Abstract
Polycyclic aromatic hydrocarbons (PAHs) are persistent organic pollutants (POPs) that are widely distributed in the environment and cause significant environmental damage. Furthermore, they endanger human health by polluting food from the natural environment and food processing. Therefore, it is necessary to accurately detect PAHs in various sample matrices, which requires precise, practical, and rapid detection methods. The purpose of this research is to develop a high sensitivity analysis method by analyzing the optimum excitation and emission wavelengths of EPA's 15 priority polyaromatic hydrocarbons in the UHPLC fluorescence detector (Acenaphthene, Anthracene, Benzo[a]anthracene, Benzo[b]fluoranthene, Benzo[k]fluoranthene, Benzo[ghi]perylene, Benzo[a]pyrene, Chrysene, Dibenzo[a,h]anthracene, Fluoranthene, Fluorene, Indeno[1,2,3-cd]pyrene, Naphthalene, Phenanthrene, and Pyrene). An average of 17–25 analyses were performed for each polyaromatic hydrocarbon, and optimized excitation and emission wavelengths were obtained. LOD levels between 2 and 90 ppt were obtained with the method created in this direction. It is worth mentioning that the limits achieved for some PAH parameters are lower than those reported in the literature after pre-concentration steps.
1 Introduction
Polycyclic aromatic hydrocarbons (PAHs) are widespread environmental contaminants, more succinctly termed PAHs or polyarenes [1]. Primarily due to forest fires, volcanic eruptions, residential wood burning, and vehicle exhaust. It can also enter water bodies through effluents from factories, sewage treatment plants and into the soil of hazardous waste landfills through effluents from storage containers. In the marine compartment, petroleum inputs are significant due to river discharges, accidental crude oil spills, ballast operations of ships, sewage disposal, offshore production, and transport [2].
PAHs which are listed as priority pollutants by the US Environmental Protection Agency (EPA) and the European Union [3–5]. The International Agency for Research on Cancer (IARC) has identified 16 PAHs [Benzo[g,h,i]perylene (BghiP), acenaphthene (Act), naphthalene (Nap), phenanthrene (Phe), anthracene (Ant), benz[a]anthracene (BaA), chrysene (Chr), fluorene (Fln), benz[b]fluoranthene (BbF), indeno[1,2,3-cd]pyrene (IcdP), carbazole (Crb), benzo[k]fluoranthene (BkF), pyrene (Py), dibenz[a,h]anthracene (DahA) benzo[a]pyrene (BaP), fluoranthene (Flt) including 6 of the 16 EPA regulated PAHs, as potential carcinogens [6, 7].
Determination of PAHs in water samples, especially drinking water, is difficult because of their low solubility, which results in low concentrations [8]. Methods currently used to detect PAHs require chromatographic separation by liquid or gas chromatography, followed by detection using mass spectrometry or fluorescence spectroscopy. Even though these methods are precision, they require significant time and resources, limiting the ability to conduct high throughput assays of large populations to measure toxicant exposure [9–13]. The selectivity is based on the fact that relatively few compounds show intrinsic fluorescence (fl) and that emission intensity depends on two variables: excitation (ex) and emission (em) wavelengths. Therefore, fluorescent compounds can be determined by their excitation, emission, or synchronous spectroscopy [14]. PAHs have good fluorescence activity, and their maximum fluorescence occurs at different Ex/Em wavelengths. Changing the Ex/Em wavelength is necessary to obtain the best detection sensitivity [15]. Programmed fluorescence switching (switching to the maximum Ex/Em wavelength of each PAH when the PAH peak crosses the fluorescence detector) is required to obtain the best sensitivity for each PAH In this study, all ex and em PAH wavelengths in the literature were compiled, and optimum ex/em wavelengths were determined for 15 Pahs. In this way, much lower limit of detection (LOD) and limit of quantification (LOQ) levels have been obtained, and method sensitivity has been improved.
2 Materials and methods
2.1 Reagents and chemicals
Analytical standard PAHs from Dr. Ehrenstorfer (Augsburg, Germany) and High Performance Liquid Chromatography (HPLC) grade solvents: acetonitrile, and water gradient grade for liquid chromatography (LC) from Merck; (München, Germany) were used. Monitor the blank to guarantee that the reagents do not contain PAH in detectable concentrations. Stock solutions contain 10 μg L−1 of individual standards. Prepare at least five calibration solutions by appropriately diluting the stock solution using acetonitrile as the solvent. The diluted solutions were stored in amber vials at 4 °C to avoid photo-degradation of PAHs.
2.2 Instrumentation
Analyses were performed using a Perkin Elmer Ultra High Performance Liquid Chromatography (UHPLC) system (Waltham, Massachusetts, USA) consisting of an FX-20 Quaternary pump, Flexar FX UHPLC, Autosampler with degasser, Flexar LC Column Oven and Flexar Fluorescence LC Detector. The data was collected and analyzed using Chromera Speciation Software.
2.3 Chromatographic conditions
The analytical column used was a UHPLC column: Brownlee Analytical PAH, 150 × 3.2 mm, 5 µm particle size, Perkin Elmer (Waltham, MA, USA); This column has been specifically designed for the analysis of priority polluting PAHs. The analytical column was kept at 25 °C during the analysis. An isocratic elution (30/70 v/v, mobile phase A and mobile phase B) was utilized. Mobile phase A consisted of water, and mobile phase B consisted of acetonitrile flow rate of 0.8 mL min−1 was used. The injection volume was 20 µL. Sampling rate is the frequency at which snapshots of an analog signal are recorded. Thus the more snapshots per second, the higher the sample rate and the better the quality. Sampling rate 5 pts/s was preferred. Selecting the baseline mode “autozero” prevents the detector from cutting peak areas at wavelength transitions. Chromatographic conditions used in optimization studies Fig. 1.
Exemplary procedure of analysis used in optimization studies
Citation: Acta Chromatographica 2023; 10.1556/1326.2023.01118
Exemplary procedure of analysis used in optimization studies
Citation: Acta Chromatographica 2023; 10.1556/1326.2023.01118
Exemplary procedure of analysis used in optimization studies
Citation: Acta Chromatographica 2023; 10.1556/1326.2023.01118
Wavelength detection studies were carried out with “medium” detector sensitivity. The reason for working at medium level is to prevent the peaks from being overloaded. In this way, all peaks were correctly observed. In addition, the energy level increases significantly in fluorescence detectors' high sensitivity and concentration work. The detector turns itself off to avoid damage. This event was observed at the data level in the study performed at ex/em 250/500 nm and given Fig. 2.
Energy level test of fluorescence detector
Citation: Acta Chromatographica 2023; 10.1556/1326.2023.01118
Energy level test of fluorescence detector
Citation: Acta Chromatographica 2023; 10.1556/1326.2023.01118
Energy level test of fluorescence detector
Citation: Acta Chromatographica 2023; 10.1556/1326.2023.01118
LOD and LOQ runs at this level have been changed to “super high”. The reason for working with super high sensitivity in LOD and LOQ work is to see the full performance of the equipment. Quantitative determination was carried out using an external calibration curve method. The calibration curves for all compounds were duplicated with a relative standard deviation (RSD) < 3%. The abundances of the selected compounds were calculated by comparing the areas of the peaks on the calibration curve with the of the peaks of the individual PAHs obtained from the HPLC fluorescence detector chromatograms. Peak identification was carried out by comparison of retention times with standards.
2.4 HPLC conditions and method optimization studies
The selection of the Ex/Em wavelength pair is essential for optimizing the sensitivity of a fluorescence detection assay. A wavelength change should be made when the fluorescence is low. At high fluorescence, wavelength changes can lead to a displacement of the baseline. Readjusting the baseline after changing the wavelength may interfere with integration and quantification. Changing the wavelengths and damping may be necessary simultaneously to obtain constant peak heights. To develop the peak lengths and areas of polyaromatic hydrocarbons, it is necessary to determine the most suitable ex and em wavelengths. Standard wavelengths used in the literature have been identified and listed for 15 PAHs. For each PAH, between 17 and 25 different ex and em values were determined from the literature, and the data in the study were obtained by analyzing them. In our study, the best analytical values for 15 PAHs were determined simultaneously with ex and em values among the values in the table. The validation study was also carried out with these data. All results in HPLC analyzes are shown by determining the maximum peak heights of the analytes in flu unit. The flu unit is the response expression of the analyte's fluorescence measurement. On the other hand, it can be considered the equivalent of the absorbance value in the HPLC system. Depending on the concentration, flu values of the analyte were evaluated according to the peak height and area. The maximum value was obtained for calculating the maximum peak heights and the area used in the quantification. As a result of the analyses carried out in the high sensitivity mode of the fluorescence detector, the peak heights in the flu unit were obtained. The optimum wavelength was determined by the highest peak area received. A calibration chart was created according to the optimally determined ex, and em values and repeatability studies were carried out.
LOD and LOQ were calculated according to the data obtained from these studies. Particularly in analyses with low legal limits, the sensitive detection limit is critical to approach these sensitive detection limits in chromatography, it is necessary to have good peak separations and high peak areas. In this sense, many studies conducted under the HPLC fluorescence detector, regardless of matrix difference, examined and verified the wavelengths used.
The obtained chromatograms were overlapped and the wavelength pair with the highest peak length was determined. The chromatograms are marked in different colors to distinguish them. There is a slight shift in the retention time of the peaks. The increase in the number of injections slightly decreased the retention in the column. Example overlapped chromatogram are given in Fig. 3.
2.5 Data description and research methodology
Since certified reference materials were used in all experimental studies, the matrix difference was ignored in detecting ex/em pairs in the literature. In this way, ex/em pairs belonging to almost every matrix were detected, the overlapping pairs were eliminated and lists were created. Studies of ex/em couples whose mobile carrier phases are acn/water were preferred not to reflect the solvent effect on the analysis results.
3 Results and discussion
Analyses were performed according to the ex/em wavelength pairs given in Table 1. With these analyzes, the changes of ex and em wavelengths on fluorescence were determined. Line structure PAHs showed better fluorescence than branched structure PAHs. The chromatograms were overlapped, and the effect of wavelength changes on the peak lengths in fluorescence (flu) was observed. The overlaid chromatograms of all PAHs are given in Fig. 4.
Excitation and emission wavelengths [15–45]
| NAP | ACT | FLN | PHE | ANT | FL | PY | BaA | CHR | BbF | BkF | BaP | DahA | BghiP | IcdP |
| 215/330 | 220/325 | 263/310 | 247/364 | 247/401 | 280/460 | 236/389 | 275/389 | 260/381 | 256/446 | 295/410 | 260/408 | 290/398 | 290/415 | 248/484 |
| 219/330 | 224/320 | 265/310 | 250/365 | 248/405 | 280/450 | 237/385 | 281/391 | 264/381 | 258/442 | 290/412 | 288/406 | 285/396 | 290/410 | 290/499 |
| 220/330 | 220/320 | 275/315 | 250/366 | 250/402 | 281/453 | 238/398 | 277/393 | 265/380 | 254/451 | 290/410 | 281/407 | 294/398 | 290/418 | 290/500 |
| 217/338 | 276/330 | 270/323 | 247/357 | 250/406 | 232/445 | 270/390 | 284/390 | 260/370 | 249/443 | 296/426 | 290/410 | 285/404 | 292/415 | 250/470 |
| 221/337 | 275/330 | 279/306 | 250/368 | 250/380 | 237/460 | 240/386 | 270/390 | 267/385 | 280/438 | 243/412 | 280/410 | 290/418 | 290/420 | 293/498 |
| 222/329 | 280/324 | 280/324 | 246/370 | 252/402 | 270/450 | 332/378 | 270/410 | 260/390 | 255/420 | 302/431 | 295/405 | 290/420 | 294/425 | 300/500 |
| 224/330 | 280/330 | 275/330 | 244/370 | 252/400 | 270/440 | 254/390 | 270/384 | 270/384 | 266/425 | 294/425 | 295/410 | 290/415 | 295/410 | 302/500 |
| 224/320 | 225/315 | 276/330 | 252/365 | 250/408 | 270/470 | 240/400 | 270/385 | 270/385 | 290/430 | 303/432 | 266/415 | 289/422 | 285/416 | 274/507 |
| 267/330 | 227/315 | 234/320 | 240/360 | 251/378 | 284/467 | 246/375 | 268/398 | 270/367 | 260/420 | 290/430 | 290/430 | 290/410 | 289/422 | 293/493 |
| 275/330 | 235/332 | 280/330 | 252/370 | 248/375 | 237/440 | 334/371 | 267/385 | 269/361 | 294/425 | 300/440 | 266/425 | 298/398 | 295/425 | 302/510 |
| 277/330 | 290/337 | 225/315 | 252/372 | 250/375 | 285/465 | 252/400 | 287/386 | 254/390 | 298/436 | 288/406 | 297/405 | 295/405 | 295/405 | 300/470 |
| 270/323 | 270/323 | 250/341 | 246/375 | 254/402 | 280/420 | 248/375 | 260/390 | 270/390 | 300/440 | 307/413 | 260/420 | 296/404 | 290/430 | 305/480 |
| 277/337 | 292/322 | 227/315 | 248/375 | 250/420 | 288/450 | 276/391 | 290/395 | 268/398 | 300/445 | 255/420 | 298/407 | 295/410 | 296/406 | 300/466 |
| 276/323 | 234/320 | 224/320 | 250/380 | 255/380 | 238/418 | 238/418 | 254/390 | 277/376 | 302/452 | 266/415 | 250/400 | 295/425 | 285/404 | 300/464 |
| 280/330 | 275/350 | 220/325 | 254/375 | 238/418 | 290/447 | 250/420 | 290/404 | 270/410 | 290/410 | 260/420 | 255/420 | 300/400 | 296/404 | 300/465 |
| 278/322 | 280/355 | 290/337 | 275/350 | 240/430 | 337/440 | 237/440 | 265/380 | 260/420 | 250/400 | 266/425 | 298/404 | 268/398 | 300/415 | 268/398 |
| 280/324 | 275/315 | 275/350 | 280/355 | 244/370 | 260/420 | 270/440 | 260/420 | 277/393 | 268/398 | 250/400 | 268/398 | 300/415 | 260/420 | 296/404 |
| 275/350 | 265/360 | 265/360 | 240/400 | 252/372 | 250/420 | - | 240/400 | 240/400 | - | 256/446 | 294/425 | 290/430 | 302/419 | 300/440 |
| 280/355 | 250/341 | 280/355 | 252/400 | 250/368 | 365/462 | - | 277/376 | 238/398 | - | 260/460 | 296/406 | 260/420 | 300/440 | 250/495 |
| 248/375 | 248/375 | 240/368 | 294/347 | 260/420 | 240/400 | - | 238/398 | 290/404 | - | 268/398 | 256/446 | 300/440 | 268/398 | 245/500 |
| - | - | 248/375 | 297/367 | - | 252/402 | - | - | - | - | - | 260/460 | 234/420 | 305/420 | 250/500 |
| - | - | - | - | 252/400 | - | - | - | - | - | 300/440 | 300/469 | 234/420 | 246/503 | |
| - | - | - | - | - | 248/375 | - | - | - | - | - | 300/470 | 300/465 | 251/510 | |
| - | - | - | - | - | - | - | - | - | - | - | - | - | 300/470 | - |
| - | - | - | - | - | - | - | - | - | - | - | - | - | 302/500 | - |
Chromatograms of analyses at different ex/em wavelengths A: Naphthalene, B: Acenaphthene, C: Fluorene, D: Phenanthrene, E: Anthracene, F: Fluoranthene, G: Pyrene, H: Benzo[a]anthracene, I: Chrysene, J: Benzo[b]fluoranthene, K: Benzo[k]fluoranthene, L: Benzo[a]pyrene, M: Dibenzo[ah]anthracene, N: Benzo[ghi]perylene, O: Indeno[1,2,3cd]pyrene
Citation: Acta Chromatographica 2023; 10.1556/1326.2023.01118
Chromatograms of analyses at different ex/em wavelengths A: Naphthalene, B: Acenaphthene, C: Fluorene, D: Phenanthrene, E: Anthracene, F: Fluoranthene, G: Pyrene, H: Benzo[a]anthracene, I: Chrysene, J: Benzo[b]fluoranthene, K: Benzo[k]fluoranthene, L: Benzo[a]pyrene, M: Dibenzo[ah]anthracene, N: Benzo[ghi]perylene, O: Indeno[1,2,3cd]pyrene
Citation: Acta Chromatographica 2023; 10.1556/1326.2023.01118
Chromatograms of analyses at different ex/em wavelengths A: Naphthalene, B: Acenaphthene, C: Fluorene, D: Phenanthrene, E: Anthracene, F: Fluoranthene, G: Pyrene, H: Benzo[a]anthracene, I: Chrysene, J: Benzo[b]fluoranthene, K: Benzo[k]fluoranthene, L: Benzo[a]pyrene, M: Dibenzo[ah]anthracene, N: Benzo[ghi]perylene, O: Indeno[1,2,3cd]pyrene
Citation: Acta Chromatographica 2023; 10.1556/1326.2023.01118
In this sense, it has been determined that the excitation value of PAHs is more decisive in fluorescence analysis. The relevant analyses results are given in Table 2.
PAH analysis results
| PAH analysis results | |||||||||||||||
| NAP | Flu | ACT | Flu | FLN | Flu | PHE | Flu | ANT | Flu | FL | Flu | PY | Flu | BaA | Flu |
| 215/330 | 185 | 220/325 | 518 | 263/310 | 930 | 247/364 | 191 | 247/401 | 745 | 280/460 | 51 | 236/389 | 135 | 275/389 | 241 |
| 219/330 | 176 | 224/320 | 400 | 265/310 | 886 | 250/365 | 180 | 248/405 | 663 | 280/450 | 50 | 237/385 | 126 | 281/391 | 240 |
| 220/330 | 166 | 220/320 | 385 | 275/315 | 443 | 250/366 | 178 | 250/402 | 653 | 281/453 | 48 | 238/398 | 113 | 277/393 | 235 |
| 217/338 | 164 | 276/330 | 210 | 270/323 | 415 | 247/357 | 176 | 250/406 | 580 | 232/445 | 44 | 270/390 | 109 | 284/390 | 211 |
| 221/337 | 139 | 275/330 | 205 | 279/306 | 391 | 250/368 | 173 | 250/380 | 544 | 237/460 | 43 | 240/386 | 108 | 270/390 | 199 |
| 222/329 | 137 | 280/324 | 198 | 280/324 | 207 | 246/370 | 172 | 252/402 | 524 | 270/450 | 43 | 332/378 | 97 | 270/410 | 194 |
| 224/330 | 110 | 280/330 | 185 | 275/330 | 200 | 244/370 | 168 | 252/400 | 520 | 270/440 | 41 | 254/390 | 89 | 270/384 | 173 |
| 224/320 | 82 | 225/315 | 183 | 276/330 | 168 | 252/365 | 166 | 250/408 | 518 | 270/470 | 41 | 240/400 | 88 | 270/385 | 170 |
| 267/330 | 73 | 227/315 | 162 | 234/320 | 135 | 240/360 | 155 | 251/378 | 468 | 284/467 | 38 | 246/375 | 62 | 268/398 | 155 |
| 275/330 | 71 | 235/332 | 155 | 280/330 | 134 | 252/370 | 149 | 248/375 | 462 | 237/440 | 36 | 334/371 | 61 | 267/385 | 154 |
| 277/330 | 66 | 290/337 | 155 | 225/315 | 115 | 252/372 | 139 | 250/375 | 415 | 285/465 | 35 | 252/400 | 59 | 287/386 | 152 |
| 270/323 | 65 | 270/323 | 142 | 250/341 | 113 | 246/375 | 138 | 254/402 | 410 | 280/420 | 25 | 248/375 | 58 | 260/390 | 120 |
| 277/337 | 60 | 292/322 | 140 | 227/315 | 105 | 248/375 | 138 | 250/420 | 326 | 288/450 | 25 | 276/391 | 57 | 290/395 | 108 |
| 276/323 | 59 | 234/320 | 128 | 224/320 | 92 | 250/380 | 110 | 255/380 | 297 | 238/418 | 18 | 238/418 | 47 | 254/390 | 105 |
| 280/330 | 56 | 275/350 | 109 | 220/325 | 85 | 254/375 | 110 | 238/418 | 267 | 290/447 | 18 | 250/420 | 23 | 290/404 | 101 |
| 278/322 | 53 | 280/355 | 95 | 290/337 | 66 | 275/350 | 50 | 240/430 | 255 | 337/440 | 15 | 237/440 | 12 | 265/380 | 77 |
| 280/324 | 50 | 275/315 | 63 | 275/350 | 34 | 280/355 | 47 | 244/370 | 229 | 260/420 | 14 | 270/440 | 10 | 260/420 | 75 |
| 275/350 | 36 | 265/360 | 40 | 265/360 | 24 | 240/400 | 37 | 252/372 | 228 | 250/420 | 13 | - | - | 240/400 | 46 |
| 280/355 | 22 | 250/341 | 34 | 280/355 | 16 | 252/400 | 37 | 250/368 | 120 | 365/462 | 8 | - | - | 277/376 | 43 |
| 248/375 | 3 | 248/375 | 4 | 240/368 | 7 | 294/347 | 17 | 260/420 | 61 | 240/400 | 4 | - | - | 238/398 | 42 |
| - | - | - | - | 248/375 | 5 | 297/367 | 14 | - | - | 252/402 | 4 | - | - | - | - |
| - | - | - | - | - | - | - | - | - | - | 252/400 | 3 | - | - | - | - |
| - | - | - | - | - | - | - | - | - | - | 248/375 | nd | - | - | - | - |
| - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - |
| - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - |
| PAH analysis results | |||||||||||||
| CHR | Flu | BbF | Flu | BkF | Flu | BaP | Flu | DahA | Flu | BghiP | Flu | IcdP | Flu |
| 260/381 | 167 | 256/446 | 139 | 295/410 | 617 | 260/408 | 317 | 290/398 | 137 | 290/415 | 76 | 248/484 | 16 |
| 264/381 | 163 | 258/442 | 138 | 290/412 | 566 | 288/406 | 312 | 285/396 | 136 | 290/410 | 74 | 290/499 | 13 |
| 265/380 | 155 | 254/451 | 127 | 290/410 | 560 | 281/407 | 311 | 294/398 | 124 | 290/418 | 73 | 290/500 | 13 |
| 260/370 | 148 | 249/443 | 125 | 296/426 | 541 | 290/410 | 294 | 285/404 | 109 | 292/415 | 73 | 250/470 | 12 |
| 267/385 | 130 | 280/438 | 111 | 243/412 | 528 | 280/410 | 289 | 290/418 | 106 | 290/420 | 72 | 293/498 | 12 |
| 260/390 | 118 | 255/420 | 106 | 302/431 | 527 | 295/405 | 228 | 290/420 | 106 | 294/425 | 64 | 300/500 | 12 |
| 270/384 | 104 | 266/425 | 106 | 294/425 | 526 | 295/410 | 222 | 290/415 | 105 | 295/410 | 64 | 302/500 | 12 |
| 270/385 | 101 | 290/430 | 102 | 303/432 | 508 | 266/415 | 207 | 289/422 | 102 | 285/416 | 61 | 274/507 | 12 |
| 270/367 | 100 | 260/420 | 99 | 290/430 | 501 | 290/430 | 207 | 290/410 | 95 | 289/422 | 59 | 293/493 | 11 |
| 269/361 | 95 | 294/425 | 87 | 300/440 | 486 | 266/425 | 189 | 298/398 | 92 | 295/425 | 59 | 302/510 | 11 |
| 254/390 | 87 | 298/436 | 81 | 288/406 | 471 | 297/405 | 187 | 295/405 | 88 | 295/405 | 54 | 300/470 | 10 |
| 270/390 | 77 | 300/440 | 76 | 307/413 | 441 | 260/420 | 180 | 296/404 | 87 | 290/430 | 52 | 305/480 | 10 |
| 268/398 | 64 | 300/445 | 75 | 255/420 | 304 | 298/407 | 171 | 295/410 | 82 | 296/406 | 50 | 300/466 | 8 |
| 277/376 | 40 | 302/452 | 62 | 266/415 | 292 | 250/400 | 170 | 295/425 | 74 | 285/404 | 49 | 300/464 | 7 |
| 270/410 | 35 | 290/410 | 61 | 260/420 | 275 | 255/420 | 168 | 300/400 | 74 | 296/404 | 45 | 300/465 | 7 |
| 260/420 | 27 | 250/400 | 44 | 266/425 | 260 | 298/404 | 166 | 268/398 | 59 | 300/415 | 38 | 268/398 | nd |
| 277/393 | 26 | 268/398 | 34 | 250/400 | 219 | 268/398 | 160 | 300/415 | 59 | 260/420 | 34 | 296/404 | nd |
| 240/400 | 16 | - | - | 256/446 | 207 | 294/425 | 160 | 290/430 | 58 | 302/419 | 31 | 300/440 | nd |
| 238/398 | 15 | - | - | 260/460 | 122 | 296/406 | 122 | 260/420 | 24 | 300/440 | 23 | 250/495 | nd |
| 290/404 | 10 | - | - | 268/398 | 120 | 256/446 | 70 | 300/440 | 22 | 268/398 | 21 | 245/500 | nd |
| - | - | - | - | - | - | 260/460 | 53 | 234/420 | 9 | 305/420 | 21 | 250/500 | nd |
| - | - | - | - | - | - | 300/440 | 48 | 300/469 | 5 | 234/420 | 12 | 246/503 | nd |
| - | - | - | - | - | - | - | - | 300/470 | 4 | 300/465 | 8 | 251/510 | nd |
| - | - | - | - | - | - | - | - | - | - | 300/470 | 5 | - | - |
| - | - | - | - | - | - | - | - | - | - | 302/500 | 1 | - | - |
*nd: not detected.
In the wavelength studies, no peak was detected in 9 wavelength pairs. These wavelength pairs belong to FL and IcdP. FL and IcdP did not peak at their respective wavelengths. In particular, the increase in the difference between emission and excitation wavelengths of IcdP resulted in a decrease in fluorescence. While BaA and BaP provide a balanced distribution regarding flu results, the highest flu value belongs to FLN. When all wavelength pairs were examined, the lowest flu values were determined to belong to IcdP. Different fluorescence radiations were detected in very close ex/em wavelength pairs. When the results of the analyzes performed at the 275/330 and 276/330 wavelengths of the FLN are examined, it is seen that a 1 nm change in the excitation value causes a 32-unit difference in flu. In the analysis results of BaA at 270/384 and 270/385 wavelengths, it was seen that a 1 nm change in the emission value caused a 3-unit difference in flu. It was observed that BkF, ANT and BaP showed good fluorescence at all detected wavelength pairs. According to the flu values taken for each PAH given in Table 2, ex and em values with the best analytical performance were accepted as optimum operating conditions.
3.1 LOD and LOQ
The limit of detection (LOD) and limit of quantification (LOQ) are terms used to describe the smallest concentration of a measurand that can be reliably measured by an analytical procedure. LOD for each analyte were calculated as being three times the average level of the baseline noise (analyzed from the injection of individual PAH containing standard solutions), and the LOQ was calculated as ten times this same level.
It is worth mentioning that the limits so far achieved for some PAH parameters are lower than those reported in the literature after pre-concentration steps. UHPLC floresence method was proposed for the analysis of fiftten PAH, and validated the developed method, optimum ex/em wavelength pairs, optimum peak height, the calibration curve equations, linear ranges, linearity coefficients (R2), limits of detection (LODs) and limits of quantification (LOQs) are summarized in Table 3.
Regression equation, linear range, LODs and LOQs (n = 7)
| Ex/Em | Flu | R2 | Regression equation | Linear range (ng L−1) | LOD (μg L−1) | LOQ (μg L−1) | |
| NAP | 215/330 | 185 | 0.999860 | y = 13.28x + 28.16 | 0.1–10 | 0.003 | 0.010 |
| ACT | 222/329 | 593 | 0.999803 | y = 16.34x + 45.11 | 0.1–10 | 0.002 | 0.007 |
| FLN | 263/310 | 930 | 0.999919 | y = 5.32x + 17.07 | 0.1–10 | 0.002 | 0.007 |
| PHE | 247/364 | 191 | 0.999733 | y = 12.5x + 29.10 | 0.1–10 | 0.009 | 0.030 |
| ANT | 247/401 | 617 | 0.999938 | y = 31.45x + 124.4 | 0.1–10 | 0.005 | 0.007 |
| FL | 280/460 | 51 | 0.999865 | y = 7.1x + 14.30 | 0.1–10 | 0.009 | 0.030 |
| PY | 236/389 | 135 | 0.999887 | y = 3.112x + 6.98 | 0.1–10 | 0.015 | 0.050 |
| BaA | 275/389 | 241 | 0.999910 | y = 7.45x + 14.20 | 0.1–10 | 0.015 | 0.050 |
| CHR | 260/381 | 167 | 0.999954 | y = 11.35x + 28.15 | 0.1–10 | 0.015 | 0.050 |
| BbF | 256/446 | 139 | 0.999877 | y = 17.10x + 37.11 | 0.1–10 | 0.015 | 0.050 |
| BkF | 295/410 | 617 | 0.999915 | y = 5.31x + 10.55 | 0.1–10 | 0.006 | 0.020 |
| BaP | 260/408 | 317 | 0.999888 | y = 3.35x+6.55 | 0.1–10 | 0.009 | 0.030 |
| DahA | 290/398 | 137 | 0.999944 | y = 6.06x+8.35 | 0.1–10 | 0.020 | 0.007 |
| BghiP | 290/415 | 76 | 0.999013 | y = 12.2x+10.56 | 0.1–10 | 0.025 | 0.083 |
| IcdP | 248/484 | 16 | 0.999801 | y = 7.28x+14.30 | 0.1–10 | 0.090 | 0.300 |
The method developed chromatographic separation and detection, was used inside an inter-laboratory study aimed at the determination of the analytes BaP, BbF, BkF, IcdIP and BghiP in drinking water samples. The Inter-laboratory test (named Water Chemistry (Aquacheck)) was organized and managed by LGC. The composition of the samples is reported in Table 4 together with the z-scores obtained. Absolute values of z-scores (|z| values) are used for assessing the acceptability of the results as follows:
|z|≤ 2 acceptable result; 2<|z|< 3 doubtful result; |z|≥3 unacceptable result. From the data shown in Table 4, it can be observed that since |z| values are included between 0.05 and 1.60 the method developed performs satisfactorily. Accuracy of the UHPLC method through z-score values inside an inter-laboratory study given Table 4. The method optimized is currently routinely used for the determination of PAHs by the laboratory in charge of the monitoring of the drinking water quality for the city of Bursa.
Accuracy of the UHPLC method through z-score values inside an inter-laboratory study
| Analyte | Results | Units | Z Score | Assigned Value | Lab. Numbers | Roboust SD | SD |
| FL | 20.7 | ng L−1 | 0.05 | 20.6 | 39 | 1.93 | 3.37 |
| BbF | 8.60 | ng L−1 | 0.52 | 7.55 | 49 | 1.053 | 1.232 |
| BkF | 8.21 | ng L−1 | 0.14 | 7.93 | 49 | 0.971 | 1.633 |
| BaP | 3.81 | ng L−1 | 0.18 | 3.72 | 49 | 0.830 | 0.786 |
| BghiP | 20.80 | ng L−1 | 1.60 | 17.60 | 48 | 2.669 | 3.076 |
| IcdP | 11.24 | ng L−1 | 0.79 | 9.67 | 49 | 1.550 | 1.816 |
4 Conclusion
An optimization study was performed to improve the sensitivity to find the specific emission and excitation wavelengths for each analyte. This task was accomplished by optimizing the excitation and emission wavelengths during the chromatographic run in correspondence with the elution of the analytes. Optimum wavelength pairs defined for PAHs as NAP 215/330, ACT 22/329, FLN 263/310, PHE 247/364, ANT 247/401, FL 2840/460, PY 236/389, BaA 275/389, CHR 260/381, BbF 256/446, BkF 295/410, BaP 260/408, DahA 290/398, BghiP 290/415 and IcdP 248/484. Improvements to the method have significantly improved its sensitivity, allowing the determination of low concentration levels associated with such a sample. It was verified that the obtained LOD and LOQ were lower for HPLC Fl, possibly due to the excellent sensitivity of this technique to detect PAH. The optimum ex and em values obtained can be studied for 15 PAHs in a wide range of samples.
Author contributions
Bİ carried out designing of the current study and coordination of the manuscript. SK carried out the experimental work and drafted the manuscript and participated in the design Bİ and SK reviewed the manuscript. All authors read and approved the final manuscript.
Funding
The authors did not receive support from any organization for the submitted work.
Availability of data and materials
All data and materials are available upon request.
Declarations
Conflict of interest
The authors declare that they have no conflict of interest.
Research involving human participants and/or animals
This research did not involve human participants or animals.
Informed consent
Informed consent obtaining for this type of study is not required.
Acknowledgements
The author Selman Kander is thankful to Bursa Water and Sewerage Administration General Directorate (BUSKİ), for providing facilities to perform this research work.
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