for SolEx TM HRP Cartridges SolEx TM HRP RSLC Columns

for SolEx TM HRP Cartridges SolEx TM HRP RSLC Columns

Product Manual for SolEx TM HRP Cartridges and SolEx TM HRP RSLC Columns Page 1 of 15 Product Manual for SolEx TM HRP Cartridges SolEx HRP (P/N 074400) SolEx TM HRP RSLC Columns SolEx HRP RSLC (P/N 075590) 2011 Dionex Corporation

Product Manual for SolEx TM HRP Cartridges and SolEx TM HRP RSLC Columns Page 2 of 15 TABLE OF CONTENTS SECTION 1 INTRODUCTION... 3 SECTION 2 OVERVIEW AND USAGE... 4 2.1. Overview...4 2.2. System Configuration and experimental workflow...5 2.3. Use of SPE Cartridges...6 2.4. Condition the SPE Cartridge / Column...7 2.5. Storage...7 2.6. Standard SolEx HRP Use Conditions...7 2.6.1. Online SPE and separation of phenols... 7 2.6.2. Online SPE and separation of endocrine disruptive compounds... 9 2.6.3. Online SPE and separation of triclosan in soapwater... 11 SECTION 3 Appendix A. Example Chromeleon Program, Dual Injection Valves in UltiMate-3000 DC Column Compartment with AS-HV... 12

Product Manual for SolEx TM HRP Cartridges and SolEx TM HRP RSLC Columns Page 3 of 15 SECTION 1 INTRODUCTION Water contaminants, including pesticides, antibiotics, pharmaceuticals, etc., are of great concern to society and must be monitored. Method detection limits (MDLs) of liquid chromatography (LC) techniques employing direct injection of samples are too high for the detection of the low levels allowed in natural waters. Therefore, water samples often require preconcentration before analysis. Solid-phase extraction (SPE) is one of the most important techniques for sample enrichment because it overcomes many of the disadvantages of liquid-liquid extraction. It is an established sample preparation technique used for sample extraction, analyte enrichment, and sample clean-up. In off-line SPE, preparing individual samples is time consuming and a new SPE cartridge must be used for each sample. Use of SPE in an online mode allows many samples to be processed without offline conditioning of the SPE device. Several of the most popular stationary phases commonly used in SPE devices contain materials that can retain organic species via adsorption. Very hydrophobic phases have the problem of de-wetting when large amounts of water samples are passed over them. This process also destabilizes the stationary phase bed so that efficiency is decreased. In addition very polar organic species do not retain well. The SolEx HRP phase is designed for fast and easy pre-concentration of contaminants prior to analysis. The SPE phase is comprised of divinylbenzene polymer with a hydrophilic bonded layer that provides a stable resin bed with no de-wetting even with 100% aqueous mobile phases. This resin shows excellent retention properties from polar to more hydrophobic analytes. This phase is provided in two 20 x 2.1 mm I.D. formats: a cartridge for HPLC system with pressures up to 600 bar and a RSLC column for pressures up to 800 bar. The convenient cartridge style is used in a holder and easily exchanged when necessary. The RSLC column style uses stainless steel hardware and is suitable for high pressure RSLC applications. RSLC Column

Product Manual for SolEx TM HRP Cartridges and SolEx TM HRP RSLC Columns Page 4 of 15 SECTION 2 OVERVIEW AND USAGE 2.1. Overview This manual describes recommended automated SPE procedures and examples of use for SPE cartridge and RSLC SPE column. A few minutes of pre-washing can be accomplished in the LC system on startup so manual cleaning is not necessary. Cartridge/ Columns Functional Group(s) Table 1 Recommended Cartridge Applications Mode of Use Typical example Applications SPE Hydrophilic Divinylbenzene Reversed Phase π-π interaction Concentration of pharmaceuticals and pesticides Table 2 SPE Products Characteristics Cartridge Type 2.1 x 20 mm SST/PEEK Column Type 2.1 x 20 mm SST Particle size 12-14 µm Resin amount 150 mg ph stability 0-14 Table 3 Operating Conditions Parameter Range Preferred Flow rate 0.2-5 ml/min 2 ml/min Solvent content 0-100% 0-100% Solvent type Acetonitrile, methanol, other ACN, MeOH tested Cartridge Backpressure 0-600 bar Up to 600 bar Columns Backpressure 0-1000 bar Up to 800 bar

Product Manual for SolEx TM HRP Cartridges and SolEx TM HRP RSLC Columns Page 5 of 15 2.2. System Configuration and experimental workflow Optimum performance is achieved when the SolEx HRP cartridge / column is used in the valve, and the sample is loaded directly onto the cartridge / column followed by elution of the retained analytes onto an analytical column. The SolEx HRP can be used with one valve and an autosampler (i.e. WPS autosampler with an injection valve) or with two independently controlled valves with an AS-DV or AS-HV high volume autosampler (no switching valve, but with an internal peristaltic pump to load sample to the injection loop in the pull mode). The configuration shown in Figure 1 can be accomplished on any system with two independently controlled two-position, six-port valves (two valves both from the column compartment or one from the column compartment, one from the autosampler) and two pumps; sample volume is controlled by the autosampler and an injection loop. Figure 1 One valve and an autosampler (with an injection valve) control of sample volume (sample loading and concentration) or Column

Product Manual for SolEx TM HRP Cartridges and SolEx TM HRP RSLC Columns Page 6 of 15 Figure 2 Flow scheme for automated online SPE-LC Sample Loading Sample Transfer Separation And Concentration Figure 2 shows the typical setup for on-line solid phase extraction liquid chromatography (SPE-LC). The experimental workflow contains the following steps: 1) Sample loading and concentration (left): the sample is injected and transferred to the SPE column with the loading pump. The sample matrix is flushed to waste, while the analytes are retained on the SPE column. At the same time the analytical LC column is equilibrated with the analytical pump. 2) Sample transfer (middle): After the sample matrix is flushed to waste, the six-port valve is switched to a position that couples the SPE cartridge / column with the analytical LC column, and the analytes are transferred. 3) Separation (right): After the transfer, the six-port valve is returned to its original position While the analytes are separated on the analytical LC column, the SPE cartridge /column can be re-equilibrated with the mobile phase on the loading pump. Note that flow across the SPE cartridge / column to the analytical column should be in the reverse direction to the flow across the SPE column when it is being loaded. This minimizes peak broadening in the analysis. 2.3. Use of SPE Cartridges SolEx online SPE phase is designed for fast and easy preconcentration of contaminants from water sample prior to HPLC analysis. The cartridge / column is placed in the valve to automatically concentrate the analytes of interest and eliminate the matrix. SolEx HRP cartridges / column generates less then 100psi backpressure with a flow rate of 1mL/min of DI water. It can be used with wide ph compatibility (0-14) and wide organic solvent content (0-100%). SolEx HRP cartridges/columns have been tested with up to 1L sample volume.

Product Manual for SolEx TM HRP Cartridges and SolEx TM HRP RSLC Columns Page 7 of 15 2.4. Condition the SPE Cartridge / Column The SPE cartridge / column is shipped in DI water. When a new cartridge / column is used for the first time, it should be washed thoroughly with the mobile phase (e.g., for 10 min at 1mL/min). When switching to a new mobile phase, make sure that the new mobile phase is compatible with the previous mobile phase in the cartridge / column to avoid clogging due to precipitation. The cartridge / column should be fully conditioned before any injection is made. 2.5. Storage Both cartridges and columns can be stored in the 1/99 to 100/0 v/v DI water/acetonitrile (or methanol) for long term storage. 2.6. Standard SolEx HRP Use Conditions The SolEx HRP SPE cartridge format is a 2.1 x 20 mm I.D stainless steel cartridge body used with a cartridge holder for easy installation in an HPLC system. The cartridges contain approximately 0.15 g of resin. Depending on the exact chemistry and types of samples treated, some cartridges can be regenerated. The SolEx HRP SPE column format is a 2.1 x 20 mm I.D stainless steel (no holder is required). The column contains approximately 0.15 g of resin. Depending on the exact chemistry and types of samples treated, some columns can be regenerated. 2.6.1. Online SPE and separation of phenols The SolEx HRP cartridge/ column can be used for a wide variety of samples. For example, the presence of phenols commonly used pharmaceuticals and pesticides in water supply are of high interest and methods are in development to provide highthroughput screening using SPE-LC-MS/MS methodologies. Figure 1 demonstrates online SPE and separation of phenols in deionized water. Figure 1 Online SPE and separation of eleven common phenols in deionized water Peaks: µg/l 1. Phenol 10 2. 4-nitrophenol 50 3. 2-chlorophenol 10 4. 2-nitrophenol 10 5. 2,4-dimethylphenol 10 6. 2,4-dinitrophenol 30 7. 4-chloro-3-methylphenol 50 8. 2,4-dichlorophenol 10 9. 4,6-dinitro-2-methylphenol 50 10. 2,4,6-trichlorophenol 30 11. Pentachlorophenol 50

Product Manual for SolEx TM HRP Cartridges and SolEx TM HRP RSLC Columns Page 8 of 15 Chromatographic conditions: SPE column: SPE-HRP cartridge, 14µm, 2.1X20mm Analytical Column: Acclaim PA, 3µm, 2.1X150mm Flow rate: 210µL/min Injection Volume: 10mL Temperature: 25 C Detection: UV, 280nm, MS Eluent: A: 25mM HAc/NH 4 Ac (1.45:1, v/v), B: Acetonitrile Gradient: Time A% B% 0.0 70 30 13.0 70 30 28.0 25 75 30.0 25 75 The method described in Figure 1 was applied to Sunnyvale tap water samples extracts, which demonstrated good recovery ranging from 5ng to 225ng as shown in Table 4. % Recovery COMPOUNDS 500ng/L 10ug/L 22.5ug/L ESI-MS/MS ESI-MS/MS, UV UV 2,4-dinitrophenol 88.3% 91.4%* 92.5% phenol Not detected 112.6% 86.5% 4-nitrophenol 100.0% 96.6% 92.8% 2-methyl-4,6-dinitrophenol 81.8% 110.0%* 98.0% 2-chlorophenol Not detected 110.7% 102.8% 2-nitrophenol 112.9% 102.7% 103.0% 2,4-dimethylphenol Not detected 95.1% 94.3% 4-chloro-3-methylphenol 92.8% 99.7% 96.2% 2,4-dichlorophenol 111.7% 103.7% 95.1% 2,4,6-trichlorophenol 92.1% 113.0% 96.3% Pentachlorophenol 94.6%** 98.7%** 97.8% *determined by ESI-MS/MS **determined by Q1 only Table 4: Recovery of phenols in Sunnyvale tap water: recovery% calculated by comparing the area responses of phenols spiked in Sunnyvale tap water to the same sample spiked in deionized water.

Product Manual for SolEx TM HRP Cartridges and SolEx TM HRP RSLC Columns Page 9 of 15 2.6.2. Online SPE and separation of endocrine disruptive compounds Endocrine disruptive compounds in sea water are another environmental concern. It has been reported that there is global contamination of sea sand and sea water with the endocrine disruptor bisphenol A. Figure 2 shows the separation of bisphenol A, β-estradiol and estrone, using the SolEx HRP cartridge for easier preconcentration. Lower detection limit can be obtained using large injection volume. Figure 2 Online SPE-LC of endocrine disruptors in deionized water, 2% sodium chloride, and half moon bay seawater Peaks: µg/l 12. Bisphenol A 5 13. β-estradiol 5 14. Estrone 5 15. Chromatographic conditions: SPE column: Analytical Column: Flow rate: Injection Volume: Temperature: Detection: Eluent: SolEx HRP cartridge, 12µm, 2.1X20mm Acclaim PA, 3µm, 2.1X150mm 300µL/min 10mL 25 C UV, 210nm, A: water B: Acetonitrile C: 100mM ph 5.4 ammonium acetate

Product Manual for SolEx TM HRP Cartridges and SolEx TM HRP RSLC Columns Page 10 of 15 Gradient: Time A% B% C% 10.0 55 35 10 20.0 30 60 10 25.0 30 60 10 25.1 55 35 10 Recovery of these three endocrine disruptive compounds in 2% sodium chloride and seawater collected at half moon bay are calculated by comparing to the area responses on analytes in deionized water. Analyte, 5 ppb 2% NaCl Seawater Bisphenol A 98.01% 97.18% β-estradiol 93.81% 93.39% Estrone 103.28% 97.81% Table 5: Recovery of endocrine disruptive compounds in 2% sodium chloride and half moon bay seawater.

Product Manual for SolEx TM HRP Cartridges and SolEx TM HRP RSLC Columns Page 11 of 15 2.6.3. Online SPE and separation of triclosan in soapwater Triclosan is a powerful antibacterial and antifungal agent. It is an ingredient added to many consumer products to reduce or prevent bacterial contamination. It may be added to antibacterial soaps and body washes, toothpastes, and some cosmetics products regulated by the FDA. Triclosan safety is currently under review by the FDA. Based on recent studies that seem to indicate it causes endocrine disruption in the body and leads to the emergence of drug-resistant "super" bacteria. Figure 3 Online SPE-LC of triclosan in deionized water, 0.0005% sodium dodecylbenzene sulfonate, and 1/500 antibacterial hand soap contains 0.15% triclosan Chromatographic conditions: SPE column: Analytical Column: Flow rate: Injection Volume: Temperature: Detection: Eluent: SolEx HRP cartridge, 12µm, 2.1X20mm Acclaim PA, 3µm, 2.1X150mm 300µL/min 10mL 25 C UV, 210nm, A: water B: Acetonitrile C: 100mM ph 5.4 ammonium acetate Gradient: Time A% B% C% 0.0 40 50 10 10.0 15 75 10 15.0 15 75 10 15.1 40 50 10

Product Manual for SolEx TM HRP Cartridges and SolEx TM HRP RSLC Columns Page 12 of 15 SECTION 3 APPENDIX A. EXAMPLE CHROMELEON PROGRAM, DUAL INJECTION VALVES IN ULTIMATE-3000 DC COLUMN COMPARTMENT WITH AS-HV The following steps in a PGM will load a loop, perform the sample preparation, load the SPE cartridge, wash the SPE cartridge, transfer the analytes from SPE column to the analytical column, and start the analysis. Both valves are installed in an UltiMate-3000 TCC column compartment. Valve nomenclature follows the default conventions of the Chromeleon Server Configuration. Column_A.SystemPressure = "LoadingPump" Column_B.SystemPressure = "MicroPump" LoadingPump.Pressure.LowerLimit = 0 [psi] LoadingPump.Pressure.UpperLimit = 5076 [psi] LoadingPump.MaximumFlowRampDown = 500 [µl/min²] LoadingPump.MaximumFlowRampUp = 500 [µl/min²] LoadingPump.%A.Equate = "%A 2mM HCOOH" LoadingPump.%B.Equate = "%B ACN" LoadingPump.%C.Equate = "%C " MicroPump.Pressure.LowerLimit = 0 [psi] MicroPump.Pressure.UpperLimit = 5076 [psi] MicroPump.MaximumFlowRampDown = 500 [µl/min²] MicroPump.MaximumFlowRampUp = 500 [µl/min²] MicroPump.%A.Equate = "%A 25mM HAc/NH4AC" MicroPump.%B.Equate = "%B ACN" MicroPump.%C.Equate = "%C" NeedleHeight = RinseVolume = Sampler.Flow = Sampler.Pressure.LowerLimit = Sampler.Pressure.UpperLimit = 10 [mm] 24.00 [ml] 2.00 [ml/min] 0 [psi] 3000 [psi] Data_Collection_Rate = 10.00 [Hz] Rise_Time = 2.00 [s] Range = 6 Smoothing = None UV_VIS_1.Wavelength = 2 UV_VIS_1.Bandwidth = UV_VIS_1.RefWavelength = Off UV_VIS_1.RefBandwidth = UV_VIS_2.Wavelength = 254 [nm] UV_VIS_2.Bandwidth = UV_VIS_2.RefWavelength = Off UV_VIS_2.RefBandwidth = UV_VIS_3.Wavelength = 280 [nm] UV_VIS_3.Bandwidth = UV_VIS_3.RefWavelength = Off UV_VIS_3.RefBandwidth = UV_VIS_4.Wavelength = 200 [nm] UV_VIS_4.Bandwidth =

Product Manual for SolEx TM HRP Cartridges and SolEx TM HRP RSLC Columns Page 13 of 15 UV_VIS_4.RefWavelength = Off UV_VIS_4.RefBandwidth = UV_VIS_5.Wavelength = 225 [nm] UV_VIS_5.Bandwidth = UV_VIS_5.RefWavelength = Off UV_VIS_5.RefBandwidth = 50 [nm] 3DFIELD.RefWavelength = 220 [nm] 3DFIELD.RefBandwidth = 50 [nm] ValveLeft = 6_1 ; this valving directs loading water through the injection loop, the SPE cartridge to waste; ValveRight = 6_1 ; this valving places SPE cartridge on line with eluent and the analytical column; 3DFIELD.MinWavelength = 3DFIELD.MaxWavelength = 3DFIELD.BunchWidth = TCC3x00_RELAY_2.State 190 [nm] 300 [nm] Off 0.000 Autozero Wait AZ_Done MicroPump.Flow = 210 [µl/min] MicroPump.%B = 30.0 [%] LoadingPump.Flow = 2000 [µl/min] LoadingPump.%B = 0.0 [%] Wait ColumnOven.Ready and MS.Ready Inject ;this Inject command of AS-HV autosampler includes the rinse of autosampler and injection loop with DI water, pumps sample through the autosampler to the injection loop using the internal peristaltic pump of AS-HV autosampler Position = GotoPosition R0 ;MS Data Acquisition On TCC3x00_RELAY_2.Duration = 2.00 MicroPump.Flow = 210 [µl/min] MicroPump.%B = 30.0 [%] LoadingPump.Flow = 2000 [µl/min] LoadingPump.%B = 0.0 [%] 0.100 ValveLeft = 1_2

Product Manual for SolEx TM HRP Cartridges and SolEx TM HRP RSLC Columns Page 14 of 15 ; this valving switch directs loading water from the loading pump through the injection loop, the SPE cartridge to waste; this step transfer the sample from injection loop to the SPE cartridge, pre-concentrate and remove the matrix; 8.000 LoadingPump.Flow = 2000 [µl/min] LoadingPump.%B = 0.0 [%] UV_VIS_1.AcqOn UV_VIS_2.AcqOn UV_VIS_3.AcqOn UV_VIS_4.AcqOn UV_VIS_5.AcqOn 3DFIELD.AcqOn ValveLeft = 6_1 ValveRight = 1_2 ; this right valving switch directs loading eluent from the micro pump through the SPE cartridge to the analytical column; this step transfer the sample from SPE cartridge to the analytical column for separation; 8.100 PrepareNextSample 10.000 LoadingPump.Flow = 2000 [µl/min] LoadingPump.%B = 90.0 [%] 13.000 MicroPump.Flow = 210 [µl/min] MicroPump.%B = 30.0 [%] ValveRight = 6_1 ; this valving switch directs eluent from the micro pump through the analytical column; this step switch the SPE cartridge offline for cleanup; 15.000 LoadingPump.Flow = 2000 [µl/min] LoadingPump.%B = 90.0 [%] 17.000 LoadingPump.Flow = 2000 [µl/min] LoadingPump.%B = 0.0 [%] 22.000 LoadingPump.Flow = 2000 [µl/min]

Product Manual for SolEx TM HRP Cartridges and SolEx TM HRP RSLC Columns Page 15 of 15 LoadingPump.%B = 0.0 [%] 24.000 LoadingPump.Flow = 210 [µl/min] LoadingPump.%B = 0.0 [%] 28.000 MicroPump.Flow = 210 [µl/min] MicroPump.%B = 75.0 [%] 30.000 LoadingPump.Flow = 2000 [µl/min] LoadingPump.%B = 0.0 [%] MicroPump.Flow = 210 [µl/min] MicroPump.%B = 75.0 [%] ;MS Data Acquisition Off UV_VIS_1.AcqOff UV_VIS_2.AcqOff UV_VIS_3.AcqOff UV_VIS_4.AcqOff UV_VIS_5.AcqOff 3DFIELD.AcqOff End