SFC中荧光检测对多环芳烃的高灵敏度检测

多环芳香烃（PAHs）是由柴油废气、煤尘和香烟烟雾等各种来源的有机化合物不完全燃烧产生的；许多是强致癌性。多环芳烃受包括美国环境保护署在内的各种组织的监管。这些规定严格控制多环芳烃对环境污染的影响及其对人类健康的影响。多环芳烃由于具有许多共轭双键的芳香族结构，表现出很强的天然荧光。这使得荧光检测的高灵敏度对于环境中经常发现的低水平是必要的。Application Note230009U High Sensitivity Detection of Polycyclic Aromatic Hydrocarbons using2/6Application NoteFluorescence Detection in SFC High Sensitivity Detection of Polycyclic Aromatic Hydrocarbons using Fluorescence Detection in SFC In t rod u ction Polycyclic Aromat i c Hyd r ocarbons (PAHs) are produced by the incom p lete combust i on of organic compounds f rom various sources i ncluding diesel exhaust， coal dust， and ciga r ette smoke； many are strongly carci n ogenic. PAHs are subject to r egulation by various organizations inc l uding the EPA (United States Environmental Protection Agency). These regulations strictly control the impact of PAHs on envi r onmental pollution and thei r effects on human health. PAHs exh i bit strong natural fluorescence d u e to the aromatic structure with many conjugated double bonds. Th i s makes t h e high se n sitivity of f luorescence detection necessary for t he low levels often found in the environment. I n th i s applicat i on n ote， 16 PAHs we r e separated by s u percri ti cal f lu i d c h romatography (SFC) and det e cted u sin g a F P-4020 fl uoresce n ce detec t or (FL detector) with a h i gh p ressure f low cell (previous l y in t roduced in LC application data No.032006U). Data was coll e ct e d u s ing b oth UV-Vis and f lu o rescence d e t e ctors for compa r ativ e p urpos e s. Keywor d s Polycyc l ic Aromat i c Hyd r ocarbons， PAHs， Ethylpyridine column， SFC ， Fluorescence detector， carc in ogenicity， UV-Vis，a b sorbance d e tector E xp e riment a l Equipment Conditions CO，Pump PU-4380 Column 2-Ethylpyridine (Princeton Chromatography)(4.6 mm l.D.x250 mm L， 5 um) Modifier Pump PU-4180 Eluent A CO， Modifier Pump Option SV Unit MX Unit Eluent B Acetonitrile Autosampler AS-4350 Gradient (A/B)， 0 min (95/5)→1.5 min (95/5)+12 min (80/20)+ 14 min (80/20) +15 min (95/5)+20 min (95/5) Column Oven CO-4060 UV/Vis Detector UV-4070 Flow Rate 3.0mL/min UV/Vis Detector Flow Cell Analytical High Pressure Column Temp. 40℃ FL Detector FP-4020 Wavelength Refer to Figure 1 FL Detector Flow Cell Analytical High Pressure Back Pressure Regulator BP-4340 Back Pressure 15 MPa Injection Volume 5uL Standard Sample M ix t ur e of 16 PAH s sta n dards in a ce t onit r il e Str uc t ure o f PA Hs 1. Naphthalene 2. Acenaphthene 3. Fluorene 4. Acenaphthylene (non-fluorescent) 5. Anthracene 6.Phenanthrene 7.Fluoranthene 9. Benzo[ajanthracene 10. Chrysene 11. Benzo[k ]fluoranthene 13. Benzo[a]pyrene 14. Dibenz[a，h]anthracene 15. Indeno[1，2，3-cd]pyrene 16. Benzo[g，h，i]perylene copyrig ht O JA S C O C o rp ora t i o n R esul t Figure 1 shows t he se p a r ation of a 16-component PAHs standard mixture wi th UV-visible detec ti on (500 p g/uL each) a nd f l uorescence detec t io n (5 pg/uL each). Since Acena p hthylene does n ot f l uoresce， i t was not detected in t he f luoresce n ce ch r omatogram a n d was co-el u t e d with F l uor e n e (p eak No. 3) ove rl a p ping in t h e UV chromatogr a m. Ta b le 1 shows th e det e ct i on l imit s of e ac h component . Wavelength changed using a t i me program Figure 1. U V a nd FL c hro m a t o g r a m o f P A H s 16-compo nen t (UV ： 500 p g/uL ， FL ： 5 p g/uL ) 1： N aphthalene ， 2： Acenap h t h e n e ， 3： F l u o r ene， 4： Ace n apht h yl ene (non-fl uo r escent )， 5： A n t h racene， 6： Ph enanthre n e， 7： F l uoran th ene， 8： Pyrene，9： Benzo[a]a n t h racene， 10C h rysene， 11： Be n zo[k ]f l uo r anthe n e， 12： Benzo [b ]fluora n t h en e ， 13： B enzo[a]pyre n e， 14： Dibenzo[a，h ]anthracene，15： Indeno[1，2，3-cd]py r ene， 16： Benzo[g.h，i ]pe r ylene T he measurement wa v elength used f or t h e UV/isibl e d e tec t o r i s t h e same as t h e exci t ation wavele n g t h of t he f luores c e n ce detecto r . The gain of t he f l uoresce n c e detec t o r i s x100. A ppl ic a tio n Lib r ary： ja s c o i n c.com /appli c a t i o ns Using the f luorescence detector， the PAHs were detected at the low detect i on l imit of 0.17 pg to 4.6 pg， and the sens i tivity ratio compared with the UV detector is approx. 20 to 400 times. F i gure 2 shows a calibration curve for the standard PAH mix t ure at concentrations f r o m 1 to 100 pg/uL measured wit h the fluorescence detector. From these results (Table 1)，excel l en t l i near i ty with a cor r elation coef f icient of 0.9997 or higher was conf i rmed for all components. Table 1. Lo w e r li m i t of det ect ion of PAHs i n e a c h d et e ctor and lin e a i t y i n FL de t ec to r Peak No. Compound UV*1 FL*2 Sensitivity Ratio*3 Correlation coefficient (FL)*4 1 Naphthalene 120 2.04 59 1.0000 2 Acenaphthene 13.6 0.293 46 0.9998 3 Fluorene*5 21.3 0.369 58 0.9999 5 Anthracene 8.67 0.166 52 0.9998 6 Phenanthrene 17.8 0.676 26 1.0000 7 Fluoranthene 58.8 2.34 25 0.9999 8 Pyrene 75.2 0.357 211 0.9998 9 Benzo[a]anthrancene 32.5 0.432 75 0.9999 10 Chrysene 31.5 1.06 30 1.0000 11 Benzo[k]fluoranthene 93.0 0.236 393 0.9997 12 Benzo[b]fluoranthene 81.9 1.46 56 0.9999 13 Benzo[alpyrene 53.6 0.233 230 0.9998 14 Dibenz[a，h]anthracene 32.6 0.737 44 1.0000 15 Indeno[1，2，3-cd]pyrene 79.2 4.61 1/ 0.9998 16 Benzo[g，h，i]perylene 65.5 0.697 94 0.9998 *1 Ca l c u lated from the measure m en t o f 500 pg/mL s t and a rd sample (S/N =3) *2 Calcula t e d from t h e mea s u r eme n t of 5 p g /mL stand a rd samp l e (S/N =3)*3 L o wer l imi t o f detect i o n o f UV detector/Lower l i m i t of d e tec t ion of F L de t ector *4 Ca l i bra ti on curve was made using s t anda r d s a t 1， 5， 10， 50，100 p g/mL *5 Th e v alue of t h e F lu o rene p e ak u sing UV det e ct ion i n cludes A c ena p h t hyle n e whi c h is co-elu t e d JASCO INC. 28600 Mary's C o ur t ， E asto n， MD 21601US A T el ： (800) 333-5272 F ax： (410) 822-7526A ppl ic a tio n Lib r ary： ja s c o i n c.com /appli c a t i o ns Figure 2. F L c hr om a to gr am of P AH s 16-c o m p o nen t s 1： N aphthalene， 2： A c e n a p h t hen e ， 3： F l uo r e n e，4： Acenaph t h y l e ne (no n -flu o r e sce n t)， 5： A nth race n e，6： P h enanthr e ne ， 7： Fl u o r an t he n e， 8： Pyren e ， 9： B enzo[a]a n t h r acene ，川机 10： Chrys e ne， 11： B enzo[k]fluoran t h e ne， 12： B en z o [b ]f luo r anthe n e， 13： Be n z o [a]p yren e ， 14： D i benzo [a，h]a n th racen e ，15： I nde n o[1，2，3-cd]pyre n e ， 16： B e nz o [g，h，i pe r ylene Measu r eme n t wavele n gt h and ga in set f or the fl u orescen c e detector i s t h e same as F igure 1. JASCO INC. 28600 Mary's C o ur t ， E asto n， MD 21601US A T el ： (800) 333-5272 F ax： (410) 822-7526A ppl ic a tio n Lib r ary： ja s c o i n c.com /appli c a t i o ns Part Number Model Description 7032-J002A PU-4380 CO， Delivery Pump 7002-J004A PU-4180 RHPLC Pump (Base Unit) 7008-H350A SV-4300 Stop valve unit 7007-H063A MX-4300-NC Dynamic Mixing Unit (for SFC/E) 7007-H146B Mixing chamber， 1.5 mL， for Analytical 7038-J002A AS-4350 SFC Autosampler 7021-J002A CO-4060 Column Oven 6990-J002A BP-4340 Automatic Back Pressure Regulator 7025-J002A UV-4070 UV/IS detector 7025-H201B Analytical high pressure flow cell 6996-J001A FP-4020 Fluorescence detector 7052-H201A Analytical high pressure flow cell 7139-J002A BS-4500-4 Bottle stand Custom order Princeton SFC 2-Ethylpyridine 60 A (4.6 mm 1.D. x250 mm L， 5 pm) 6688-H564A LC-NetCG CG cable (x6) 6925-H262A Preheat coil 1/16"，0.8 um I.D. x5mL 7059-J012A ChromNAV V2 ChromNAV 2.0 Chromatography Data System SFC tubing kit 7001-H405B Maintenance tool kit JASCO INC. 28600 Mary's C o ur t ， E asto n， MD 21601US A T el ： (800) 333-5272 F ax： (410) 822-7526A ppl ic a tio n Lib r ary： ja s c o i n c.com /appli c a t i o ns