HPLC-FLD Analysis

In this method the wine sample is filtered through a 0.45 am PTFE cartridge and analysis is performed by direct injection into the HPLC column using a system equipped with a fluorimetric detector operating at excitation and emission wavelengths 225 and 320 nm, respectively 

HPLC-fluorimetric detector (excitation wavelength 225 nm emission wavelength 

25 °C isocratic elution with mobile phase composed of 50 mM NaH2P04 (pH 

40)/acetonitrile/methanol 65 30 5 (v/v/v) flow rate 1.5mL/min injection volume 

Franke, K.E and Christensen, L.P. (1990) Elevated putrescine levels in grapevine leaves that display symptoms of potassium deficiency, Am. J. Enol. Vitic., 41, 121-125. 

---

FMAE was compared with pressurized-liquid, Soxhlet, and ultrasonic extraction for the analysis of sewage sludges. The optimum conditions for FMAE were determined to be 30 W, for 10 min in 30 ml of acetone HEX (1 1, v/v). Recoveries for certified reference marine sediment, CRM 1941a, were 75, 61, and 56% for FLA, B[b]F, and B[a]P, respectively. Activated copper bars were added to samples to remove sulfur. Mean recovery by FMAE was 70% compared to traditional Soxhlet extraction. FMAE recoveries exceeded those for ultrasonic extraction but better results were obtained by pressurized-liquid extraction (PEE) with the best results obtained by Soxhlet extraction. All extracts required column cleanup, concentration, and solvent exchange for RP-HPLC-FLD analysis. 

Rellan S, Gago-Martinez A (2007) Improved conditions for the application of solid phase microextraction prior to HPLC-FLD analysis of anatoxin-a. J Sep Sci 30 2522-2528. doi 10.1002/jssc.200700103... 

PUF has also been used as a solid-phase to sorb PAHs from water samples. PUF sorbents were batch-equilibrated with PAH standard solutions, removed, squeezed with a filter paper, air-dried, and subjected to solid-matrix luminescence (SML). PAH was converted to 3,4 benzopyrene for luminescence analysis. " A level of 8ngmU PAH was detected in sewage water from a petroleum refinery. More recently, cotton was applied as a solid-phase sorbent in the online investigation of tap water, sewage treatment plant effluent, and river water in Beijing, China. Water samples, of 100-ml volume and 10% 2-propanol, to prevent sorption on container walls, were preconcentrated using a precolumn attached to an HPLC-FLD system. Recoveries between 92 and 119% were reported at ngU spike levels for FLU, PHN, FLA, and B[k]F in real water samples. 

Polydimethylsiloxane rods (1 mm X 10 mm) are an effective sorbent for PAHs from aquatic solutions.Each rod is placed in a vial with only 15 ml of sample and shaken for 3 h. The rods are then extracted with 100 ml of ACN H20 (4 1, v v) with 10 min of sonication prior to analysis. When HPLC-FLD is utilized for analysis, recoveries of 62 to 97% were obtained with limits of detection of 0.1 to 1.2 ng 1 when a total of four desorptions were combined. The method was applied to the semiquantitative screening of scrubber dust slurry from copper processing. Further quantitation by pressurized liquid extraction-HPLC-FLD showed that the concentration of B[k]F and PHN were 8.9 mg kg and 135.6 mg kg respectively. Since the PDMS rod is thicker than the PDMS coating on typical stir bars, longer equilibration times are required, yet the rods are inexpensive and disposable. 

PHN, ACE, PYR, CHY, B[a]P, and benzo[e]pyrene were separated in a 50 mM borate buffer (pH 9) containing a mixture of 20 mM neutral methyl-(3-cyclodextrin (M(3CD) and 25 mM anionic sulfobutylether-(3-cyclodextrin (SB(3CD) at 30 kV and 30°C. " B[a]P and benzo[e]pyrene were successfully resolved with the other compounds in under 11 min in a 50-cm effective length of capillary without micelles in the mobile phase. The system was also less sensitive to temperature and separation potential. LIE detection with excitation at 325 nm at 2.5 mW from a He/Cd laser coupled to an optical fiber allowed for detection limits in the sub ppb range. The method described above was applied to the analysis of contaminated soil that had been extracted by supercritical CO2 for 20 min at 120°C and collected in methanol/DCM. ° Of the 16 EPA PAH mixtures, eleven compounds were detectable in the low ppb range. Ten of the eleven detectable compounds were measured in the soil extract. When compared to RP-HPLC, CE values were slightly lower but only six compounds were detected by HPLC-FLD. No direct relationship between PAH molecular size, polarity, or volatility with migration order was observed and B[b]F/B[k]F isomers were readily separated. 

Consequently, analysis of PAHs is mainly accomplished by GC-MS in simple or tandem mode (GC-MS/MS) and HPLC coupled with fluorescence detector (HPLC-FLD) since most of the PAHs are strong fluorophores. Multiple reaction monitoring (MRM) in a triple quadrupole GC-MS/MS is inherently more selective and sensitive than either scan or selected ion monitoring (SIM) as many matrix interferences are minimized or even removed. For this reason various groups have used tandem MS (Matozzo et al. 2010 Xia et al. 2012), however sufficient detection was accomplished also with GC-MS-SIM (Webster et al. 2006). HPLC-UV has also been used with less sensitivity as expressed by LOD values, although in one work LOD values with UV detection are reported quite low (Cravo et al. 2012). PAHs as in almost all UV or FLD methods were identified on the basis of retention time and quantification on an external standard method. 

An important work on the LC-APPI-MS/MS field was developed for the analysis of 12 PAHs in sediment samples (Moriwaki et al. 2004). In this context APPI was performed in positive mode aiming to overcome the low sensitivity of poor fluorescence PAHs. Extraction of PAHs from sediments was conducted with ultrasonic agitation and the mixture was purified on a silica cartridge. PAHs were eluted with a mixture of hexane toluene (9 1) and separated on a reversed phase ODS column with a runtime of 20 min. From mass spectrometry point of view the positive molecular ions were observed as the main peaks for all PAHs. Limits of determination ranged from 6 to 91 ng/g thus they were comparable with LODs presented in HPLC-FLD analyses. The method had also acceptable recovery values (90-116%). 

The LOD valnes vary in most reports of HPLC-FLD methods from 0.01 to 35 ng/g. The latter proves that HPLC-FLD is antagonizing GC-MS methods since the LOD values are comparable. Studies with HPLC-FLD demonstrating LODs in the pg/g scale do not exist, which implies that GC-MS and GC-MS/MS are superior to HPLC-FLD. However if the goal of the lab is only the analysis of PAHs and not the inclusion of other contaminants HPLC-FLD is a reliable tool. However if the analyses intends to be expanded to other contaminants (such as pesticides) GC-MS or LC-MS are the instrumentation of choice. 

A study of residual analysis of thirty pesticides and their transformation products was based on SPE on-line with HPLC-UVD or post-column derivatization with o-phthalaldehyde (73) and fluorescence detection (FLD), according to EPA method 531.1 and others. The method allowed determination of many pesticides in river and well waters at 0.01 to 0.5 -ig/L levels195. An automatized procedure was proposed for determination... 

The CLD methods for HPLC using isoluminol (190) with microperoxidase catalysis, for determination of lipid hydroperoxides in clinical fluids, have been reviewed. Determination of phospholipids hydroperoxides by luminol (124) CL has been reviewed . A fast RP-HPLC method (retention times 1 to 2 min) for determination of hydroperoxides and other peroxide compounds includes UVD, which is not always effective, and CLD, attained on injection of luminol (124), the CL reagent (Scheme 3), hemin (75a), a catalyst, and NaOH to raise the pH of the solution. A FLD cell may act as CLD cell if the excitation source is turned off. The selectivity of CLD is of advantage over UVD in industrial analysis thus, for example, UVD of a sample from a phenol production line based on cumene oxidation (equation 13) shows peaks for cumyl hydroperoxide (27), unreacted cumene, cumyl alcohol and acetophenone, whereas CLD shows only the 27 peak. The... 

---