Chapter 1 Introduction 1.1 Principle of CSIA 1 1.2 Gas Chromatography- Isotope Ratio Mass Spectrometry 2 1.3 Calibration of GC-IRMS 3 1.3.1 Materials 3 1.3.2 Vacuum test of IRMS system 3 1.3.3 Mass resolution test of IRMS 3 1.3.4 System suitability test of GC-IRMS 4 1.3.5 Linearity test of ion source 4 1.3.6 Calibration of reference gas 5 1.3.7 Accuracy test of GC-IRMS 6 1.4 Applications of CSIA 7 1.4.1 Forensic science 7 1.4.2 Foods 7 1.4.3 Drugs. 7 1.4.4 Environmental forensics 8 1.4.5 Natural attenuation 9 1.5 Thesis structure 9 1.6 References 11 Chapter 2 Compound Specification Carbon Isotope Analysis of Organic Solvents by GC-IRMS for Source Identification in Environmental Forensics 2.1 Introduction 20 2.2 Experimental 20 2.2.1 Materials 20 2.2.2 Sampling 21 2.2.3 Gas Chromatograph Isotope Ratio Mass Spectrometer 22 2.2.4 Organic solvents analysis by GC-IRMS 22 2.2.5 Industrial waste analysis by GC-IRMS 24 2.2.6 Data processing 26 2.3 Results and discussion 28 2.3.1 Method validation 28 2.3.2 □13C database of organic solvents 28 2.3.3 Data processing. 29 2.3.4 Source identification 29 2.4 Conclusions 32 2.5 References 33 Chapter 3 Biodegradation of Chlorinated Hydrocarbon in Groundwater at a Pollution Site Using GC-IRMS CSIA 3.1 Introduction 51 3.2 Experimental 52 3.2.1 Materials 52 3.2.2 Instrument 53 3.2.3 Method 54 3.2.4 Simulated weathering experiment based on microbial degradation set up 57 3.2.5 Sampling 58 3.2.6 Investigation of contaminants in groundwater at the pollution site 59 3.2.7 Feasibility of using CSIA in natural attenuation study 59 3.3 Results and discussion 59 3.3.1 Analytical results 59 3.3.2 Biodegradation mechanism 61 3.3.3 Case study of a pollution site 63 3.4 Conclusions 65 3.5 References 66 Chapter 4 Apportionment of Polycyclic Aromatic Hydrocarbons in Sediments at Kaohsiung Harbor, Taiwan Using GC-IRMS CSIA 4.1 Introduction 81 4.2 Experimental 83 4.2.1 Materials 83 4.2.2 Instrument 84 4.2.3 Pretreatment method 85 4.2.4 GC-MS quantitative analysis 87 4.2.5 GC-IRMS CSIA 88 4.2.6 Sampling 90 4.2.7 PAHs source apportionment procedure 91 4.3 Results and discussion 92 4.3.1 Pretreatment results 92 4.3.2 PAHs content in sediments 93 4.3.3 □13C values of 16 PAHs in sediments 95 4.3.4 PAHs source apportionment 96 4.4 Conclusions 98 4.5 References 101 Chapter 5 Developing CSIA with GC-IRMS for Tracing Origins of Methamphetamines in Urines from Suspected Drug Abusers 5.1 Introduction 124 5.2 Experimental 125 5.2.1 Materials 125 5.2.2 GC-IRMS analysis of abused drugs 125 5.2.3 GC-IRMS analysis of urinary samples 130 5.3 Results and discussion 132 5.3.1 Method validation 132 5.3.2 □13C and □15N of abused drugs and urinary samples 133 5.3.3 Data processing 134 5.3.4 Source identification 134 5.4 Conclusions 135 5.5 References 136 Fig. 1-1 Schematic diagram of the GC-IRMS instrument……………………………… 12 Fig. 1-2 Picture of the GC-IRMS instrument…………………………………………….12 Fig. 1-3 Stainless steel gas cylinder as sampler……………………………………… ….13 Fig. 1-4 The mass spectrum of IRMS mass resolution test………………………………13 Fig. 1-5 Diagram of CO2 reference On/Off test…………………………………………..14 Fig. 1-6 GC-IRMS chromatogram of FID test sample………………………………… ..14 Fig. 1-7 The linearity test diagram of CO2 reference gas (r□ 0.99)………………………15 Fig. 1-8 The linearity test diagram of N2 reference gas (r□ 0.99)………………………..15 Fig. 1-9 GC-IRMS chromatogram of Mix C calibrator…………………………………..16 Fig. 1-10 GC-IRMS chromatogram of acetanilide calibrator…………………………….16 Fig. 1-11 Diagram of thesis structure……………………………………………………..17 Fig. 2-1 GC-IRMS schematic diagram…………………………………………………...34 Fig. 2-2 Components: dichloromethane, chloroform, benzene and trichloroethylene might interfere with each other when 8 components were analyzed simultaneously….34 Fig. 2-3 The total procedure of source identification for industrial wastes………………35 Fig. 2-4 Components: cis 1,2-dichloro ethylene and 1,2-dichloro ethane when 2 components were analyzed simultaneously……………………………………..36 Fig. 2-5 XY scatter diagram of 54 d13C values for 54 organic solvents…………………37 Fig. 2-6 D940144 industrial waste sample bar chart……………………………………..38 Fig. 2-7 D940149 industrial waste sample bar chart……………………………………..38 Fig. 2-8 D940150 industrial waste sample bar chart……………………………………..38 Fig. 2-9 Factor analysis of 40 industrial waste samples 2D diagram…………………….39 Fig. 2-10 Source identification results with one way ANOVA for 54 organic solvents □13C database………………………………………………………………………...40 Fig. 2-11 Relationship of □13C values (Y-axis) and mixing ratio of two chloroforms (solvent-15 and solvent-16) with different source…………………………………..42 Fig. 2-12 Relationship of d13C values (Y-axis) and mixing ratio of two tetrachloro ethylenes (solvent-25 and solvent-27) with different source…………………..42 Fig. 3-1 SPME GC-IRMS schematic diagram……………………………………………68 Fig. 3-2 Apparatus of head space SPME extraction……………………………………...68 Fig. 3-3 Location of pollution site is on northwest of Taiwan (R.O.C.)………………….69 Fig. 3-4 The distribution of sampling wells from up to down gradient in pollution site and surrounding area…………………………………………………………………69 Fig. 3-5 Evalution procedure of CSCIA technology uses for natural attenuation in pollution site……………………………………………………………………..70 Fig. 3-6 The primer pair of 563F and 1357R directed PCR analysis. The chromosomal DNA was extracted by commercial kit and analyzed by PCR for each sample PCR product amplified by primers of 16S rRNA was run on 2 % agarose gel. The positive signals appeared in samples EX2 and MW122………………………….71 Fig. 3-7 The primer pair of tce-f and tce-r directed PCR analysis. The chromosomal DNA was extracted and analyzed by PCR for each sample PCR product amplified by primers of tceA was run on 2 % agarose gel. The positive signals were observed in samples EX2, EX13, A6 and MW122………………………………………..71 Fig. 3-8 The nested PCR analysis of 16S rRNA. The chromosomal DNA was extracted and analyzed by PCR for each sample PCR product amplified by the universal primers of 16S rRNA, eub-f and eub-r and re-amplified by the D. ethenogenes specific 16S rRNA primers, 563f and 1357r. After running on 2 % agarose gel, the nested PCR products could be observed in sample EX2, EX13 and MW108………………………………………………………………………….72 Fig. 3-9 HS-GC-FID chromatogram of PCE, TCE, cis-1,2-DCE, VC and IS in 4-Mix….72 Fig. 3-10 SPME-GC-IRMS chromatogram of PCE, TCE, cis-1,2-DCE, trans-1,2-DCE, 1,1,1-TCA and p-DCB…………………………………………………………73 Fig. 3-11 Simulated sample (spiking PCE 80 □g/ml) HS-GC-FID chromatogram………73 Fig. 3-12 Simulated sample (spiking PCE 80 □g/ml) SPME-GC-IRMS chromatogram...74 Fig. 3-13 The relationship of peak areas of PCE, TCE, cis-1,2-DCE, VC and sampling time in simulated sample-1 (941122)………………………………………….74 Fig. 3-14 The relationship of □13C values of PCE, TCE, cis-1,2-DCE, VC and sampling time in simulated sample-1 (941122)…………………………………………..75 Fig. 3-15 Concentration data in studying report in April, 2003 followed NIEA W7853.53B method………………………………………………………………………….75 Fig. 3-16 Concentrations data in 6 samples in this study obtained from HS-GC-FID method………………………………………………………………………….76 Fig. 3-17 Contaminants PCE and daughter product TCE, cis-1,2-DCE and VC in four wells WM122, EX13 and A6 (S: pollution source; D: down gradient well in pollution site; DD: down gradient well in surrounding area)………………….76 Fig. 3-18 □13C of PCE enriched in down gradient well…………………………………..77 Fig. 3-19 □13C of cis-1,2-DCE enriched in down gradient well………………………….77 Fig. 3-20 □13C of TCE enriched in down gradient well…………………………………..78 Fig. 3-21 HS-GC-MS TIC chromatogram of groundwater sampled from down gradient MW122 well…………………………………………………………………...78 Fig. 4-1 The chemical structure of 16 PAHs, 6 biomarkers and surrogate structure….103 Fig. 4-2 Pretreatment procedure of sediments samples……………………………….104 Fig. 4-3 Location of Kaohsiung harbor in Taiwan……………………………………105 Fig. 4-4 27 sediments samples were sampled in Kaohsiung harbor sampling locations were divided into 3 areas, one is Lin-hai industrial park, second is main chanel, third is fourth channel…………………………………………105 Fig. 4-5 Procedure of source identification for PAHs in sediments…………………..106 Fig. 4-6 Two GC-IRMS chromatograms of simulated samples (sand blank spiked 16 PAHs and sand blank spiked 16 PAHs and DRO) purifying after stage 3…..107 Fig. 4-7 Two GC-IRMS chromatograms of simulated samples after stage 2 and Soxhelt extraction/ column clean up………………………………………………….107 Fig. 4-8 Three GC-IRMS chromatograms of 1 ml purified extract from stages (stage 1, stage 2 and stage 3) pretreatment of 5 g real sediments sample (M-4), respectively………………………………………………………………….108 Fig. 4-9 GC-MS SIM chromatograms of 16 PAHs and 2 surrogates (2-fluorobiphenyl and 4,4’-dibromooctafluorobiphenyl)……………………………………….109 Fig. 4-10 GC-MS TIC chromatograms of 6 biomarkers and Nap-d8 (IS) internal standard……………………………………………………………………..109 Fig. 4-11 GC-MS SIM/ GC-IRMS/ GC-FID chromatogram of 16 PAHs and 2 SS…..110 Fig. 4- 12 Tree diagram for 16 PAHs contents in seventeen sediments sample due to Euclidean distance statistic analysis and five groups were divided at linkage distance 4.2………………………………………………………………. 111 Fig. 4- 13 The identification results of TE purified extract GC-MS TIC chromatogram from NIST 98 database searching.………………………………………….111 Fig 4-14 The identification results of FB-1 purified extract GC-MS TIC chromatogram from NIST 98 database searching……………………………………………112 Fig. 4-15 The GC-IRMS chromatogram and GC-FID chromatogram of 5 g CP (CPC Corp. Taiwan in Lin-hai industrial park) sediments sample……………….112 Fig. 4-16 Broken-line diagram of □13C values to six kinds of PAHs for comparison of four sediments samples (CB-1, CB-2, CS-1 and CS-2)……………………112 Fig. 5- 1 Flow chart of pretreatment for 8 controlled drugs…………………………..137 Fig. 5- 2 Apparatus of head space type SPME (solid phase micro extration)………...138 Fig. 5- 3 500 □g/ml, 2 ml 13C GC-IRMS chromatogram of 8 mixing drugs…………..139 Fig. 5- 4 2000 □g/ml, 2 ml 15N GC-IRMS chromatogram of 8 mixing drugs ………...139 Fig. 5-5 3Mix-1 (Dexamethamphetamine,(±)MDMA, Ketamine) 15N GC-IRMS Chromatogram……………………………………………………………….139 Fig. 5- 6 3Mix-1 (Dexamethamphetamine,(±)MDMA, Ketamine) 13C GC-IRMS Chromatogram……………………………………………………………….140 Fig. 5- 7 13C HS-SPME GC-IRMS chromatogram of U-1163………………………..140 Fig. 5- 8 15N HS-SPME GC-IRMS chromatogram of U-1163………………………..141 Fig. 5- 9 Tree diagram for 13C, 15N of methamphetamine in 11 waste urine samples fromdrug abuser due to Euclidean distance statistic analysis and five groups were divided at linkage distance 1.50………………………………………..141 Fig. 5- 10□□13C, □15N isotope fingerprint of 11 controlled drug standards……………142 Fig. 5- 11 One way ANOVA analysis results of 3 methamphetamines with different source for □13C and □15N …………………………………………………...142 Fig. 5- 12 One way ANOVA analysis results of 2 ketamines with different source for□□13C and □15N ……………………………………………………………..143 Fig. 5- 13 □13C, □15N isotope fingerprint of 3 methamphetamine standards and 11 methamphetamine contents in urine sample from drug abuser……………143 Fig. 5- 14 One way ANOVA results of □13C and □15N of 3 methamphetamines standards from different sources and 11 methamphetamines in urine sample…………………………………………………………………….144 Table 1- 1 Relative abundances of naturally occurring isotopes of elements commonly analyzed by IRMS………………………………………………………… .18 Table1- 2 Standard deviation of mean □13C value (n=10) of CO2 reference On/Off test ………………………………………………………………………….18 Table1- 3 Standard deviation of mean □13C value (n=10) of C-13, C-14, C-15 for GC-IRMS analysis of FID test sample……………………………………...19 Table 2- 1 Source information and □13C values of 54 organic samples ………………43 Table 2- 2 Source information and □13C values of component in 20 industrial wastes offered by EAL…………………………………………………………….45 Table 2- 3 3 set of GC-IRMS methods were used for determination of □13C value of 54 organic solvents…………………………………………………………….47 Table 2- 4 43 identified components via NIST 98 in 40 industrial wastes from GC-MS TIC chemical fingerprint..…………………………………………………48 Table 2- 5 Contribution of 43 components in industrial waste to factor1 and factor 2 for factor analysis………………………………………………………………50 Table 3- 1 Test results of 20 samples from pollution site and surrounding area……….79 Table 3- 2 Concentrations (□g/ml) and □13C value (‰) for chlorinated ethylene from monitoring well in pollution site and surrounding area……………………..80 Table 4- 1 GPS coordinates, water content and TOC of 27 sediments samples from Kaohsiung harbor………………………………………………………..113 Table 4- 2 Recovery (%) of 16 PAHs in sand blank after each purification step…......114 Table 4- 3 The retention times order, selected qualifier and quantifier ions (m/z) of 16 PAHs and 6 Biomarkers…………………………………………………115 Table 4- 4 Identification results of 16 PAHs and 5 Biomarkers in six sediments samples (M-1~6) with GC-MS…………………………………………………...116 Table 4- 5 16 PAHs contents and 6 Biomarker of seventeen sediments samples from Kaohsiung harbor………………………………………………………..117 Table 4- 6 □13C value of 16 PAHs in eleven sediments samples from Kaohsiung harbor……………………………………………………………………120 Table 4- 7 Source apportionment of 16 PAHs in sediments for Kaohsiung harbor by specific PAHs contents ratio due to Figure 5 procedure………………… 122 Table 4- 8 Differentiation of 16 PAHs in eleven sediments samples with One way ANOVA at 95% confidential interval…………………………………….123 Table 5- 1 Physical property, chemical structure and abbreviation of 8 controlled drugs…145 Table 5- 2 □13C and □15N values of 11 controlled drug standards from different source….146 Table 5- 3 □13C and□□15N values of 11 urine samples from drug abuser……………...146