NETCHEM Remote Access Laboratory Guide Quality assessment through internal methods for determination of Cr(VI) concentration in natural water In this exercise, you will: Learn about quality assessment concept as an important part of quality control program in an analytical laboratories. Learn about four internal methods of quality assessment for Cr(VI) determination. Gain experience in practical application of internal methods of quality assessment. ______________________________________________________________________________________________________ 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 A quality assurance program has two principal components: quality control and quality assessment. Quality control directives explain how an analysis should be properly conducted. (they do not indicate whether the system is under statistical control). Quality assessment component indicate if the system is under statistical control.   The goal of quality assessment is to detect when the system has moved out of statistical control; and, if possible, to suggest why a loss of statistical control has occurred so that corrective actions can be taken. The methods of quality assessment are divided into two categories: internal methods and external methods. The most useful methods for quality assessment are those that are coordinated by the laboratory and that provide the analyst with immediate feedback about the system’s state of statistical control. Internal methods of quality assessment are the analysis of duplicate samples, the analysis of blanks, the analysis of standard samples, and spike recoveries. ______________________________________________________________________________________________________ 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 Analysis of Duplicate Samples is related with the determination of the precision of an analysis. The duplicate samples are taken from a single gross sample (also called a split sample). The results from the duplicate samples, X1 and X2, are evaluated by determining the difference, D, or the relative difference, Dr, between the samples D = X1 – X2 and comparing the results with accepted given values.   The Analysis of Blanks The most common blank is a method, or reagent blank, in which an analyte-free sample, usually distilled water, is carried through the analysis using the same reagents, glassware, and instrumentation. This is to identify and correct systematic errors due to impurities in the reagents and contamination in the glassware and instrumentation. ______________________________________________________________________________________________________ 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 Analysis of Standards The analysis of a standard containing a known concentration of analyte also can be used (in the absence of a standard reference material in the lab), to monitor a system’s state of statistical control. The method is verified by periodically analyzing one of the calibration standards.   Spike Recoveries One of the most important quality assessment tools is the recovery of a known addition, or spike, of analyte to a blank or a sample. To determine a spike recovery, the sample is split into two portions, and a known amount of a standard solution of the analyte is added to one portion. The concentration of the analyte is determined for both the spiked, Cm+add, and unspiked Cm portions, I, and the percent recovery, %R, is calculated as %𝑅= 𝐶 𝑚+𝑎𝑑𝑑 − 𝐶 𝑚 𝐴 𝑥100 where A is the concentration of the analyte added to the spiked portion. ______________________________________________________________________________________________________ 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 Hexavalent chromium compounds are genotoxic carcinogens. Chromate (a typical form of chromium(VI) at neutral pH) is transported into cells where is reduced first to pentavalent chromium (chromium(V)) then to trivalent chromium (chromium(III)) without the aid of any enzymes. The resultant chromium(III) forms stable complexes with nucleic acids and proteins. This causes strand breaks and Cr-DNA adducts which are responsible for mutagenic damage. The correlations between the occurrence of human diseases and abnormal accumulations of Cr(VI) has led to the development of several analytical methods for it’s determination. One of the techniques used to determine specifically Cr(VI) in natural water is Sf UV-Vis. This method is based on the absorbance (λ=540nm), of the red- violet complex that chromium (VI) forms with diphenyl carbazide in acidic medium. The interaction is very sensitive, ε=40000(g/l)-1cm-1. For the total chromium determination is required the oxidation of all possible states of Cr into Cr(VI). ______________________________________________________________________________________________________ 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.

Material For this lab exercise, you will need the following material: Equipment Spectrophotometer UV VIS Glass cuvette Computer connected to Sf UV VIS ______________________________________________________________________________________________________ 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.

Assessories Reagents Volumetric flask 50 mL. Mikropipet (100-1000μL). Pipettes (2ml) Reagents Stndard solution of Cr(VI), 10mg/L Phosphoric acid solution, H3PO4 1:1 Acetone solution of diphenylcarbazide, 0,25% ______________________________________________________________________________________________________ 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.

Dilute all solutions up to 50ml Procedure: 1. Prepare the standard solutions of Cr(VI) to create the calibration graph according to the data in the following table: Use solution1 for blank corrections before the measurements and measure the absorbance (λ=540nm) of each standard solutions and fill the table using glass cuvette. Create the calibration graph in an excel sheet and find: Equation of the normal calibration curve, Sensitivity and Correlation coefficient solution reagent 1 2 3 4 5 100mg/L Cr(VI) (ml) 0.1 0.3 0.6 0.9 H3PO4 1:1 (ml) 0.25% DK (ml) Dilute all solutions up to 50ml C (mg/L) 0.2 1.2 1.8 A (λ=540nm) ______________________________________________________________________________________________________ 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: 2. Preparation of sample solutions for quality assessment of Cr(VI) determination. Analysis of Duplicate Samples Two aliquots from the sample are needed. Add 5 ml sample in each two 50ml volumetric flask and proceed in the same way as with standard solutions: add 3ml H2SO4 1:1 and 2 ml DK 25% and dilute with distilled water up to 50ml. Measure the absorbance in λ=540nm using the blank corrections first. Calculate the concentration of Cr(VI) in both sample solutions and find the difference, D: 𝐷=𝐶1−𝐶2 and the relative difference Dr: 𝐷 𝑟 = 𝐷 𝐶1+𝐶2 2 𝑥100 Consult the results with proper statistical table in the textbook or discus with professor. ______________________________________________________________________________________________________ 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: ii) Spike Recoveries To determine a spike recovery, two diluted sample solutions are needed. Introduce in each 50 ml volumetric flasks 3 ml sample. In one of them add 0.3ml standard solution of Cr(VI) 100mg/L and than proceed in the same way as with standard solutions: add 3ml H2SO4 1:1 and 2 ml DK 25% and dilute up to 50ml. Using calibration graph (equation of linear calibration), determine the concentrations of Cr(VI) in the unspiked sample (Cm) and in the spiked sample (C(m+add)), and calculate the percent recovery, %R as: %𝑅= 𝐶 𝑚+𝑎𝑑𝑑 − 𝐶 𝑚 𝐴 𝑥100 where A is the concentration of the Cr(VI) added to the spiked portion. Consult the results with proper statistical table in the textbook or discus with professor. ______________________________________________________________________________________________________ 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.

______________________________________________________________________________________________________ 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 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: Majlinda Vasjari Date of creation: September, 2018. 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.