METHOD 8032 ACRYLAMIDE BY GAS CHROMATOGRAPHY

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1 METHOD 8032 ACRYLAMIDE BY GAS CHROMATOGRAPHY 1.0 SCOPE AND APPLICATION 1.1 Methd 8032 is used t determine trace amunts f acrylamide mnmer in aqueus matrices. This methd may be applicable t ther matrices. The fllwing cmpund can be determined by this methd: Cmpund Name CAS N. a Acrylamide a Chemical Abstract Services Registry Number. 1.2 The methd detectin limit (MDL) in clean water is µg/l. 1.3 This methd is restricted t use by, r under the supervisin f, analysts experienced in the use f gas chrmatgraphs and skilled in the interpretatin f gas chrmatgrams. Each analyst must demnstrate the ability t generate acceptable results with this methd. 2.0 SUMMARY OF METHOD 2.1 Methd 8032 is based n brminatin f the acrylamide duble bnd. The reactin prduct (2,3-dibrmprpinamide) is extracted frm the reactin mixture with ethyl acetate, after salting ut with sdium sulfate. The extract is cleaned up using a Flrisil clumn, and analyzed by gas chrmatgraphy with electrn capture detectin (GC/ECD). 2.2 Cmpund identificatin shuld be supprted by at least ne additinal qualitative technique. Analysis using a secnd gas chrmatgraphic clumn r gas chrmatgraphy/mass spectrmetry may be used fr cmpund cnfirmatin. 3.0 INTERFERENCES 3.1 N interference is bserved frm sea water r in the presence f 8.0% f ammnium ins derived frm ammnium brmide. Impurities frm ptassium brmide are remved by the Flrisil clean up prcedure. CD-ROM Revisin 0

2 4.0 APPARATUS AND MATERIALS 4.1 Gas chrmatgraphic System Gas chrmatgraph suitable fr n-clumn injectins with all required accessries, including detectr, analytical clumns, recrder, gases, and syringes. A data system fr measuring peak heights and/r peak areas is recmmended Clumn: 2 m x 3 mm glass clumn, 5% FFAP (free fatty acid plyester) n mesh acid washed Chrmsrb W, r equivalent Detectr: electrn capture detectr. 4.2 Kuderna-Danish (K-D) apparatus Cncentratr tube - 10 ml graduated (Kntes K r equivalent). A grund glass stpper is used t prevent evapratin f extracts Evapratin flask ml (Kntes K r equivalent). Attach t cncentratr tube with springs, clamps, r equivalent Snyder clumn - Three ball macr (Kntes K r equivalent) Snyder clumn - Tw ball micr (Kntes K r equivalent) Springs - 1/2 inch (Kntes K r equivalent). 4.3 Separatry funnel ml. 4.4 Vlumetric flask (Class A) ml, with grund glass stpper; 25 ml, amber, with grund glass stpper. 4.5 Syringe - 5 ml. 4.6 Micrsyringes - 5 µl, 100 µl. 4.7 Pipets (Class A). 4.8 Glass clumn (30 cm x 2 cm). 4.9 Mechanical shaker. 5.0 REAGENTS 5.1 Reagent grade chemicals shall be used in all tests. Unless therwise indicated, it is intended that all reagents shall cnfrm t the specificatins f the Cmmittee n Analytical Reagents f the American Chemical Sciety, where CD-ROM Revisin 0

3 such specificatins are available. Other grades may be used, prvided it is first ascertained that the reagent is f sufficiently high purity t permit its use withut lessening the accuracy f the determinatin. 5.2 Organic-free reagent water. All references t water in this methd refer t rganic-free reagent water, as defined in Chapter One. 5.3 Slvents Ethyl acetate, C H CO C H. Pesticide quality, r equivalent Diethyl ether, C H OC H. Pesticide quality, r equivalent Must be free f perxides as indicated by test strips (EM Quant, r equivalent). Prcedures fr remval f perxides are prvided with the test strips. After cleanup, 20 ml f ethyl alchl preservative must be added t each liter f ether Methanl, CH OH. Pesticide quality, r equivalent Benzene, C H. Pesticide quality, r equivalent Acetne, CH COCH. Pesticide quality, r equivalent Saturated brmine water. Prepare by shaking rganic-free reagent water with brmine and allwing t stand fr 1 hur, in the dark, at 4 C. Use the aqueus phase. 5.5 Sdium sulfate (anhydrus, granular), Na2SO 4. Purify by heating at 400 C fr 4 hurs in a shallw tray, r by precleaning the sdium sulfate with methylene chlride. If the sdium sulfate is precleaned with methylene chlride, a methd blank must be analyzed, demnstrating that there is n interference frm the sdium sulfate. 5.6 Sdium thisulfate, Na S O, 1 M aqueus slutin Ptassium brmide, KBr, prepared fr infrared analysis. 5.8 Cncentrated hydrbrmic acid, HBr, specific gravity Acrylamide mnmer, H C:CHCONH, electrphresis reagent grade, 2 2 minimum 95% purity Dimethyl phthalate, C H (COOCH ), 99.0% purity Flrisil (60/100 mesh): Prepare Flrisil by activating at 130 C fr at least 16 hurs. Alternatively, stre Flrisil in an ven at 130 C. Befre use, cl the Flrisil in a desiccatr. Pack 5 g f the Flrisil, suspended in benzene, in a glass clumn (Sec. 4.8) Stck standard slutins Prepare a stck standard slutin f acrylamide mnmer as specified in Sec When cmpund purity is assayed t be 96% CD-ROM Revisin 0

4 r greater, the weight can be used withut crrectin t calculate the cncentratin f the stck standard. Cmmercially prepared standards can be used at any cncentratin if they are certified by the manufacturer r by an independent surce Disslve mg f acrylamide mnmer in rganic-free reagent water in a 100 ml vlumetric flask, and dilute t the mark with rganic-free reagent water. Dilute the slutin f acrylamide mnmer s as t btain standard slutins cntaining mg/l f acrylamide mnmer Calibratin standards Dilute the acrylamide stck slutin with rganic-free reagent water t prduce standard slutins cntaining mg/l f acrylamide. Prir t injectin the calibratin standards are reacted and extracted in the same manner as envirnmental samples (Sec. 7) Internal standards The suggested internal standard is dimethyl phthalate. Prepare a slutin cntaining 100 mg/l f dimethyl phthalate in ethyl acetate. The cncentratin f dimethyl phthalate in the sample extracts and calibratin standards shuld be 4 mg/l. 6.0 SAMPLE COLLECTION, PRESERVATION, AND HANDLING 6.1 See the intrductry material t this chapter, Organic Analytes, Sec PROCEDURE 7.1 Brminatin Pipet 50 ml f sample int a 100 ml glass stppered flask. Disslve 7.5 g f ptassium brmide int the sample, with stirring Adjust the ph f the slutin with cncentrated hydrbrmic acid until the ph is between 1 and Wrap the flask with aluminum fil in rder t exclude light. Add 2.5 ml f saturated brmine water, with stirring. Stre the flask and cntents in the dark, at 0 C, fr at least 1 hur After reacting the slutin fr at least an hur, decmpse the excess f brmine by adding 1 M sdium thisulfate slutin, drpwise, until the clr f the slutin is discharged Add 15 g f sdium sulfate, using a magnetic stirrer t effect vigrus stirring. CD-ROM Revisin 0

5 7.2 Extractin Transfer the slutin int a 150 ml separatry funnel. Rinse the reactin flask three times with 1 ml aliquts f rganic-free reagent water. Transfer the rinsings int the separatry funnel Extract the aqueus slutin with tw 10 ml prtins f ethyl acetate fr 2 min each, using a mechanical shaker (240 strkes per min). Dry the rganic phase with 1 g f sdium sulfate Transfer the rganic phase int a 25 ml amber vlumetric flask. Rinse the sdium sulfate with three 1.5 ml prtins f ethyl acetate and cmbine the rinsings with the rganic phase Add exactly 100 µg f dimethyl phthalate t the flask and make the slutin up t the 25 ml mark with ethyl acetate. Inject 5 µl prtins f this slutin int the gas chrmatgraph. 7.3 Flrisil cleanup: Whenever interferences are bserved, the samples shuld be cleaned up as fllws Transfer the dried extract int a Kuderna-Danish evapratr with 15 ml f benzene. Evaprate the slvent at 70 C under reduced pressure, and cncentrate the slutin t abut 3 ml Add 50 ml f benzene and subject the slutin t Flrisil clumn chrmatgraphy at a flw rate f 3 ml/min. Elute the clumn first with 50 ml f diethyl ether/benzene (1:4) at a flw rate f 5 ml/min, and then with 25 ml f acetne/benzene (2:1) at a flw rate f 2 ml/min. Discard all f the first eluate and the initial 9 ml prtin f the secnd eluate, and use the remainder fr the determinatin, using dimethyl phthalate (4 mg/l) as an internal standard. NOTE: Benzene is txic, and shuld be nly be used under a ventilated labratry hd. 7.4 Gas chrmatgraphic cnditins: Nitrgen carrier gas flw rate: 40 ml/min Clumn temperature: 165 C. Injectr temperature: 180 C Detectr temperature: 185 C. Injectin vlume: 5 µl 7.5 Calibratin: Inject 5 µl f a sample blank (rganic-free reagent water carried thrugh all sample strage, handling, brminatin and extractin prcedures) Prepare standard slutins f acrylamide as described in Sec Brminate and extract each standard slutin as described in Secs. 7.1 and 7.2. CD-ROM Revisin 0

6 Inject 5 µl f each f a minimum f five standard slutins: ne shuld be near the detectin limit; ne shuld be near, but belw, the expected cncentratins f the analyte; ne shuld be near, but abve, the expected cncentratins f the analyte Prepare a calibratin curve using the peak areas f the standards. If the calibratin curve deviates significantly frm a straight line, prepare a new calibratin curve with the existing standards, r, prepare new standards and a new calibratin curve. See Methd 8000, Sec , fr additinal guidance n calibratin by the internal standard methd Calculate the respnse factr fr each standard accrding t Equatin 1. (P s) (M is) RF = Equatin 1 (P is) (M A) RF = Respnse factr P s = Peak height f acrylamide M = Amunt f internal standard injected (ng) is P is = Peak height f internal standard M A = Amunt f acrylamide injected (ng) Calculate the mean respnse factr accrding t Equatin 2. n 3 RF i=1 RF = Equatin 2 n RF = Mean respnse factr RF = Respnse factrs frm standard analyses (calculated in Equatin 1) n = Number f analyses 7.6 Gas chrmatgraphic analysis: Inject 5 µl prtins f each sample (cntaining 4 mg/l internal standard) int the gas chrmatgraph. An example GC/ECD chrmatgram is shwn in Figure The cncentratin f acrylamide mnmer in the sample is given by Equatin 3. (P A) (M is) [A] = Equatin 3 (P is) (RF) (V i) (V s) [A] = Cncentratin f acrylamide mnmer in sample (mg/l) CD-ROM Revisin 0

7 P = Peak height f acrylamide mnmer A M = Amunt f internal standard injected (ng) is V s = Ttal vlume f sample (ml) P = Peak height f internal standard is RF = Mean respnse factr frm Equatin 2 V i = Injectin vlume (µl) 8.0 QUALITY CONTROL 8.1 Refer t Chapter One and Methd 8000 fr specific quality cntrl prcedures. 9.0 METHOD PERFORMANCE 9.1 The fllwing perfrmance data have been generated under the cnditins described in this methd: The calibratin curve fr Methd 8032 is linear ver the range 0-5 µg/l f acrylamide mnmer The limit f detectin fr an aqueus slutin is µg/l The yields f the brminated cmpund are % and %, at frtificatin cncentratins f 1.0 and 5.0 µg/l, respectively. 9.2 Table 1 prvides the recveries f acrylamide mnmer frm river water, sewage effluent, and sea water. 9.3 The recvery f the brminatin prduct as a functin f the amunt f ptassium brmide and hydrbrmic acid added t the sample is shwn in Figure The effect f the reactin time n the recvery f the brminatin prduct is shwn in Figure 3. The yield was cnstant when the reactin time was mre than 1 hur. 9.5 Figure 4 shws the recvery f the brminatin prduct as a functin f the initial ph frm 1 t The yield was cnstant within this ph range. The use f cnventinal buffer slutins, such as sdium acetate - acetic acid slutin r phsphate slutin, caused a significant decrease in yield REFERENCES 1. Hashimt, A., "Imprved Methd fr the Determinatin f Acrylamide Mnmer in Water by Means f Gas-Liquid Chrmatgraphy with an Electrncapture Detectr," Analyst, 101: , CD-ROM Revisin 0

8 TABLE 1 RECOVERY OF ACRYLAMIDE FROM WATER SAMPLES AS 2,3-DIBROMOPROPIONAMIDE Overall Acrylamide a Amunt f 2,3-DBPA /µg Brminatin Recvery f Cefficient Sample Mnmer Recvery Acrylamide f Matrix Spiked/µg Calculated b Fund b % b Mnmer, % Variatin Standard River Water Sewage Effluent Sea Water a b 2,3-Dibrmprpinamide Mean f five replicate determinatins CD-ROM Revisin 0

9 Figure 1 Typical gas chrmatgrams f the brminatin prduct btained frm aqueus acrylamide mnmer slutin: A. Untreated B. With Flrisil cleanup BL. Chrmatgram f blank, cncentrated five-fld befre gas chrmatgraphic analysis. Peaks: 1. 2,3-Dibrmprpinamide 2. Dimethyl phthalate 4-7. Impurities frm ptassium brmide Sample size = 100 ml; acrylamide mnmer = 0.1 µg CD-ROM Revisin 0

10 Figure 2 Effect f (A) ptassium brmide and (B) hydrbrmic acid n the yield f brminatin. Sample size = 50 ml; acrylamide mnmer = 0.25 µg CD-ROM Revisin 0

11 Figure 3 Effect f reactin time n the brminatin. Reactin cnditins: 50 ml f sample; 0.25 µg f acrylamide mnmer; 7.5 g f ptassium brmide; 2.5 ml f saturated brmine water Extractin cnditins: 15 g f sdium sulfate; extractin at ph 2; slvent = 10 ml f ethyl acetate (X2) CD-ROM Revisin 0

12 Figure 4 Effect f initial ph n the brminatin. Reactin and extractin cnditins as in Figure 3. The ph was adjusted t belw 3 with cncentrated hydrbrmic acid, and t 4-5 with dilute hydrbrmic acid. Reactin at ph 6 was in distilled water. ph 7.35 was achieved by careful additin f dilute sdium hydrxide slutin. The brken line shws the result btained by the use f sdium acetate - acetic acid buffer slutin. CD-ROM Revisin 0

13 METHOD 8032 ACRYLAMIDE BY GAS CHROMATOGRAPHY CD-ROM Revisin 0

14 METHOD 8032 cntinued CD-ROM Revisin 0

METHOD 3620A FLORISIL COLUMN CLEANUP

METHOD 3620A FLORISIL COLUMN CLEANUP 1.0 SCOPE AND APPLICATION 1.1 Flrisil, a registered trade name f the Flridin C., is a magnesium silicate with acidic prperties. It is used fr general clumn chrmatgraphy