flexible tubing that is free of phthalate plasticizer (Fluran TM tubing is acceptable, PVC tubing is not.) c. Sam ple at 1 L/min for a total sample of at least 1 L. d. Using a disposable pipet, transfer the entire conte nts of the impinger to a 20-mL vial with a PTFE-lined screw cap. Secure screw cap with sealing band. e. Store samples in a refrigerator and ship the samples with ice packs. f. Obtain bulk sa m ples and M SDS s as des cribed in step 1.f. g. Obta in a m inim um of three field controls and three fie ld blanks per sam ple set or at least one fie ld control and one field blank for every ten samples. These controls and blanks are necessary for distinguishing be twee n artifacts a nd iso cyanates .

NIOSH Manual of Analytical Methods (NMAM), Fourth Edition ISOCYANAT ES, TOTA L (MAP): METHO D 5525, Issue 1, dated 15 March 2003 - Page 4 of 17 3. Impinger + filter sampling a. Sam ple with an im pinger followed by a M AP-coated filter whe n im pinger sa m pling is d esired (e.g ., a highly reactive aerosol) but particles < 2 µm are suspected to be present (e.g., condensation or com bus tion ae rosol). Particles < 2 µm are n ot collected efficiently by impingers [1,2]. b. Attach the inlet of the filter cassette to the outlet of the impinger and the outlet of the filter ca sse tte to the pum p with flexible tu bing. The portion of the tubing connecting the impinger to the filter should be free of phthalate plasticizer (Fluran TM tubing is acc epta ble, PVC tubing is not.) c. Sam ple at a rate of 1 L/min for a total sample size of at least 1 L. d. After sampling, treat the impinger solution as described in step 1.d. If a combined measure of the total isocyanate from the two stage s is de sired, place the filter in the im pinger sa m ple vial. If se para te m eas urem ents of the two sta ges are desired, place the filter in a separate ja r contain ing MAPacetonitrile extraction solution as in step 1.d. e. Store samples in a refrigerator and ship the samples with ice packs. f. Obtain bulk sa m ples and M SDS s as in step 1.f. g. Obtain a minimum of three field controls and three field blanks as described in steps 1.g. and 2.g.

SAMPLE PREPARATION: NOT E 1: NOT E 2:

For correct sample preparation, follow directions below for type of sampler used: glass fiber filter (4); im pinger (5); impinger followed by glass fiber filter (6). Re frigera te all sam ples imm ediately upon rec eipt.

4. Filter and filter extract a. For vapor-only samples received without in-field extraction, follow the procedure in step 1.d. of filter sampling and allow the samples to extract overnight in a refrigerator before proceeding. b. For samples that are extracted in-field, acetylate with 5 µL of acetic anhydride upon receipt of samples. For sam ples that are ex trac ted in the lab, acetylate with 5 µL of acetic anhydride after overnight extra ction. Let these re act at least tw o ho urs at room tem pera ture or overnight in the refrigerator before proceeding. c. Attach the outlet of a 0.45-µm PTFE Luer-lock syringe filter to the S PE vacuum m anifold with disposable liner. Place an empty polypropylene syringe barrel in the inlet of the syringe filter. Add the entire extra ction s olution into the syringe barrel. d. Force the sam ple through the PT FE filter us ing either positive or negative pressure and collect in a glass vial with a PTFE-lined cap. 5. Impinger a. Attach a 6-mL SPE cartridge containing 500 mg silica gel to a solid-phase extraction (SPE) vacuum manifold with disposable liners. b. Condition the SPE cartrid ge with 2 m L butyl benzoate, bringing the liquid level down to the top of the sorbent bed. c. Add a 5.0-m L aliquot of sample solution to the SPE cartridge, adjusting the vacuum of the SPE m anifold to obtain a flow of a pprox. 1 to 2 m L/m in and sto pping when the liquid level reaches the top of the sorb ent. d. Add 6 m L methylene chloride to th e SPE cartrid ge. Elu te th e cartridge at 1 to 2 m L/m in until th e liquid level reach es the top of the sorb ent. D iscard all elua te collected up to this po int. e. Add 3 mL of 9:1 acetonitrile-methanol to the SPE cartridge. Elute at approx.1-2 mL/m in until the liquid level re aches th e top of the sorbent. C ollect th e eluate in a pre-weighed glass vial. f. Add 3 m L of m etha nol to the SP E ca rtridge. Elute and collect the eluate in the same vial as in step 5.g. g. Use a stream of nitrogen to reduce the volum e to exactly 1.0 mL. Rec onstitute to volum e if necessary by addition of acetonitrile. Determine exact volume using the sample weight and the density of acetonitrile (0.786 g/m L). 6. Impinger + Filter a. Follow steps under Sample Preparation step 5 for the impinger part of the sample. b. Follow steps under Sample Preparation step 4 for the filter part of the sample.

NIOSH Manual of Analytical Methods (NMAM), Fourth Edition ISOCYANAT ES, TOTA L (MAP): METHO D 5525, Issue 1, dated 15 March 2003 - Page 5 of 17 CALIBRATION AND QUALITY CONTRO L: 7. Ca librate with six working standards in the range of interest. Prepare liquid standards with concentrations in terms of m oles isocyanate group per liter (normality) in the range 3 x 10 -5 N to 2 x 10- 8 N. Intersperse standards am ong sam ples. For filter samples and impingers, liquid standards should consist of the MAP derivative(s) of the app ropriate diisocyanate m onom er(s) in acetonitrile. A concentrated stock solution of the MAP derivatives of isocyanate monom ers can be made in methylene chloride. (Solubility: 1x10 -3N to 5x10 -3N). See AP PE NDIX C for synthe sis of M AP deriva tives to be used as standards. For filter samples only, to m imic sam ples in artifac t content, a MAP impregnated filter similar to the filter used for the sam ple ca n be place d in 5.0 mL of each level of standard, and then treated the same as the samples starting at step 4. 8. Prepare calibration graphs (response vs. no rm ality of stan dard solution) for both the UV and fluorescence dete ctors . a. The calibration curve prepared from either the UV or the FL detector may be used to quantify the m onom er. A quadratic fit may be used for the fluorescence calibration curve if curvature is seen at the low end. b. A linear calibration curve using UV peak area must be used to quantify the oligomer because the UV respon se fo r m ono m er an d oligom er is the sam e wh ile the FL res pon se is not. 9. Analyze two solvent blanks at the beginning of each sample set. Using the second blank chromatogram, subtract this chromatogram electronically from sam ple chromatograms. This improves the UV baseline of sample chromatograms and facilitates accurate integration of peaks in low level samples. Analyze one add itional so lvent blank within the sam ple se t. 10. Analyze a minimum of three field controls and three field blanks or at least one field control and one fie ld blank for every ten samples. Use the field controls and field blanks to identify non-isocyanate peaks (generally reagent artifact peaks) that are likely to appear in the samples. If the blanks are consistent and the sam ple set is analyzed soon after sampling, subtraction of a field blank instead of the solvent blank from the sam ple data m ay ma ke interpretation of the data simpler. 11. Analyze bulk isocyanate products whenever available, but if oligomer is to be quantified, analysis of the bulk products used at the worksite must be done. Bulk isocyanate chromatograms are useful for qualitative confirmation of p eaks observe d in the sam ple chro m atog ram s. It is also advisable to an alyze bulk non-isocyanate products (such as the polyol portion of a two-part spray system) in the same m anner to ensure that they give rise to no interfering com pounds that may be m istaken for isocyanates. Bulk prod ucts m ust be deriva tized with M AP prio r to analysis. A n appropriate dilution of the bulk in methylene chloride bas ed o n the m anu facturer’s NC O c onte nt, followed by imm ediate derivatization of an aliquot of this diluted bulk in 5 x 10-4 M MAP in aceton itrile m ust be done. After rea ctin g overnight, ac etyla te th is bulk-MAP rea ctio n m ixture with acetic anhydride as in step 4.b. AP PE NDIX D is a protoc ol fo r a b ulk dilution/derivatization with MAP that works well for most representative bulks. This bulk-MAP sample can then be analyzed on the HPLC. 12. Analyze three qu ality contro l blind sp ikes per s am ple se t.

MEASUREMENT: 13. Set HP LC and detectors to conditions an d se ttings g iven on pa ges 552 5-1 a nd 5 525 -2. Note th at in addition to a mobile phase flow of 1.5 mL/min, there is also a post column addition of 0.7 mL/min of 65:35 (v/v) ace tonitrile-4.4 N phos pho ric acid. Keep the column at constant temperature in an oven at 30 oC. Equilibrate the entire system for at least forty minutes before the sta rt of the daily analysis, us ing m obile phase B (65:35 (v/v) acetonitrile-pH 1.6 buffer), running post-column acid mobile phase also. 14. The HP LC grad ient pro gram can be custo m ized to optim ize the se para tion of the particular isoc yanate species. An example of a gradient program to start with is: 0 - 4 min: 100% m obile phase A (65:35 (v/v) acetonitrile-pH 6 b uffer) 4 - 17 min: Linear gradient from 100% mobile phase A to 100% mobile phase B (65:35 (v/v) acetonitrile-pH 1.6 bu ffer) 17 - 30 min: Hold 100% mobile phase B 30 - 36 min: Re-equilibrate at 100% m obile phase A NOTE 1: A gradient that provides better resolution for aliph atic isocyanate produ cts is given in Bello et al. [7].

NIOSH Manual of Analytical Methods (NMAM), Fourth Edition ISOCYANAT ES, TOTA L (MAP): METHO D 5525, Issue 1, dated 15 March 2003 - Page 6 of 17 NOTE 2:

For isocyanate products that essentially contain only monom er, the gradient can be truncated after the monomer elutes. After truncation, a two minute hold using pH 1.6 mobile phase before return ing to initial co nditions will ens ure b ase line stability for the ne xt sa m ple run. NOTE 3: If there is an interfering peak in the area of interest that is not influenced by pH, changing the length of hold at the beginning of the run should move the interference peak away from the area of interest. NOTE 4: For multi-component monom ers such as TDI and HMDI and for multi-component isocyanate products that contain monom er and oligom er, ch anging the hold at the beginning, changing the gradient ramp, placing a hold in the middle of the ramp, or a combination of all of the abo ve m ay help in sep aration of the individual co m pon ents for a s pec ific prod uct. 15. Injec t a 30-µL sam ple aliqu ot. 16. Mo nom er m eas urem ent: Isoc yanate spe cies for which pure ana lytical stand ards are a vailable, suc h as diisocyanate monom ers, can be measured by comparing the response of the peak at the correct retention time with the calibration curve generated by analyzing standards. Either UV peak height or area or fluorescence peak height or area can be us ed for quantification of m ono m er. T he fluo rescence detec tor is m ore sensitive, s o it is usually better for measuring monom ers at low levels. Peak height is usually preferable to peak area, especially in the presence of closely-eluting interfering peaks. Confirmation of the identity of the monom er is achieved by comparing the FL/UV response ratio of the sample peak to that of the standard giving sim ilar resp ons e. NOTE: The FL/UV ratio may change somewhat at low levels. The sample peak should give a FL/UV response ratio within 15% of the ratio for the standard peak of comparable size. 17. Oligom er m easurem ent (total isocyanate): Isoc yanate species for which pure analytical standards are not available, such as oligomeric isocyanates, must be measured by using the UV are a of the sam ple peak(s) and the slope of the linear portion of the calibration curve generated by analyzing monom er standards. Frequently, numerous isocyanate species elute as an envelope of poorly res olved peaks. In this case, rather than attempting to integrate peaks individually, the entire chromatogram is integrated over the area o f interest. Because the fluorescence baseline is not disturbed by the gradient and the fluorescence detector is selective for MAP-derivatized compounds, the sample fluorescence chromatogram can be very useful for determ ining when to begin and end integration of peaks in the UV chromatogram. The fluorescence chromatogram is used qualitatively to confirm the presence of MAP gro ups in the eluting species. The fluorescence response varies too much from com pound to compound to quantify isocyanate species for which standards are unavailable. However, the excitation and emission wavelengths that have been chosen make the detection very selective for MAP derivatives. Experience has shown that MAP-derivatized isocyanates will have a FL /UV ratio of approxim ately 0.33 to 2 times that of the M AP-derivatized mo nom er.

CALCULATIONS: 18. Monom ers: Ge nera te a calibration graph usually using fluorescence, plotting peak area or peak height as a function of concentration (no rm ality, isocyanate milliequivalents per milliliter) of monom er standards. Determine the normality of the analyzed sample aliquot from the calibration graph.

NIOSH Manual of Analytical Methods (NMAM), Fourth Edition ISOCYANAT ES, TOTA L (MAP): METHO D 5525, Issue 1, dated 15 March 2003 - Page 7 of 17 a. For impinger samples use the following formula:

whe re: M = N= W teq = Vf = Vt = V al =

m ass of isocyanate sp ecies (µg/sam ple), the normality (isocyanate milliequivalents per milliliter) of the analyzed sample solution as take n from the calibration curve, the equivalent weight of the m onomeric isocyanate in milligrams per isocyanate m illiequ ivalent, the final volum e of S PE solution in m illiliters, the 15-mL volume of the total sample, and the vo lum e of the aliquot analyzed.

b. For filter samples use the following formula:

where: M = N= W teq = Vf =

mass of isocyanate species (µg/sample) the normality (isocyanate m illiequ ivalents per milliliter) of the analyzed sam ple solution as taken from the calibration curve, the equivalent w eight of the monom eric isocyanate in milligrams per isocyanate milliequivalent, and the final volume of the sample solution in milliliters.

19. Oligom ers (total isocyanate): Identification of oligomers is best done by following the flow chart presented in Appendix E. T he bulk chrom atogram is used as the comparison standard when identifying which peaks to include in the quantification. Quantifica tion of oligomers m ust be acco m plished using a UV area calibration curve generated from analyzin g m onom er sta ndards, preferably using the m onom er on which the oligom er is based. Generate a calibration curve , plotting UV peak are a as a fun ctio n of concentratio n (n orm ality, isocyanate m illiequ ivalents per milliliter) of monom er standards. Use the slope of the linear portion of the calibration curve to determine the normality of a single compone nt, a gro up of c om ponents , or all isocyanate com pon ents in the analyzed sam ple aliqu ot.

where: N= UV = slopecal =

the norm ality of sam ple co m pon ent(s ), the UV area of sample component(s), and the slope of the calibra tion curve in units of U V area per norm ality.

The method m easures normality of oligomeric isocyanate group in the analyzed sample. It cannot m easure µg per sample unless the molecular weight of the component is known. Generally, there is a m ixture of compounds of different and unknown molecular weights. To deal in micrograms per sample, use the form ulae given above for m onom er quantification and the equivalent weight of the particular monom er of interest. This value is the monom er mass equivalent - the mass of the monom er that would conta in the same number of isocyanate groups as w as fou nd in the sam ple. Alte rnatively, the micrograms of isocyanate group per sample can be calculated using the formulae and entering 42 for the equivalent weight.

NIOSH Manual of Analytical Methods (NMAM), Fourth Edition ISOCYANAT ES, TOTA L (MAP): METHO D 5525, Issue 1, dated 15 March 2003 - Page 8 of 17 EVALUATION OF METHOD: Shelf Life of MAP , MAP Solutions and M AP-C oated Filters: Pu re, so lid M AP is stab le for years when stored in a freezer. Concentrated solutions of MAP (concentrated stocks or filter-spiking solutions) are not very stable and should be kept in the freezer for a maximum of one week [7,8]. MAP -coated glass-fiber and qu artz-fiber filters are stable for 1 week at room temperature and 6 months in the freezer [7,8]. Artifacts derived from MAP are present in the chromatogram of MAP-coated filter sam ples. Although the levels are low, these impurities dictate the method LOD when they coelute with an analyte. The level of artifacts observed is not correlated well with storage time. Room temperature storage results in greate r artifact levels than sto rage in the freezer. MAP is known to be light sensitive. It is not clear what role exposure to light plays in the formation of the chromatographic interferences. It is recomm ended that exposure to light be minimized when handling MAP solutions and MAP-coated filters. No formal study has been conducted reg arding the shelf life of im pinger solutions of MAP in butyl benzoate. However, no problems have been observed when the impinger solutions are used within one week of being m ade. Im pinger sam ples are noted to have fe we r artifacts than filter sam ples. A ltho ugh this may be due in part to greater stability of the dilute M AP solutions versus M AP on filters , it ma y also be attribu table to the generally lower amount of reagent used in impinger samples as well as the solid-phase extraction procedure to which impinger sa m ples are s ubje cted . Stability of MAP-Isocyanate Derivatives: Pure, solid MAP-derivatized isocyanate monom ers are stable for years when stored in the freezer. MAPderivatized m onom ers and oligom ers of alip hatic isocyanate s are very stab le in aceto nitrile (the sam ple solve nt) for at least one year [7]. Samples containing TDI-MAP gave results 16-24% lower than the original value s wh en re ana lyzed afte r stora ge fo r nine m onth s in the freezer [8]. Filte r M ate rials: Com parison of quartz-fiber and glass-fiber filters showed no significant differences in recovery of MAPderivatized monom ers from the filters. Com parison of stainless steel and cellulose backup pads showed no significant reagent loss with either. Neither backup material contributed to the artifact production in the filter sam ple [8]. Filter Versus Impinger Sampling: A side-by-side comparison of MAP-coated IOM filters and MAP impingers, conducted in autobody shops using aliphatic isocyanate products [9], showed no significant difference in the performance of these two samplers. It can be concluded from this stu dy that losses of relatively slow-curing aliphatic isocyanates collected on filters due to curing reactions are minimal when filters are extracted in the field imm ediately after sampling. It also appears from this stu dy that th e IO M’s bette r as piratio n effic iency for rela tively large aerosol did not result in increased collection efficiency compared to the midget impinger. This suggests th at an inhalable sampler is not necessary in this type of environm ent. In general, the approp riate choice of sam pler - filter, impinger, or im pinger an d filter in se ries - is d ictated by the particular expos ure s cen ario [1,2 ]. PEEK Versus Stainless Steel HPLC Columns: PEEK and stainless steel HPLC columns were compared with respect to efficiency, tailing, and carryover. No significant differences in performance were observed [8]. Therefore, stainless steel columns are considered acceptable. However, because MAP derivatives are known to adsorb onto sta inless ste el surfaces, it is recomm ended that the frits of the column be constructed of more inert material, i.e., PEEK or titanium. pH Gradient: Method 5525 uses pH -gradient H PL C to enable m easurem ent of rela tively weak ly retained monofunctional isocyanates and diisocyanate monomers as well as relatively strongly retained oligomeric isocyanates in the sam e analysis. Several characteristics of the pH gradient have been evaluated [10]. The pH grad ient is selective, only accelerating strongly basic compounds (all com pounds containing a MA P group a re strongly basic). The strength of the pH grad ient w as evaluate d by com paring the degree to which retention is NIOSH Manual of Analytical Methods (NMAM), Fourth Edition ISOCYANAT ES, TOTA L (MAP): METHO D 5525, Issue 1, dated 15 March 2003 - Page 9 of 17 shortened in the strong mobile phase relative to the weak m obile phase. A model monofunctional isocyanate (octyl isocyanate) eluted 14 times faster and a model diisocyanate (MDI) eluted several hundred times faster using the strong mobile phase. Re-equilibration time is only a few minutes when switching from the strong m obile phas e ba ck to the weak m obile phas e. Detector baselines are very stable during the gradient. The fluorescence baseline is unchanged and the UV253 baseline is disturbed less than 1 mV . The UV response of MAP derivatives has been found to be independent of the pH gradient. However, the fluorescence response is highly dependent on the pH of the m obile phase. This is corrected by post-column addition of a cid to the m obile phas e prior to detection . Co m pou nd-to -Co m pou nd U V R esp ons e Va riability: Evaluation of UV response for several MAP-derivatized isocyanates, including monofunctional isocyanates and diisocyanates [5,7] showed a compound-to-compound RSD of 3.0-3.5% at the maxim um 254-256 nm and an RSD of 1.0-2.2% at the less intense but more selective wavelengths of 352 nm , 368 nm , and 388 nm . In other studies, the UV response of MDI triisocyanate oligomer varied from 6% higher than the mean of several m onom ers [7] to ab out 10% lower than the M DI m ono m er [8]. Th e o verwhe lm ing m ajority of the da ta sug ges ts very little variability in UV response among MAP-derivatized isocyanate compounds, supporting the validity of quantifying non-monom eric isocyanate species by using the UV area of the monom er standard. Reco very of MAP-D erivatized Mon om ers: Quartz-fiber and glass fiber filters were spiked with HDI-MAP, 2,4-TDI-MAP, and MDI-MAP at 630 ng NCO group per compon ent per filter along with 500 :g MAP. Recovery ranged from 97-98% for the quartz-fiber filters and 97-9 9% for the glass -fiber filters [8]. In another study, MAP-coated quartz-fiber filters were spiked with a mixture of HDI, 2,4-TDI, and MDI at nominal levels of 21, 63, 210, 630, and 2100 ng NCO group per component. Re co veries averaged 93% for HDI, 91% for 2,4-TDI, and 92% for MDI with no correlation between recovery and spiking level over the range studied. As part of an International Organization for Standardization (ISO) intercomparison stu dy, tw o com m ercial pro ducts conta ining only m onom eric isocyanates were studied [8]. Levels spiked on filters were 2100, 1050, 525, and 105 ng N CO group per filter, corresponding to 2X , 1X, 0.5X, and 0.1X the U.K STEL level. For Desm odur H (HDI monom er), the mean recoveries were 104% at 2X, 103% at 1X, 102% at 0.5X, and 99% at 0.1X. For D esm odur T -80 (a m ixture of 2,6-TDI and 2,4-TDI), the mean recoveries were 101% at 2X, 104% at 1X, 103% at 0.5X, and 108% at 0.1X. Be llo et al. found HDI monom er recovery averaging 108% in the range of 0.45 to 42 ng NCO per filter and, in a se para te study, a m ean reco very of 100 % in the ra nge of 8 to 250 ng N CO per filter [7]. Recovery of Total Isocyanate Group: Losses of isocyanates can occur at several stages in the sampling and analytical pro cess [1,2 ]. Be llo et al. evaluated total reactive isocyanate group (TRIG) recoveries from spiked filters and impingers as well as recoveries from HPLC analysis [7]. Desmodur N3300 (HDI isocyanurate) was derivatized with MAP and spiked onto quartz-fiber filters. Ex tractio n and analysis fo und re covery ranging from 91-9 3% com pare d to directly analyzed product for spiking levels ranging from 90 ng to 2.34 :g NCO per filter. Sim ilarly, a bulk product containing HDI isocyanurate and IPDI oligomers was derivatized with MAP, spiked into MAPcontaining impingers, processed by solid-phase extraction, and analyzed by HPLC . The recove ry of the TR IG varied from 92-106% for the HDI isocyanurate in the range 18-2 500 ng NCO/mL and 76-89% for the IPDI oligomer in the range of 16-1900 ng NCO/m L. Recoveries from HPLC analysis were determined by comparison with isocyanate conte nt obta ined by bulk p roduct titration . The pro ducts investiga ted and their respective recoveries were Bayer N100 (HDI biuret) 75%, Bayer N3300 (HDI isocyanurate) 94%, Bayer N3400 (HDI isocyanurate + uretidinedione) 95%, Bayer Z4470 (IPDI oligomers) 100%, and B26-2 (HDI isocyanurate + IPDI oligomers) 96%. These HPLC recoveries were conducted at high analyte levels. It is recognized that quantitative recoveries of complex products are not possible at low levels because many components fall below the lim it of detection [7]. As part of an International Organization for Standardization (ISO) intercomparison study, two comm ercial products containing oligomeric isocyanates we re stu died [8]. Le vels spiked on filters were 2100, 1050, 525, and 105 ng NCO group per filter, corresponding to 2X, 1X, 0.5X, and 0.1X the U.K. STEL level. For Desmodur N3300 (HDI isocyanurate), the mean recoveries were 52% at 2X, 43% at 1X, 35% at 0.5X, and 28% at 0.1X. These results are very different than those obtained by Bello et al. [7] and require re-exam ination. For Sup rasec 50 30 (polymeric M DI), the mean recoveries were 65% at 2X, 62% a t 1 X, 5 9% at 0.5 X, and 54% at 0.1X . Stu dies are underway to account for m issing isocyanate in oligomeric products. NIOSH Manual of Analytical Methods (NMAM), Fourth Edition ISOCYANAT ES, TOTA L (MAP): METHO D 5525, Issue 1, dated 15 March 2003 - Page 10 of 17 Limit of Detection/Limit of Quantification: The limit of detection (LOD) and limit of quantification (LOQ) were determined by spiking several MAPderivatized m onom ers onto MAP filters in the range of 0.01 to 3.0 nanoe quiva lents, e xtrac ting the filters with acetonitrile, acetylating and ana lyzing by HP LC. The m etho d LO D is e stim ated to be abo ut 0.2 nan oeq uivalen ts NCO per sam ple and the LOQ 0.5 nanoequivalents NCO per sample for both fluorescence height and UV area [8]. As an example, these are equivalent to an LOD of 17 ng per sample and an LOQ of 42 ng per sample for HDI monom er. Bello et al. estimated the method detection limit to be 8 ng per sam ple for HDI monom er [9]. The instrumental LOD is substantially lower than the method detection limit, but is not achievable because of low levels of reagent-derived artifacts that are present in the chromatogram . Occ asionally an analyte peak will coelute with a relatively large artifact peak, raising the LOD and LOQ substan tially. Our experience is that IPDI m onom er isom ers coelute with som e HD I oligom ers when bo th are present in a sa m ple. T his m akes the IPD I m ono m ers, which are typically pres ent at very low levels, unm eas urab le.

REFERENCES: [1]

[2]

[3]

[4]

[5]

[6] [7]

[8]

[9]

[10]

[11] [12] [13]

NIOSH [1998]. Chapter K: De term ination of a irborne isocyanate exp osu re. In: O’Connor PF, ed. NIOSH Ma nua l of Analytical Metho ds, 4 th ed. C incinn ati, OH : U.S. Dept. of Health and Human Services, Public Health Service, Centers for Disease Control and Prevention, National Institute for Occupational Safety and Health, DHHS (NIOSH). DHHS/NIOSH Publication #98-119. Streicher RP, Reh CM, Key-Schwartz RJ, Schlecht PC, Cassinelli ME, O’Connor PF [2000]. Determ ination of airborne isocyanate exposure: considerations in method selection. American Ind ustrial Hyg iene Ass ociation Journal 61: 544-556. NIOSH [1994]. Method 55 22, Isocyanates. In: Eller PM, Cassinelli ME, eds. NIOSH Man ua l of Analytical Me thod s, 4 th ed. Cincinnati, OH: U .S. Dept. of He alth an d Hum an S ervices, P ublic H ealth Service, Centers for D isease C ontrol and Prevention, National Institute for Occ upational Safety and Health, DHHS (NIOSH) Publication No. 94-113. NIOSH [1994]. Method 5521, Isocyanates, Monom eric. Issue 2. In: Eller PM, Cassinelli ME, eds. NIOSH Manual of A nalytica l Methods, 4 th ed. Cincinnati, OH: U.S. Dept. of Health and Human Services, Pub lic Health Service, Cente rs for Diseas e Contro l and P reve ntion, Na tional Institute for Occupational Safety and Health, DHHS (NIOSH) Publication No. 94-113. Streicher RP, Arnold JE, Ernst MK, Coope r CV [1996]. Development of a novel derivatization reagent for the sampling and analysis of total isocyanate group in air and comparison of its pe rform anc e with tha t of several estab lished rea gents . Am erican Industrial H ygiene As sociation Journal 57: 905-913. For inform ation o n availability of MAP, contact Robert Streicher, NIOSH M ail Stop R-7, 4676 Co lum bia Parkw ay, Cincinnati, OH 45226, tel: 513-841-4296 , E-m ail: rstreicher@c dc.gov. Be llo D, Streicher RP, W oskie SR [2002]. Eva luation of the NIO SH Draft Method 5525 for Determination of the Tota l Re active Iso cyanate G roup (TRIG) in A ir in Autobody Repair Shops. Journal of Environmental Monitoring 4: 351-360. Streicher RP, Ernst MK, W illiamson GY. Unpublished data. Cincinnati, OH: U.S. Dept. of Health and Hum an Services, Public Health Service, Centers for Disease Control and Prevention, National Institute for Occupational Safety and Health. Bello D, Streicher RP, Liu YC, Sparer J, Youngs F, W oskie SR [2002]. Field comparison of impingers and treated filters for sampling of total aliphatic isocyanates using the MAP reagent. American Ind ustrial Hyg iene Ass ociation Journal 63: 790-796. Streicher RP, Ernst MK, W illiamson G Y [2000]. Use of pH-gradient HPLC for the analysis of di- and polyisocyanates. Presented at the 32 nd Central Regional Meeting of the Am erican C hem ical Society, Covington, KY, May 18. Unless otherwise specified, properties were obtained from the Aldrich H andbook o f Fine Ch em icals and Laboratory Equipment, 2000-2001. W oods G [1987]. The IC I Po lyureth anes Book . New York, NY: IC I Po lyureth anes and John W iley & Sons, Inc. Inte rnatio nal Ch em ical Safety Card # 0278, http://hazard.c om /m sds/m f/cards/file/02 78.htm l.

NIOSH Manual of Analytical Methods (NMAM), Fourth Edition ISOCYANAT ES, TOTA L (MAP): METHO D 5525, Issue 1, dated 15 March 2003 - Page 11 of 17 [14] [15]

Inte rnatio nal Ch em ical Safety Card # 0499, http://hazard.c om /m sds/m f/cards/file/04 99.htm l. Dharmarajan V, M yer HE [19 91]. Hazard Evaluation and Control of Comm ercial Isocyanate Products. Professional Development Course #60, presented at the American Industrial Hygiene Conference and Exhibition, Salt Lake City, UT, May 19.

METHOD WRITTEN BY: Robert P. Streicher, Ph. D., M. Kathleen Ernst and George Y. W illiamson, NIOSH/DART

NIOSH Manual of Analytical Methods (NMAM), Fourth Edition ISOCYANAT ES, TOTA L (MAP): METHO D 5525, Issue 1, dated 15 March 2003 - Page 12 of 17 TABLE 1. EXPOSURE LIMITS, PROPERTIES, CAS AND RTECS NUMBERS Isocyanates

Expo sure Limits µg /m 3*

Properties [11]

OSHA

NIOSH

AC GIH

(PEL)

(REL)

(TLV)

Toluene 2,6-diisocyanate (2,6-TDI)

no PEL

lowest feasible (Ca)

liquid; d 1.225 g/mL @ 25oC; MP no TLV 19.5-21.5/C; VP 3.33 Pa (0.025 mm Hg) @ 25oC [12]

Toluene 2,4-diisocyanate (2,4-TDI)

liquid; d 1.214 g/mL @ 25oC; MP 20lowest 22oC, VP 3.33 Pa (0.025 mm Hg) @ feasible 36 140 (Ca) 140 STEL 25oC [12] (ceiling)

4,4'-Methylenebis(phenyl isocyanate) (MDI)

50; 200 200 (10 min (ceiling) ceiling)

51

34

no PEL

35; 140 (10 min ceiling)

Isophorone diisocyanate (IPDI)

45

no PEL

45; 180 (10 min ceiling)

4,4'-Methylenebis(cyclohexyl isocyanate) (HMDI)

No REL; 110 (10 min no PEL ceiling)

1,6-Hexamethylene diisocyanate (HDI)

1,5-Naphthalene diisocyanate (NDI)

no PEL

40;70 (10 min ceiling)

54

CAS #

RTECS #

91-08-7

CZ6310000

584-84-9

CZ6300000

solid (fused); d 1.180g/mL @ 25oC; MP 42-44oC; VP 6x10-4 Pa (4.5x10-6 mm Hg) @ 25oC [12]

101-68-8 NQ9350000

liquid; d 1.04 g/mL @ 25oC; BP 255oC; 7 Pa (0.05 mm Hg) @ 25oC [13]

822-06-0 MO1740000

liquid; mixture of isomers, d 1.049 @ 25oC; MP -60oC [14]; VP 0.04 Pa @ 20oC (0.0003 mm Hg) [12] 4098-71-9 NQ9370000

liquid; mixture of isomers, d 1.066 g/mL @ 25oC; VP 0.13 Pa @ 20oC (0.001 mm Hg) [15]

5124-30-1 NQ9250000

solid flakes; MP 129-131oC [12]; VP 0.4 Pa @ 25oC (0.003 mm Hg) [12]

3173-72-6 NQ9600000

no TLV

1 ppb = 7.1 2 µg /m 3 TD I, 10.24 µg/m 3 MD I, 6.88 µ g/m 3 HD I, 9.09 µ g/m 3 IPD I, 10.73 µg/m 3 HM DI, 8.60 µg/m 3 NDI

NIOSH Manual of Analytical Methods (NMAM), Fourth Edition ISOCYANAT ES, TOTA L (MAP): METHO D 5525, Issue 1, dated 15 March 2003 - Page 13 of 17

TABLE 2. SYNONY MS O F COM MO N ISOCYANATES Isocyan ate Acronym

Com mon Chem ical N am e

2,4-TDI

Toluene 2,4-d iisocyanate

2,4-Diisocyanatotoluene; 2,4-diisocyanato-1-methylbenzene; isocyanic acid, 4-methyl-m-phenylene ester; methyl-m-phenylene isocyanate; 4-methyl-1,3-phenylene diisocyanate; tolylene 2,4-d iisocyanate

2,6-TDI

Toluene 2,6-d iisocyanate

2,6-Diisocyanatotoluene; 2,6-diisocyanato-1-methylbenzene; 2-m ethyl-1,3-phe nylene diisocyanate; tolylene 2,6-d iisocyanate

MDI

Methylenebis(phenyl isocyanate)

4,4'-Diisocyanatodiphenylmethane; 4,4'-diphenylmethane diisocyanate; isocyanic acid, m ethylene di-p-phenylene ester; 1,1 '-m eth ylenebis(4-isocyanatob enzen e); 4,4 '-m eth ylenebis(phenyl isocyanate); m eth ylenebis(4-phenyl isocyanate); m eth ylenediphenyl diisoc yanate; m ethylenedi-p-phe nylene diisocyanate

HDI

Hexam ethylene diisoc yanate

1,6-D iisocyanato hex ane ; 1,6-H exa ned iol diisocyanate; HM DI; hexane-1,6-diisocyanate ; 1,6 -hexylene diisocyanate; is ocyanic acid, hexam ethylene ester

IPDI

Isophorone diisoc yanate

5-Isocyanato-1-(isocyanatomethyl)-1,3,3-trimethylcyclohexane; 3-isocyanatom eth yl-3,5,5 -trim eth ylcyclohexylisocyanate; is ocyanic acid, me thylene(3,5,5-trimethyl-3,1-cyclohexylene) ester; isophoro ne d iam ine diiso cyanate

HMDI

4,4’-Methylenebis(cyclohexyl isocyanate)

Hydrogenated MDI

Synonym s

NIOSH Manual of Analytical Methods (NMAM), Fourth Edition ISOCYANAT ES, TOTA L (MAP): METHO D 5525, Issue 1, dated 15 March 2003 - Page 14 of 17 TABLE 3. EXAMPLES OF EXPOSU RE SCENERIOS AND WH ICH SAMPLER TO CH OOSE Sam pling Section in Method

Sam pler

Ex posure Sc enario

GFF: 0.1 mg MAP/cm 2

1

Vapor only (e.g., flexible foam m anufacture using TD I mon om er) Aerosols of aliphatic isocyanates (e.g., autobody refinishing using HDI- and IPDI-based polyisocyanates) Aerosols of arom atic isocyanates <2 µ m (e.g., con den sation aero sol fro m hea ting M DI)

Impinger: 1x10 -4 M MAP in butyl benzoate (2.76 mg MAP per 100 mL)

2

Aerosols of arom atic isocyanates >2 µ m (e.g., spraying of MDI polyisocyanate or cutting partially-cured MD I-bas ed p rodu ct)

Impinger + GFF: MAP impinger followed by MAP on GFF

3

Aerosols of aromatic isocyanates both <2 µm and >2 µm (m ixture of mechanically generated aerosol and condensation aerosol, e.g., heating and cutting partially-cu red M DI-b ase d pro duc t)

NIOSH Manual of Analytical Methods (NMAM), Fourth Edition ISOCYANAT ES, TOTA L (MAP): METHO D 5525, Issue 1, dated 15 March 2003 - Page 15 of 17

APPENDIX A: PREPARATION OF PHO SPHATE-FORMATE BU FFER AND MO BILE PHASE 100 mM PHO SPHATE - 100 mM FOR MATE BUFFER Add 46.12 g 85% Phosphoric Acid (~27 mL) and 15.08 m L Form ic Acid to 3700 m L deionized hi-pure water. The initial pH should be ~1.56. Titrate with Triethylamine (TEA) to a pH of 6.0 using a standardized pH m eter. This will take between 115 and 120 mL of TEA. After the pH adjustment to pH 6.0, add additional deionized hi-pure water to bring the final volume to 4.0 L. Sp lit into two 2L-po rtions. The first portion is adju sted to pH 1.6 with concentrated HCl, monitoring the pH by pH m eter. T his shou ld take be twee n 30 and 35 m L of c onc entra ted H Cl. The second portion remains at pH 6.0, but must have a volume of hi-pure water added that is equal to the volume of H Cl ad ded to the first portion. This will ensure that the concentration of salts in the two different pH buffers are the same resulting in less baseline change during the gradient run. Store all buffers in the refrigerator until mixed with acetonitrile. MO BILE PH ASE: 65 :35 A CE TO NIT RILE-P HO SPHA TE /FO RM AT E BUF FE R (F or bo th pH 6.0 and pH 1.6) Mix 650 mL acetonitrile and 350 mL buffer. Total volume will be less than 1000 mL. The m obile phase made from pH 6.0 buffer is designated mobile phase A. The m obile phase made from pH 1.6 buffer is designated m obile phas e B.

APP EN DIX B: PREPARATION OF PHOSPH ORIC ACID AND MOBILE PHASE FOR POST-COLUMN ADDITION 4.4 N PH OS PH OR IC A CID Add 50 mL phosphoric acid (85%) to 400 mL deionized hi-pure water and adjust to 500 mL. 65:35 ACETONITRILE-(4.4 N PHOSPHO RIC ACID) MOBILE PHASE Mix 650 mL acetonitrile with 350 mL of 4.4 N phosphoric acid.

NIOSH Manual of Analytical Methods (NMAM), Fourth Edition ISOCYANAT ES, TOTA L (MAP): METHO D 5525, Issue 1, dated 15 March 2003 - Page 16 of 17

APPENDIX C: SYNTHESIS OF M AP DERIVATIVES Chem ical

MW

EQ. W T.

MAP Derivative MW

MAP Derivative EQ. W T.

MAP: 1-(9-Anthracenylmethyl)piperazine

276 .4

276 .4

HD I: Hex am ethylene diisocyanate

168 .2

84.1

721 .0

360 .5

TDI: Tolylene diisocyanate (toluene diisocyanate)

174 .2

87.1

727 .0

363 .5

MDI: 4,4'-Methylenebis(phenyl isocyanate)

250 .3

125 .2

803 .1

401 .6

HMD I: 4,4'-Methylenebis(cyclohexyl isocyanate)

262 .4

131 .2

815 .2

407 .6

MDI Trimer (MDI Triiso)

381 .4

127 .1

121 0.6

403 .5

IPD I: Isoph oron e diisocyanate

222 .3

111 .2

775 .1

387 .5

PhICN : Phe nyl isocyanate

119 .1

119 .1

395 .5

395 .5

BuICN : Butyl isoc yanate

99.1

99.1

375 .5

375 .5

318 .4

318 .4

AcMAP: Acetylated MAP

2 MAP + 1 diisocyanate yields 1 MAP-diisocyanate derivative 1. 2. 3. 4. 5. 6. 7. 8. 9. 10.

Theoretical yield 1 meq: range from 360-408 m g. W eigh 1 m eq of d iisocyanate to fou r de cim al places. Dissolve in 10 m L of toluene. W eigh 1.2 meq of MAP (20% excess) to four decimal places. Dissolve in 20 mL of toluene. W hile stirring the M AP solution, add the diisocyanate solution dropw ise ov er a 1 0 to 15 m inute p eriod. Continue to stir for at least one ho ur. Tightly cover the solution and store overnight in the freezer to promote maximum precipitation. Us ing a B uec hne r funnel with filter pap er, filter the solution to collect the prec ipitate. W ash several times with small amounts of cold toluene to remove any remaining MAP. W ash several tim es with sm all am ounts of c old hexane to displace the toluene. Continu e to pull air throu gh the so lid for se vera l minutes to dry. Trans fer the solid derivative into a preweighed vial with T eflon cap. Using the vacuum pum p, apply vacuum until a stable weight is obtained.

NIOSH Manual of Analytical Methods (NMAM), Fourth Edition ISOCYANAT ES, TOTA L (MAP): METHO D 5525, Issue 1, dated 15 March 2003 - Page 17 of 17 APPENDIX D: PREPARATION OF B ULK DERIVATIVES 1. Dilute bulk 1/10 in methylene chloride by wt/wt. Quickly proceed to step 2. a. W eigh disposable 7 m L vial em pty. b. Add about 0.5 g bulk, weigh vial again. c. Add 4.5 g (3.4 mL) m ethylene chloride by volume addition, weigh vial again. Use this total weight of 1:10 dilution in calculating final concentration of bulk solution. Density of methylene chloride is 1.325 g/m L. 2. W eigh 1.0 mL of the 1:10 dilution of bulk to determine density. Use this density in calculating the final concentration of bulk solution. 3. Dilute the solution resulting from Step 1 1:100 (10 :L into 990 :L) in methylene chloride by vol/vol. Imm ediately proceed to step 4. 4. Add 25 :L of the solution resulting from Step 3 to 975 :L acetonitrile containing 5X10 -4 N MAP ( 1:40 dilution). 5. Let react overnight in dark. 6. Add 5 :L acetic anhydride. Allow to react at least two hours at room tem perature or overnight in the refrigerator before proceeding. 7. Analyze by HPLC, injecting 30 :L. 8. Total dilution is about 1:40,000. 9. Final bulk solution concentration (C B, g/mL):

where: W tbulk = W ttot = Dens 1:10 = DF =

the the the the

weight of sample (Step 1) combined weight of sample and methylene chloride (Step 1) density of the initial sample preparation (Steps 1 and 2) dilution factor (Steps 3 and 4) (as presented above, DF = 0.00025)

NIOSH Manual of Analytical Methods (NMAM), Fourth Edition