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 as a cleanup prcedure prir t sample analysis by gas chrmatgraphy. 1.2 General applicatins: Cleanup f pesticide residues and ther chlrinated hydrcarbns; the separatin f nitrgen cmpunds frm hydrcarbns; the separatin f armatic cmpunds frm aliphatic-armatic mixtures; and similar applicatins fr use with fats, ils, and waxes (Flridin). Additinally, Flrisil is cnsidered gd fr separatins with sterids, esters, ketnes, glycerides, alkalids, and sme carbhydrates (Grdn and Frd). 1.3 Specific applicatins: This methd includes guidance fr cleanup f sample extracts cntaining the fllwing analyte grups: phthalate esters; nitrsamines; rganchlrine pesticides; nitrarmatics; halethers; chlrinated hydrcarbns; and rganphsphrus pesticides. 2.0 SUMMARY OF METHOD 2.1 The clumn is packed with the required adsrbent, tpped with a water adsrbent, and then laded with the sample t be analyzed. Elutin is effected with a suitable slvent(s) leaving the interfering cmpunds n the clumn. The eluate is then cncentrated (if necessary). 3.0 INTERFERENCES 3.1 A reagent blank shuld be perfrmed fr the cmpunds f interest prir t the use f this methd. The level f interferences must be belw the methd detectin limit befre this methd is perfrmed n actual samples. 3.2 Mre extensive prcedures than thse utlined in this methd may be necessary fr reagent purificatin. 4.0 APPARATUS AND MATERIALS 4.1 Beaker - 500 ml. 4.2 Chrmatgraphic clumn - 300 mm lng x 10 mm ID r 400 mm lng x 20 mm ID, as specified in Sectin 7.0; with Pyrex glass wl at bttm and a Tefln stpcck. NOTE: Fritted glass discs are difficult t decntaminate after highly cntaminated extracts have been passed thrugh. Clumns withut frits may be purchased. Use a small pad f Pyrex glass wl t CD-ROM 3620A - 1 Revisin 1
retain the adsrbent. Prewash the glass wl pad with 50 ml f acetne fllwed by 50 ml f elutin slvent prir t packing the clumn with adsrbent. 4.3 Kuderna-Danish (K-D) apparatus. 4.3.1 Cncentratr tube - 10 ml, graduated (Kntes K-570050-1025 r equivalent). Grund-glass stpper is used t prevent evapratin f extracts. 4.3.2 Evapratin flask - 500 ml (Kntes K-570001-0500 r equivalent). Attach t cncentratr tube with springs, clamps, r equivalent. 4.3.3 Snyder clumn - Three ball macr (Kntes K-503000-0121 r equivalent). 4.3.4 Snyder clumn - Tw ball micr (Kntes K-569001-0219 r equivalent). 4.3.5 Springs - 1/2 inch (Kntes K-662750 r equivalent). 4.4 Muffle furnace. 4.5 Reagent bttle - 500 ml. 4.6 Water bath - Heated, with cncentric ring cver, capable f temperature cntrl (+5 C). The bath shuld be used in a hd. 4.7 Biling chips - Slvent extracted, apprximately 10/40 mesh (silicn carbide r equivalent). 4.8 Erlenmeyer flasks - 50 and 250 ml. 4.9 Tp-lading balance - 0.01 g. 5.0 REAGENTS 5.1 Organic-free reagent water - All references t water in this methd refer t rganic-free reagent water, as defined in Chapter One. 5.2 Flrisil - Pesticide residue (PR) grade (60/100 mesh); purchase activated at 1250 F (677 C), stred in glass cntainers with grund-glass stppers r fil-lined screw caps. 5.2.1 Deactivatin f Flrisil - fr cleanup f phthalate esters. T prepare fr use, place 100 g f Flrisil int a 500 ml beaker and heat fr apprximately 16 hr at 40 C. After heating, transfer t a 500 ml reagent bttle. Tightly seal and cl t rm temperature. When cl add 3 ml f rganic-free reagent water. Mix thrughly by shaking r rlling fr 10 min and let stand fr at least 2 hr. Keep the bttle sealed tightly. CD-ROM 3620A - 2 Revisin 1
5.2.2 Activatin f Flrisil - fr cleanup f nitrsamines, rganchlrine pesticides and PCBs, nitrarmatics, halethers, chlrinated hydrcarbns, and rganphsphrus pesticides. Just befre use, activate each batch at least 16 hr at 130 C in a glass cntainer lsely cvered with aluminum fil. Alternatively, stre the Flrisil in an ven at 130 C. Cl the Flrisil befre use in a desiccatr. (Flrisil frm different batches r surces may vary in adsrptive capacity. T standardize the amunt f Flrisil which is used, the use f lauric acid value is suggested. The referenced prcedure determines the adsrptin frm hexane slutin f lauric acid (mg) per g f Flrisil. The amunt f Flrisil t be used fr each clumn is calculated by dividing 110 by this rati and multiplying by 20 g (Mills). 5.3 Sdium sulfate (granular, anhydrus), 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. 5.4 Eluting slvents 5.4.1 Diethyl ether, C H OC H - Pesticide quality r equivalent. 2 5 2 5 Must be free f perxides, as indicated by test strips (EM Quant r equivalent). Prcedures recmmended fr remval f perxides are prvided with the test strips. After cleanup, 20 ml ethyl alchl preservative must be added t each liter f ether. 5.4.2 Acetne, CH COCH - Pesticide quality r equivalent. 3 3 5.4.3 Hexane, C H - Pesticide quality r equivalent. 6 14 5.4.4 Methylene chlride, CH Cl - Pesticide quality r equivalent. 2 2 5.4.5 Pentane, CH (CH ) CH - Pesticide quality r equivalent. 3 2 3 3 5.4.6 Petrleum ether (biling range 30-60 C) - Pesticide quality r equivalent. 6.0 SAMPLE COLLECTION, PRESERVATION, AND HANDLING 6.1 See the intrductry material t this chapter, Organic Analytes, Sectin 4.1. 7.0 PROCEDURE 7.1 Phthalate esters 7.1.1 Reduce the sample extract vlume t 2 ml prir t cleanup. The extract slvent must be hexane. 7.1.2 Place apprximately 10 g f deactivated Flrisil (Sectin 5.1.1) int a 10 mm ID chrmatgraphic clumn. Tap the clumn t settle CD-ROM 3620A - 3 Revisin 1
the Flrisil and add apprximately 1 cm f anhydrus sdium sulfate t the tp. 7.1.3 Preelute the clumn with 40 ml f hexane. The rate fr all elutins shuld be abut 2 ml/min. Discard the eluate and, just prir t expsure f the sdium sulfate layer t the air, quantitatively transfer the 2 ml sample extract nt the clumn using an additinal 2 ml f hexane t cmplete the transfer. Just prir t expsure f the sdium sulfate layer t the air, add 40 ml f hexane and cntinue the elutin f the clumn. Discard this hexane eluate. 7.1.4 Next, elute the clumn with 100 ml f 20% ethyl ether in hexane (v/v) int a 500 ml K-D flask equipped with a 10 ml cncentratr tube. Cncentrate the cllected fractin as needed. N slvent exchange is necessary. Adjust the vlume f the cleaned-up extract t whatever vlume is required (10 ml fr Methd 8060) and analyze by gas chrmatgraphy. Cmpunds that elute in this fractin are: Bis(2-ethylhexyl) phthalate Butyl benzyl phthalate Di-n-butyl phthalate Diethyl phthalate Dimethyl phthalate Di-n-ctyl phthalate 7.2 Nitrsamines 7.2.1 Reduce the sample extract vlume t 2 ml prir t cleanup. 7.2.2 Add a weight f activated Flrisil (nminally 22 g) predetermined by calibratin (Sectin 5.1.2) int a 20 mm ID chrmatgraphic clumn. Tap the clumn t settle the Flrisil and add abut 5 mm f anhydrus sdium sulfate t the tp. 7.2.3 Pre-elute the clumn with 40 ml f ethyl ether/pentane (15:85) (v/v). Discard the eluate and, just prir t expsure f the sdium sulfate layer t the air, quantitatively transfer the 2 ml sample extract nt the clumn using an additinal 2 ml f pentane t cmplete the transfer. 7.2.4 Elute the clumn with 90 ml f ethyl ether/pentane (15:85) (v/v) and discard the eluate. This fractin will cntain the diphenylamine, if it is present in the extract. 7.2.5 Next, elute the clumn with 100 ml f acetne/ethyl ether (5:95) (v/v) int a 500 ml K-D flask equipped with a 10 ml cncentratr tube. This fractin will cntain all f the nitrsamines listed in the scpe f the methd. 7.2.6 Add 15 ml f methanl t the cllected fractin, cncentrate as needed using pentane t prewet the K-D clumn and set the water bath at 70 t 75 C. When the apparatus is cl, remve the Snyder clumn and rinse the flask and its lwer jint int the cncentratr tube with 1 t 2 ml f pentane. CD-ROM 3620A - 4 Revisin 1
7.3 Organchlrine pesticides, halethers, and rganphsphrus pesticides (see Tables 1 and 2 fr fractinatin patterns f cmpunds tested) 7.3.1 Reduce the sample extract vlume t 10 ml prir t cleanup. The extract slvent must be hexane. 7.3.2 Add a weight f activated Flrisil (nminally 20 g), predetermined by calibratin (Sectin 5.1.2), t a 20 mm ID chrmatgraphic clumn. Settle the Flrisil by tapping the clumn. Add anhydrus sdium sulfate t the tp f the Flrisil t frm a layer 1 t 2 cm deep. Add 60 ml f hexane t wet and rinse the sdium sulfate and Flrisil. Just prir t expsure f the sdium sulfate t air, stp the elutin f the hexane by clsing the stpcck n the chrmatgraphic clumn. Discard the eluate. 7.3.3 Adjust the sample extract vlume t 10 ml with hexane and transfer it frm the K-D cncentratr tube t the Flrisil clumn. Rinse the tube twice with 1-2 ml hexane, adding each rinse t the clumn. 7.3.4 Place a 500 ml K-D flask and clean cncentratr tube under the chrmatgraphic clumn. Drain the clumn int the flask until the sdium sulfate layer is nearly expsed. Elute the clumn with 200 ml f 6% ethyl ether in hexane (v/v) (Fractin 1) using a drip rate f abut 5 ml/min. All f the halethers are in this fractin. Remve the K-D flask and set aside fr later cncentratin. Elute the clumn again, using 200 ml f 15% ethyl ether in hexane (v/v) (Fractin 2), int a secnd K-D flask. Perfrm a third elutin using 200 ml f 50% ethyl ether in hexane (v/v) (Fractin 3), and a final elutin with 200 ml f 100% ethyl ether (Fractin 4), int separate K-D flasks. 7.3.5 If necessary, cncentrate the eluates by standard K-D techniques using the water bath at abut 85 C (75 C fr Fractin 4). Adjust the final vlume t whatever vlume is required (1-10 ml). 7.4 Nitrarmatics and isphrne 7.4.1 Reduce the sample extract vlume t 2 ml prir t cleanup. 7.4.2 Add a weight f activated Flrisil (nminally 10 g) predetermined by calibratin (Sectin 5.1.2) int a 10 mm ID chrmatgraphic clumn. Tap the clumn t settle the Flrisil and add abut 1 cm f anhydrus sdium sulfate t the tp. 7.4.3 Pre-elute the clumn with methylene chlride/hexane (1:9) (v/v) at abut 2 ml/min. Discard the eluate and, just prir t expsure f the sdium sulfate layer t the air, quantitatively transfer the sample extract nt the clumn using an additinal 2 ml f hexane t cmplete the transfer. Just prir t expsure f the sdium sulfate layer t the air, add 30 ml f methylene chlride/hexane (1:9) (v/v) and cntinue the elutin f the clumn. Discard the eluate. 7.4.4 Elute the clumn with 90 ml f ethyl ether/pentane (15:85) (v/v) and discard the eluate. This fractin will cntain the diphenylamine, if it is present in the extract. CD-ROM 3620A - 5 Revisin 1
7.4.5 Next, elute the clumn with 100 ml f acetne/ethyl ether (5:95) (v/v) int a 500 ml K-D flask equipped with a 10 ml cncentratr tube. This fractin will cntain all f the nitrsamines listed in the scpe f the methd. 7.4.6 Add 15 ml f methanl t the cllected fractin, cncentrate using pentane t prewet the K-D clumn, and set the water bath at 70 t 75 C. When the apparatus is cl, remve the Snyder clumn and rinse the flask and its lwer jint int the cncentratr tube with 1 t 2 ml f pentane. 7.4.7 Next, elute the clumn with 30 ml f acetne/methylene chlride (1:9) (v/v) int a 500 ml K-D flask equipped with a 10 ml cncentratr tube. Cncentrate the cllected fractin, while exchanging the slvent t hexane. T exchange the slvent, reduce the elutin slvent t abut 10 ml. Add 50 ml f hexane, a fresh biling chip, and return the reassembled K-D apparatus t the ht water bath. Adjust the final vlume f the cleaned-up extract t whatever vlume is required (1-10 ml). Cmpunds that elute in this fractin are: 2,4-Dinitrtluene 2,6-Dinitrtluene Isphrne Nitrbenzene. 7.5 Chlrinated hydrcarbns 7.5.1 Reduce the sample extract vlume t 2 ml prir t cleanup. The extract slvent must be hexane. 7.5.2 Add a weight f activated Flrisil (nminally 12 g) predetermined by calibratin (Sectin 5.1.2) int a 10 mm ID chrmatgraphic clumn. Tap the clumn t settle the Flrisil and add abut 1 t 2 cm f anhydrus sdium sulfate t the tp. 7.5.3 Preelute the clumn with 100 ml f petrleum ether. Discard the eluate and, just prir t expsure f the sdium sulfate layer t the air, quantitatively transfer the sample extract t the clumn by decantatin and subsequent petrleum ether washings. Discard the eluate. Just prir t expsure f the sdium sulfate layer t the air, begin eluting the clumn with 200 ml f petrleum ether and cllect the eluate in a 500 ml K-D flask equipped with a 10 ml cncentratr tube. This fractin shuld cntain all f the chlrinated hydrcarbns: 2-Chlrnaphthalene 1,2-Dichlrbenzene 1,3-Dichlrbenzene 1,4-Dichlrbenzene Hexachlrbenzene Hexachlrbutadiene Hexachlrcyclpentadiene Hexachlrethane 1,2,4-Trichlrbenzene. CD-ROM 3620A - 6 Revisin 1
7.5.4 Cncentrate the fractin, using hexane t prewet the clumn. When the apparatus is cl, remve the Snyder clumn and rinse the flask and its lwer jint int the cncentratr tube with hexane. Adjust the final vlume f the cleaned-up extract t whatever vlume is required (1-10 ml). 8.0 QUALITY CONTROL 8.1 Refer t Chapter One fr specific quality cntrl prcedures and Methd 3600 fr cleanup prcedures. 8.2 The analyst shuld demnstrate that the cmpunds f interest are being quantitatively recvered befre applying this methd t actual samples. 8.3 Fr sample extracts that are cleaned up using this methd, the assciated quality cntrl samples shuld als be prcessed thrugh this cleanup methd. 9.0 METHOD PERFORMANCE 9.1 Table 1 indicates the distributin f chlrinated pesticides, PCB's, and halethers in varius Flrisil clumn fractins. 9.2 Table 2 indicates the distributin f rganphsphrus pesticides in varius Flrisil clumn fractins. 10.0 REFERENCES 1. Grdn, A.J. and R.A. Frd, The Chemist's Cmpanin: A Handbk f Practical Data, Techniques, and References (New Yrk: Jhn Wiley & Sns, Inc.), pp. 372, 374, and 375, 1972. 2. Flridin f ITT System, Flrisil: Prperties, Applicatin, Bibligraphy, Pittsburgh, Pennsylvania, 5M381DW. 3. Mills, P.A., "Variatin f Flrisil Activity; Simple Methd fr Measuring Absrbent Capacity and its use in Standardizing Flrisil Clumns," Jurnal f the Assciatin f Official Analytical Chemists, 51, 29, 1968. 4. U.S. Fd and Drug Assciatin, Pesticides Analytical Manual (Vlume 1), July 1985. 5. U.S. EPA 40 CFR Part 136, "Guidelines Establishing Test Prcedures fr the Analysis f Pllutants Under the Clean Water Act; Final Rule and Interim Final Rule and Prpsed Rule," Octber 26, 1984. CD-ROM 3620A - 7 Revisin 1
TABLE 1 DISTRIBUTION OF CHLORINATED PESTICIDES, PCBs, AND HALOETHERS INTO FLORISIL COLUMN FRACTIONS Percent Recvery by Fractin a Parameter 1 2 3 Aldrin 100 -BHC 100 -BHC 97 -BHC 98 -BHC 100 Chlrdane 100 4,4'-DDD 99 4,4'-DDE 98 4,4'-DDT 100 Dieldrin 0 100 Endsulfan I 37 64 Endsulfan II 0 7 91 Endsulfan sulfate 0 0 106 Endrin 4 96 Endrin aldehyde 0 68 26 Halethers R Heptachlr 100 Heptachlr epxide 100 Txaphene 96 PCB-1016 97 PCB-1221 97 PCB-1232 95 4 PCB-1242 97 PCB-1248 103 PCB-1254 90 PCB-1260 95 a Eluant cmpsitin: Fractin 1-6% ethyl ether in hexane Fractin 2-15% ethyl ether in hexane Fractin 3-50% ethyl ether in hexane R = Recvered (n percent recvery data presented). SOURCE: U.S. EPA and FDA data. CD-ROM 3620A - 8 Revisin 1
TABLE 2 DISTRIBUTION OF ORGANOPHOSPHORUS PESTICIDES INTO FLORISIL COLUMN FRACTIONS Percent Recvery by Fractin a Parameter 1 2 3 4 Azinphs methyl 20 80 Blstar (Sulprfs) ND ND ND ND Chlrpyrifs >80 Cumaphs NR NR NR Demetn 100 Diazinn 100 Dichlrvs NR NR NR Dimethate ND ND ND ND Disulftn 25-40 EPN >80 Ethprp V V V Fensulfthin ND ND ND ND Fenthin R R Malathin 5 95 Merphs V V V Mevinphs ND ND ND ND Mnchrtphs ND ND ND ND Naled NR NR NR Parathin 100 Parathin methyl 100 Phrate 0-62 Rnnel >80 Stirphs (Tetrachlrvinphs) ND ND ND ND Sulftepp V V TEPP ND ND ND ND Tkuthin (Prthifs) >80 Trichlrnate >80 a Eluant cmpsitin: Fractin 1-200 ml f 6% ethyl ether in hexane Fractin 2-200 ml f 15% ethyl ether in hexane Fractin 3-200 ml f 50% ethyl ether in hexane Fractin 4-200 ml f 100% ethyl ether R = Recvered (n percent recvery infrmatin presented) (U.S. FDA). NR = Nt recvered (U.S. FDA). V = Variable recvery (U.S. FDA). ND = Nt determined. SOURCE: U.S. EPA and FDA data. CD-ROM 3620A - 9 Revisin 1
METHOD 3620A FLORISIL COLUMN CLEANUP CD-ROM 3620A - 10 Revisin 1
METHOD 3620A cntinued CD-ROM 3620A - 11 Revisin 1