POLYSTYRENE AS A MEDIUM IN REVERSE-PHASE SEPARATION OF POLYCYCLIC AROMATIC HYDROCARBONS A thesis submitted in partial fulfillment of the requirements for the degree of Master of Science By MADHAVI MANTHA B.S., Sambhalpur University, 1995 M.S., Sambhalpur University, 1997 2008 Wright State University WRIGHT STATE UNIVERSITY SCHOOL OF GRADUATE STUDIES May 27, 2008 I HEREBY RECOMMEND THAT THE THESIS PREPARED UNDER MY SUPERVISION BY Madhavi Mantha ENTITLED Polystyrene as a Medium in Reverse-Phase Separation of Polycyclic Aromatic Hydrocarbons BE ACCEPTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF Master of Science . ______________________________ Audrey E. McGowin, Ph.D. Thesis Director ______________________________ Kenneth Turnbull, Ph.D. Department Chair Committee on Final Examination _________________________________ Audrey E. McGowin, Ph.D. _________________________________ Eric Fossum, Ph.D. _________________________________ Steven Higgins, Ph.D. _________________________________ Joseph F. Thomas, Jr., Ph.D. Dean, School of Graduate Studies ABSTRACT Mantha, Madhavi M.S., Department of Chemistry, Wright State University, 2008. Polystyrene as a Medium in Reverse-Phase Separation of Polycyclic Aromatic Hydrocarbons. The purpose of the research was to design a method using high-performance liquid chromatography (HPLC) that would separate the sixteen polycyclic aromatic hydrocarbons (PAHs) designated by Environmental Protection Agency (EPA) as priority pollutants on two different kinds of polystyrene (PS) in the reversed phase. One was a zirconia-based column coated with polystyrene. The other was a polystyrene divinylbenzene (PS-DVB) column. The idea behind using these columns was to take advantage of extreme retention of PAHs by these columns by using high temperatures or temperature programming in order to reduce the amount of organic modifier in the mobile phases to perform analytical separations and achieve faster LC separations. Separation of the sixteen PAHs was attempted using PS-coated zirconia and the optimum conditions consisted of thermal programming as well as mobile phase gradient by running the sample at 60oC for the first 15 minutes and then at 50oC for the next fifteen minutes and using 30% acetonitrile (ACN) from 0-10 minutes, 30-50% ACN from 10-15 minutes and 50-75% ACN from 15-30 minutes. Co-elution of geometric isomers, benz(a)anthracene with chrysene and benzo(b)fluoranthene with benzo(k)fluoranthene was observed. The optimum conditions using the PS-DVB column were temperature 70oC isothermal for 5 min, 70-110oC gradient at 5.33oC min-1 for the next 7.5 min and iii 110oC isothermal for the next 17.5 min, mobile phase 50-90% ACN, remaining -1 H2O:MeOH 1:1 (v/v), flow rate 2 mL min . Again co-elution of geometric isomers, benz(a)anthracene with chrysene and benzo(b)fluoranthene with benzo(k)fluoranthene was observed. The last three anlaytes, dibenz(a,h)anthracene, indeno(1,2,3,cd)pyrene and benzo(ghi)perylene had better separation with one peak and two shoulders, but could not be resolved completely. No combination of mobile phases containing THF, ACN, H2O and/or MeOH and no temperature program was able to completely resolve the EPA 16 PAHs better than typical silica-based C-18 columns. The PS columns did, however show potential for separation of PAHs and their environmental derivatives with two to three rings, such as those found in the vapor phase in the environment. iv TABLE OF CONTENTS Page Chapter 1 Introduction Overview….……………………………………………………………………………….1 Sources .......................................................................................................................……..1 Physical Properties ....................................................................................................……...3 Mobility of PAHs in the Environment…………………………………………………….4 Biodegradation of PAHs ..............................................................................................……4 Photodegradation of PAHs ........................................................................................……..5 Human Exposure and Health effects of PAH ............................................................……..6 Regulations ................................................................................................................……..7 Measurement of PAHs in the environment ................................................................……..7 Advantages of applying elevated temperatures and mobile phase gradient in LC Separations ..................................................................................................................……8 Silica-Based C-18 Columns ...............................................................................................10 Other Chromatographic Columns ......................................................................................10 Polymer-Based Stationary Phases......................................................................................11 Polystyrene-divinylbenzene Stationary Phases .......................................................... .......12 Chemically-modified Polystyrene-divinylbenzene Stationary Phases..............................13 Polymer-coated Zirconia-based Stationary Phases............................................................15 Current Research................................................................................................................16 v TABLE OF CONTENTS (CONTINUED) Page Chapter 2 Experimental Instrument ......................................................................................................................18 Reagents and Standards .................................................................................................19 Zirchrom-PS Column .....................................................................................................21 PS-DVB Column............................................................................................................21 Experimental Parameters ...............................................................................................22 Chapter 3 Results and Discussion Zirchrom PS-Column Method Optimization………………………………...………...24 Method Development Using Different Mobile Phases and Conditions………….…….27 Discussion on Zirchrom Column .................................................................................. .29 Effect of Temperature Programming and Mobile Phase Gradient on Resolution……..30 Sample Calculations........................................................................................................31 PS-DVB Column (53mm x 7mm) Method Development……………………………...31 Method Optimization ......................................................................................................32 PS-DVB Column (53mm x 10 mm) ...............................................................................37 Study of two- and three-ring PAHs ................................................................................42 PS-DVB Column Discussion ..........................................................................................43 Chapter 4 Conclusion ........................................................................................... .49 References……………………………………………………………………………51 vi LIST OF FIGURES Figure Page 1.1 Chemical Structure of the carcinogen benzo(a)pyrene diol epoxide ....................5 1.2. Chemical Structure of PS-DVB resins used in PD-DVB column .......................12 2.1 Instrumentation HPLC system ..........................................................................19 3.2 Chromatogram of 5 ppm PAHs 16 using Zirchrom column at 80oC, 20-80% ACN:H2O (v/v) ...................................................................................................26 3.3 Chromatogram of 5 ppm PAHs 16 using Zirchrom column at 60oC for 15 min, o 50 C for 15 min, 30-75% ACN:H2O (v/v).. .......................................................27 3.4 Chromatogram of 5 ppm PAHs 16 using Zirchrom column at 25oC, 40-80% ACN, remaining H2O:MeOH 19:1 (v/v).... .........................................................28 3.5 Chromatogram of 5 ppm PAHs 16 using PS-DVB column at 110oC, 50-90% ACN:H2O (v/v) ...................................................................................................35 3.6 Chromatogram of 5 ppm PAHs 16 using PS-DVB column at 100oC for 10 min, o 100-150 C for 17 min, 50-90% ACN:H2O (v/v). ..............................................36 3.7 Chromatogram of 5 ppm PAHs 16 using PS-DVB column at 60oC, 50% ACN 50% MeOH (v/v) .................................................................................................36 3.8 Chromatogram of 5 ppm PAHs 16 using PS-DVB column at 70oC for 5 min, 70- o o 110 C for 7.5 min, 110 C for 17.5 min 50-90% ACN:H2O (v/v). .....................37 3.9 Chromatogram of 5 ppm PAHs 16 using PS-DVB column at 40-80oC for 20 min, o 80 C for 20 min, 50% ACN, 50% THF:H2O 1:1 (v/v). .....................................39 3.10 Chromatogram of 5 ppm PAHs 16 using PS-DVB column at 80oC, 50-75% ACN, remaining MeOH:H2O 1:1 (v/v). .............................................................39 3.11 Chromatogram of 10 ppm PAHs 10 using PS-DVB column at 80-100oC, 50- 90% ACN, remaining MeOH:H2O 1:1 (v/v). ....................................................43 3.12 Textures of copolymer of styrene and divinylbenzene .......................................46 vii LIST OF TABLES Table Page 1.1 List of Analytes ....................................................................................................2