Course: Methodology of instrumental analysis of water and air NETCHEM Comparison of extraction techniques (SPE and LLE) for the detection of PAH in water by HPLC Author: PhD Ivana Mihajlović, assistant professor Technical processing: Nikola Maoduš Institution: University of Novi Sad, Faculty of Technical Sciences, Department of Environmental Engineering, Trg Dositeja Obradovića 6, 21000 Novi Sad Course: Methodology of instrumental analysis of water and air ______________________________________________________________________________________________________ This project has been funded with support from the European Commission. This publication reflects the views only of the authors, and the Commission cannot be held responsible for any use which may be made of the information contained therein.

Background Educational objectives 1.To learn how to prepare sample by liquid-liquid extraction (LLE) and solid-phase extraction (SPE) for HPLC analysis 2. To develop a method for HPLC analysis 3. To analyze HPLC chromatograms 4. To master skills in identifying PAHs from the water sample 5. To gain knowledge on the influence of sample preparation techniques to the results of PAH analysis

Equipment and materials Chemical substances: Dichloromethane (HPLC grade), Acetonitrile (HPLC grade), Methanol (HPLC grade), Sodium sulphate (anhydrous) Distilled water (miliQ quality) Internal standard (phenanthrene d-10) EPA PAH Mix A Laboratory equipment and consumables: Digester, Kuderna-Danish apparatus (KD), Water bath, Separation funnel (2 L), Glass beakers (100 mL), Spoon, Glass vials (2 mL and 4 mL), Stands and clamps, Automatic pipette (5 mL and 1000 μL), Graduated cylinders (50 mL), Volumetric flasks (1 L) Cartridges C18 for SPE (500 mg/6 mL) SPE Vacuum Manifold SPE large volume sampler Vacuum pump Personal Protection Equipment: Eyeshields, Faceshields, Gloves, Complete suit protecting against chemicals, Respirator filter or respiratory protective device ______________________________________________________________________________________________________ This project has been funded with support from the European Commission. This publication reflects the views only of the authors, and the Commission cannot be held responsible for any use which may be made of the information contained therein.

Procedure: Liquid-liquid extraction: Spike the water sample with EPA PAH mix A. Spike the water sample with internal standard solution (phenathrene d-10). Shake the volumetric flask to homogenize the sample. Transfer prepared sample into the separation funnel and add 20 mL of solvent (dichloromethane). Shake the separation funnel for 20 minutes. Separate the extract (solvent) to glass beaker. Repeat extraction procedure with 20 mL of dichloromethane. Add 1 spoon (approximately 5g) of anhydrous sodium sulphate to remove water from the extract. Transfer separated extract to the Kuderna-Danish apparatus. Evaporate the extract to dryness. Dissolve the extract with 1 mL of acetonitrile. Transfer the extract to vial for HPLC analysis. ______________________________________________________________________________________________________ This project has been funded with support from the European Commission. This publication reflects the views only of the authors, and the Commission cannot be held responsible for any use which may be made of the information contained therein.

Procedure: Solid-phase extraction: Spike the water sample with EPA PAH mix A. Spike the water sample with internal standard (IS) solution (phenathrene d-10). Shake the volumetric flask to homogenize the sample. Place SPE cartridges C18 on vacuum manifold. Condition the cartridge with 5 mL of dichloromethane, 5 mL of methanol and 5 mL of distilled water. Be careful not to dry cartridge! Load the water sample using SPE Large Volume Sampler. Be careful not to dry cartridge! Rinse the cartridge with 3 ml of methanol/water mixture (1:1, v/v). Dry the cartridge using a vacuum for 20 minutes. Elute the cartridge in vial with 4 ml of dichloromethane. Evaporate the extract in a gentle flow of nitrogen to dryness. Transfer the sample to vial with 1 mL of acetonitrile for HPLC analysis. ______________________________________________________________________________________________________ This project has been funded with support from the European Commission. This publication reflects the views only of the authors, and the Commission cannot be held responsible for any use which may be made of the information contained therein.

High-performance liquid chromatography(HPLC) analysis Set the HPLC parameters for PAH analysis: Method Flow Stop time Gradient Time (min) A % B % Mobile phases Wavelength (nm) PAH 0.9 mL/min 32 min 0.00 55 45 02.00 35 65 10.00 15 85 20.00 0 100 27.00 0 100 29.00 55 45 A – ultrapure water B – acetonitrile (ACN) A: 215 B: 255 C: 275 D: 280 E: 230 F: 254 Column : Zorbax Extend C18 (Agilent Technologies) Size 4.6 x 150 mm Particle size 5-Micron Temperature: 35 ◦C Size of sample loop - 900 µL ______________________________________________________________________________________________________ This project has been funded with support from the European Commission. This publication reflects the views only of the authors, and the Commission cannot be held responsible for any use which may be made of the information contained therein.

Data analysis Analyze standard solutions (PAH mix A) and create calibration curve (area of PAH standard/area of IS vs. conc. of PAH standard/conc. of IS) for quantification of PAH concentrations. Calculate recoveries of compunds extracted by LLE and SPE. Discuss results obtained by extraction of PAH in water using LLE and SPE. ______________________________________________________________________________________________________ This project has been funded with support from the European Commission. This publication reflects the views only of the authors, and the Commission cannot be held responsible for any use which may be made of the information contained therein.

Prerequisites Students should: familiarize themselves with basics of HPLC technique familiarize themselves with Team Viewer software (a guide is provided at NETCHEM page), see materials uploaded at the course's Moodle page (http://mdl.netchem.ac.rs/course/view.php?id=24)

Remote access classroom-laboratory   DESCRIPTION OF REMOTE ACCESS NETCHEM COMMUNICATION SIDES (NOTE: NETCHEM Communication is defined as event that involves all kinds of internet interactions (in real time and not in real time) between participants via devices (PCs, laptops, tablets and mobile phones)) host side (NOTE: Host side of NETCHEM Communication is defined as PC who invites other users to join the session) Participant’s PC in the classroom guest side (NOTE: Guest side of NETCHEM Communication is defined as PC who joins the invitation to session) Participant’s PC in the laborotory COMMUNICATION SOFWARE Team Viewer Meeting: No Remote control: Yes Meeting and Remote control simultaneously: No Skype Call 1:1: No Conference Call: Yes COMUNICATION HARDWARE on host side 1 PC for each participant on guest side 1 PC, headset with microphone and camera INFORMATION EXCHANGE TYPE Educational (one side is dominantly receptive) Yes Place of Educator participant: guest side Number of educator(s): 1 Place of student participant: host side Number of student participant(s): up to 5 Consultative (two sides are equal in giving-receiving information) Number of host side participant(s): Number of guest side participant(s):

Remote Access Connection Instructions What makes these labs different and unique from other classroom experiments is that we have incorporated a section in each activity to remotely characterize your samples from your classroom. Request a remote lab session specifying information such as: the day, the time, and the instrument you are interested in using by visiting our web site: http://netchem.ac.rs/remote-access You will see the list of partners with the instruments provided to chose from. You will be contacted by a Remote Access staff member to set up a test run to ensure you are set up properly and have the required infrastructure. Send samples or verify the in-house sample you would like us to prepare and load for characterization. Send your samples to the Remote Access center that you chose on your request. There are two communications soft-ware packages, that will allow us to communicate instructions and answer questions during the session. - TeamViewer: You can obtain a free download at: https://www.teamviewer.com/en/index.aspx - Skype ______________________________________________________________________________________________________ This project has been funded with support from the European Commission. This publication reflects the views only of the authors, and the Commission cannot be held responsible for any use which may be made of the information contained therein.

Remote Access Connection Instructions You will need: Computer with administrator access to install plug-ins and software An internet connection Speakers Microphone Projector connected to the same computer Web browser (Firefox preferred) During the test run you can refer to this guide to perform the following steps, but it’s very important that you only proceed with these steps during your scheduled times. You may interfere with other remote sessions and potentially damage equipment if you log in at other times. Open and logon to your Zoom/Team-viewer account. You will be given the access code to enter at the time of your test and then again during the remote session. If you are using the Zoom software, Remote Access staff will give you the access code. If you are using the Team-viewer software, Remote Access staff will give you the ID & password. You should soon see the Remote Access desktop and at this point you can interact with the icons on the screen as if it were your desktop. Switch to full screen mode by selecting the maximize screen option in the top right corner of the screen. Upon completion of the session, move your mouse to the top right corner of the screen, and click on the X to disconnect the remote session. It will ask if you want to end the remote session. Click Yes. ______________________________________________________________________________________________________ This project has been funded with support from the European Commission. This publication reflects the views only of the authors, and the Commission cannot be held responsible for any use which may be made of the information contained therein.

Author, Editor and Referee References This remote access laboratory was created thanks to work done primarily at University of Niš. Contributors to this material were: dr Ivana Mihajlović Refereeing of this material was done by: _____________________ Editing into NETCHEM Format and onto NETCHEM platform was completed by: ______________ ______________________________________________________________________________________________________ This project has been funded with support from the European Commission. This publication reflects the views only of the authors, and the Commission cannot be held responsible for any use which may be made of the information contained therein.

References and Supplemental Material The NETCHEM platform was established at the University of Nis in 2016-2019 through the Erasmus Programme. Please contact a NETCHEM representatives at your institution or visit our website for an expanded contact list. The work included had been led by the NETCHEM staff at your institution. ______________________________________________________________________________________________________ This project has been funded with support from the European Commission. This publication reflects the views only of the authors, and the Commission cannot be held responsible for any use which may be made of the information contained therein.