BASICS OF ANALYTICAL CHEMISTRY AND CHEMICAL EQUILIBRIA

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3 BASICS OF ANALYTICAL CHEMISTRY AND CHEMICAL EQUILIBRIA

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5 BASICS OF ANALYTICAL CHEMISTRY AND CHEMICAL EQUILIBRIA BRIAN M. TISSUE Virginia Tech Department of Chemistry Blacksburg, VA

6 Cover Design: Wiley Cover Illustration: Courtesy of Brian M. Tissue Copyright 2013 by John Wiley & Sons, Inc. All rights reserved. Published by John Wiley & Sons, Inc., Hoboken, New Jersey. Published simultaneously in Canada. No part of this publication may be reproduced, stored in a retrieval system, or transmitted in any form or by any means, electronic, mechanical, photocopying, recording, scanning, or otherwise, except as permitted under Section 107 or 108 of the 1976 United States Copyright Act, without either the prior written permission of the Publisher, or authorization through payment of the appropriate per-copy fee to the Copyright Clearance Center, Inc., 222 Rosewood Drive, Danvers, MA 01923, (978) , fax (978) , or on the web at Requests to the Publisher for permission should be addressed to the Permissions Department, John Wiley & Sons, Inc., 111 River Street, Hoboken, NJ 07030, (201) , fax (201) , or online at Limit of Liability/Disclaimer of Warranty: While the publisher and author have used their best efforts in preparing this book, they make no representations or warranties with respect to the accuracy or completeness of the contents of this book and specifically disclaim any implied warranties of merchantability or fitness for a particular purpose. No warranty may be created or extended by sales representatives or written sales materials. The advice and strategies contained herein may not be suitable for your situation. You should consult with a professional where appropriate. Neither the publisher nor author shall be liable for any loss of profit or any other commercial damages, including but not limited to special, incidental, consequential, or other damages. For general information on our other products and services or for technical support, please contact our Customer Care Department within the United States at (800) , outside the United States at (317) or fax (317) Wiley also publishes its books in a variety of electronic formats. Some content that appears in print may not be available in electronic formats. For more information about Wiley products, visit our web site at Library of Congress Cataloging-in-Publication Data: Tissue, Brian M., 1961 Basics of analytical chemistry and chemical equilibria / Brian M. Tissue. pages cm Published simultaneously in Canada Title page verso. Includes index. ISBN (cloth) 1. Chemistry, Analytic Textbooks. 2. Chemical equilibrium Textbooks. I. Title. QD75.22.T dc Printed in the United States of America

7 CONTENTS PREFACE ix I QUANTITATIVE ANALYSIS USING REACTIONS THAT GO TO COMPLETION 1 1 MAKING MEASUREMENTS Introduction / GLP and a Few Other Important Acronyms / Precision and Random Error / Discarding a Suspected Outlier / Calibration / Maintaining Accurate Results / 43 Practice Exercises / 48 2 SAMPLE PREPARATION, EXTRACTIONS, AND CHROMATOGRAPHY Sampling and Control Samples / Sample Preparation / Solvents and Solutions / Introduction to Solubility / Extraction / Stationary Phases / 82 v

8 vi CONTENTS 2.7 Solid-Phase Extraction (SPE) / Introduction to Chromatography / Immunoassays / 96 Practice Exercises / 97 3 CLASSICAL METHODS Introduction / Review of Chemical Reactions / Reactions in Aqueous Solution / Gravimetry / Titration / Titration Curves / Coulometry / 134 Practice Exercises / MOLECULAR SPECTROSCOPY Introduction / Properties of EM Radiation / Electromagnetic Spectrum / Spectroscopic Transitions / UV/Vis Absorption Spectroscopy / UV/Vis Instrumentation / Beer Lambert Law / Molecular Fluorescence / 167 Practice Exercises / 171 II REACTIONS THAT DO NOT GO TO COMPLETION. EQUILIBRIA IN AQUEOUS SOLUTIONS ACID BASE EQUILIBRIA AND ACTIVITY Acids and Bases / Weak Acids and Weak Bases / Water and K w / Acid Strength / The Concept of Activity / Acid Base Equilibrium Calculations / 212 Practice Exercises / 218

9 CONTENTS vii 6 BUFFER SOLUTIONS AND POLYPROTIC ACIDS Buffer Solutions / Alpha Fraction Plots / Weak Acid Titration Curve / Polyprotic Acids / 233 Practice Exercises / COMPLEXATION AND PRECIPITATION EQUILIBRIA Complex Terminology / Complex Equilibria / Competing Equilibria / Stepwise Complexation / Precipitate Equilibrium / Molar Solubility / Precipitation and Competing Equilibria / 282 Practice Exercises / 287 III INSTRUMENTAL METHODS AND ANALYTICAL SEPARATIONS ELECTROANALYTICAL CHEMISTRY Introduction / Standard Reduction Potentials / Using Half Reactions / Background on Spontaneous Reactions and Equilibrium / Reaction Energies, Voltages, and the Nernst Equation / Electrochemical Cells / Potentiometry / Ion-Selective Electrodes (ISE) / Voltammetry / 328 Practice Exercises / ATOMIC SPECTROMETRY Atomization / Atomic Absorption Spectrometry (AAS) / Atomic Emission Spectrometry (AES) / Introduction to Mass Spectrometry (MS) / 357

10 viii CONTENTS 9.5 ICP-MS Measurements / Summary / 362 Practice Exercises / ANALYTICAL SEPARATIONS Thin-Layer Chromatography / Chromatogram Terminology / Column Selection / High Performance Liquid Chromatography / Gas Chromatography / Molecular Mass Spectrometry / Electrophoresis / 398 Practice Exercises / 402 INDEX 407

11 PREFACE This text will introduce you to analytical chemistry: the science of making quantitative measurements. Quantifying the individual components in a complex sample is an exercise in problem solving. An effective and efficient analyst will have expertise in sampling, sample processing, and method validation; the chemistry that can occur in a sample before and during analysis; selecting an appropriate analytical method; and proper record keeping, data analysis, and reporting of results. I do not attempt to be comprehensive in this text. Samples that require analysis are so diverse that it is not possible to describe every sample preparation protocol, separation method, and measurement technique. These details are contained in handbooks and method compilations, many of which are now accessible from online sources. This text emphasizes the fundamental chemical and physical concepts that underlie the analytical methods. With an understanding of the fundamental concepts, a scientist faced with a difficult analysis can apply the most appropriate techniques, identify when a particular problem cannot be solved with existing methods, and develop new analytical methods. The proficient analyst will also be alert to interferences and problems in analytical measurements and recognize when an answer might not be correct. I organize the discussion of the core principles of analytical chemistry into three parts: Part I: analytical concepts such as calibration and uncertainty, sample preparation, classical (wet-chemical) methods, and molecular UV/Vis spectroscopy ix

12 x PREFACE Part II: chemical equilibria involving acids, bases, complexes, and insoluble precipitates Part III: electroanalytical methods, atomic and mass spectrometry, and chromatographic separations The analytical methods in Part I rely on reactions that go to completion. Part II is a detailed treatment of chemical equilibria reactions in which reactants and products coexist. Equilibrium is critical to the functioning of many aspects of chemical, biochemical, and environmental systems. Part III describes the most common instrumental methods of analysis, illustrating many of the tools of the trade for making quantitative measurements. Even if your future career veers away from science, you will find the problem-solving and graphical data analysis skills developed in this text to be useful. Many of the topics in this text follow directly from first-year college chemistry. You will want access to a general chemistry text or online resource to refresh your memory of the principles that underlie the different types of reactions and analytical methods. The level of this text presumes that you know Basic math Basic chemistry Reaction types Algebra Exponential functions Calculating and plotting in a spreadsheet Predicting properties based on the periodic table The nature of chemical compounds Stoichiometry and balancing reactions Acid base Complexation Precipitation Reduction and oxidation (redox) The beginning of each chapter lists learning outcomes that serve as a brief outline to help categorize new material. After completing a chapter, make a concept map to help yourself see the big picture and underlying concepts. You will often encounter a repeat of concepts in the text. Making connections with prior material makes learning analytical concepts much easier. Treating every topic as something new becomes overwhelming. Each chapter contains sample calculations and practice exercises. I assume that your goal is success. Achieving success requires skills, and acquiring skills takes practice. Variables and constants are italicized to not be confused with other text. As much as possible, I use the conventions and terminology in the IUPAC

13 PREFACE xi Compendium of Chemical Terminology. 1 You will find other symbols in other books and resources, so use the context to decipher the differences. Relevant spreadsheets and links to useful resources are available at or Blacksburg, VA Brian M. Tissue May See IUPAC Compendium of Chemical Terminology the Gold Book. Available at Accessed 2013 Feb 13.

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15 PART I QUANTITATIVE ANALYSIS USING REACTIONS THAT GO TO COMPLETION

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17 CHAPTER 1 MAKING MEASUREMENTS Learning Outcomes Describe aspects of good laboratory practice (GLP). Use correct terms to describe analytical measurements and data. Calculate analyte concentration from measurement results. Use statistical formulas to express the precision of analytical measurements. Use calibration methods to obtain accurate results. 1.1 INTRODUCTION There are few areas in our modern life in which the quantity of substances is not important. Industries and government agencies spend substantial resources to determine and monitor the safe levels of chemicals in foods, pharmaceuticals, and the environment. Setting permissible levels of contaminants is based on quantitative results from toxicology studies, and raising or lowering a level has significant costs and consequences. Similarly, companies compete for sales by providing high quality goods at the lowest price. Optimizing industrial processes depends on making decisions and analyzing measurements correctly. You might not make many measurements yourself, but you may rely on data and quantitative results to make decisions. Everything from choosing one product over another based on a product label, voting for one candidate versus another Basics of Analytical Chemistry and Chemical Equilibria, First Edition. Brian M. Tissue John Wiley & Sons, Inc. Published 2013 by John Wiley & Sons, Inc. 3

18 4 MAKING MEASUREMENTS based on their views on environmental regulation, and to making life decisions such as beginning daily doses of a cholesterol-lowering drug. Think about the last time that you had a medical checkup. Did the doctor determine your health by just looking at you? Modern medicine relies on a variety of technological tools and clinical analyses. We all hope that the doctor and the clinical technicians analyzing our samples were paying attention when they took analytical chemistry! Why should we analyze things? Think about the last meal or snack that you ate. If it was a packaged item, the Nutrition Facts label shows the amount of fats, carbohydrates, protein, vitamins, and minerals in the item. I bet you prefer that the companies perform quality checks of the contents so that you get your money s worth. No doubt you also want an independent agency, say the FDA or USDA, 1 to check that there is not too much of a mineral, or contaminants such as Pb or rat poison, that could make the food unhealthy. Even for fresh food, we still rely on biological and chemical testing to ensure that the food is safe. On this point, we immediately come to the reality that not every single piece of food is tested. Ensuring a safe food supply relies on validated procedures to keep food safe as well as on inspections and tests to sample a representative amount of the food supply. When you make a measurement, or make a decision based on someone else s measurement, do you trust the value? This chapter introduces the terminology and statistical tools used to describe and assess quantitative results. Some of the details will be new to you, but they all fit into a framework for collecting and reporting quantitative measurements. Table 1.1 begins building our vocabulary of measurement science and data handling concepts by defining general terms. In quantitative analysis, we want a measurable detector signal that we can relate to an analyte concentration. Doing so can be quite involved, and Chapter 2 discusses various sample preparation methods to isolate an analyte from interferences so that it can be measured. What mechanisms are available to detect an analyte? I can think of only three general strategies to detect and quantitate analytes: measuring a physical property, using electromagnetic radiation, called spectroscopy, and measuring an electric charge or current. Table 1.2 lists some examples in each of these categories. We will discuss most of these methods, so do not worry if they are unfamiliar. Chapter 3 discusses classical methods that rely on physical measurements, Chapters 4 and 9 cover spectroscopic techniques, and Chapter 8 describes electroanalytical chemistry. 1 U.S. Food and Drug Administration and U.S. Department of Agriculture. See What FDA Regulates, Other countries and trade blocs have similar agencies.

19 INTRODUCTION 5 TABLE 1.1 Term Measurement Terms Definition Sample Sample Test portion Analyte Qualitative analysis Quantitative analysis Detector Signal Sensitivity Selectivity v. To collect one or more samples. n. Substance of interest, assumed to be representative of remaining substance that is not collected. May refer to an unprocessed field sample or to a laboratory sample that has undergone one or more sample preparation steps. Test portion is the preferred term for laboratory samples. n. A portion of a collected sample that is processed and measured. n. The chemical species to be identified or quantitated. It might exist as a pure substance or as one constituent of a multicomponent sample. n. Making measurements to determine the identity, structure, or physical properties of a substance. n. Making measurements to determine the amount of an analyte in a sample. n. Device that responds to the presence of analyte, usually generating an electrical output. n. The detector output that is displayed or recorded. n. The change in detector signal versus change in analyte concentration. n. The discrimination of an analyte versus other components in the sample. TABLE 1.2 Measurement Strategies Physical Property Spectroscopy Electric Charge or Current Mass or volume Absorption Electrical conductivity Density Emission Electrical potential, for example, ph meter Refractive index Scattering Voltammetry (reduction and oxidation current) Freezing point depression Mass spectrometry (ion current) Thermal conductivity Although I list only three general detection strategies, each of these general categories encompass a multitude of analytical techniques. For example, spectroscopic methods have been developed to use most of the electromagnetic spectrum, including X-ray, ultraviolet (UV), visible (Vis), infrared, and radio waves. The different regions of the electromagnetic spectrum interact with matter differently and provide different types of information. This text concentrates on quantitative methods for aqueous solution, but there are many other spectroscopic techniques for quantitative measurements of solids and to identify or to determine physical properties. These general categories vary in sensitivity and selectivity. Measurements based on a physical property are usually less sensitive than spectroscopic or charge-based instrumental methods. The methods based on physical methods are useful for fairly high concentrations of analytes and for preparing standards

20 6 MAKING MEASUREMENTS TABLE 1.3 SI Base Units Unit of Name Symbol Length Meter m Mass Kilogram kg Time Second s Electric current Ampere A Thermodynamic temperature Kelvin K Amount of substance Mole mol Luminous intensity Candela cd to calibrate instrumental methods. When coupled with separations, detectors based on physical methods are very important to be able to detect all analytes in the sample. Spectroscopic and electroanalytical methods can be extremely sensitive and selective for specific analytes. To choose among any one of these three general strategies for a given analytical problem depends on the nature of the analyte and the sample matrix. New analytical methods and instruments are developed continuously. The breadth of research and development in analytical chemistry is too extensive to convey through just a few examples. For an overview of current research topics in analytical sciences, browse the Technical or Preliminary Programs of upcoming analytical chemistry conferences. 2 Concentration Units Most of the quantitative methods that we will discuss are aimed toward determining the concentration of an analyte in a sample. Concentration is the quantity of one substance, the analyte divided by the total quantity of all substances in the sample. Concentration is different from an amount, and we often interconvert between the two. Table 1.3 lists the SI base units that are used to derive other units. 3 In addition to these units, we drop or add the prefixes listed in Table 1.4 to indicate numerical factors. Common units that we work with in addition to the mole and kg are mmol (millimole), mg (milligram), and g (gram). From the SI base units, we derive other useful units, and some are listed in Table 1.5. The SI unit for an amount of a substance is the mole. A mole is a fixed number of something. If you have one mole of fish, you have fish (that s a lot of fish). The mole is determined from the number of atoms in 12 g of carbon-12 ( 12 C). We cannot count individual atoms or molecules easily, so the physical means of measuring an amount is to determine a mass and convert it to 2 EAS (Eastern Analytical Symposium), FACSS (Federation of Analytical Chemistry and Spectroscopy Societies), or Pittcon (Pittsburgh Conference on Analytical Chemistry and Applied Spectroscopy), 3 SI is the abbreviation for The International System of Units. See /cuu/units/index.html for more information.

21 INTRODUCTION 7 TABLE 1.4 Common Numerical Prefixes Prefix Name Factor Prefix Name Factor p Pico k Kilo 10 3 n Nano 10 9 M Mega 10 6 μ Micro 10 6 G Giga 10 9 m Milli 10 3 T Tera TABLE 1.5 SI Derived Units Unit of Name Symbol Area Square meter m 2 Volume Cubic meter m 3 Volume Liter (=0.001 m 3 ) l Concentration Kilogram per cubic meter kg/m 3 Concentration Moles per liter M (mol/l) Density Mass per volume ρ (g/ml) Relative density (specific gravity) Ratio of a density to a reference d (unitless) moles. The unified atomic mass unit or atomic mass constant is 1/12 of the mass of a carbon-12 atom and has units of u (amu is common in older literature). For large biomolecules, the equivalent unit of Dalton (Da) is common. The atomic weight or standard atomic weight of an element is the relative mass to the unified atomic mass unit. Atomic weights are therefore relative, unitless quantities. Because most elements consist of multiple isotopes, the atomic weight is the weighted average based on the abundance of the different isotopes. We will ignore the variability of atomic weights in this course, but it can be significant for some light elements and enriched radioactive materials. We will also not show conversion of unitless atomic weights to proportionality factors when converting between amount and mass. For our calculations, we will use atomic or molecular formula weights with units of gram per mole (g/mol). As a final practical note, mass and weight are not the same thing. Mass is an intrinsic property of a substance, but weight is a force that is dependent on mass and gravity. Modern analytical balances can be calibrated to read mass accurately, so we will follow common usage and assume that any weights that we measure are equal to mass. Different types of analytical methods will measure concentration, volume, or mass. Similarly, different types of detectors respond to analytes in different ways, that is, respond to either the analyte concentration or the analyte amount. Part of the common language that we need are conventions for describing amounts and concentration in different orders of magnitudes. Table 1.6 lists a number of common units that are used for analytical results. 4 4 Note that parts per thousand is sometimes abbreviated as ppt in older literature, and should not be confused with parts per trillion.

22 8 MAKING MEASUREMENTS TABLE 1.6 Common Concentration Units Name Symbol Description Molality m Moles solute/kilogram solvent (mol/kg) Molarity M Moles solute/liter solution (mol/l) Mole fraction X Moles solute/moles total (unitless) Percentage % Parts per hundred, often as weight percent Parts per thousand Parts per million ppm (mg/kg or mg/l) Parts per billion ppb (μg/kg or μg/l) Parts per trillion ppt (ng/kg or ng/l) The reason for multiple concentration units and the numerical prefixes is convenience. We can quote everything as percentage, but doing so can lead to awkward expression of values. For example, the EPA 5 lead limit of 15 ppb would be written as the awkward value %. Some units will be accompanied by explanatory text, for example a percentage might be listed as a weight percentage or percentage v/v (volume per volume). For ppm, ppb, and ppt units, the per l definitions are equivalent to per kg only for dilute aqueous solution, that is, at a solution density of 1.0 kg/l. There are many other units for specialized measurements. Water hardness is usually quoted as the equivalent amount of calcium carbonate in mg/l or grains/gallon (1 grain/gallon = 17.1 mg/l). The salinity of salt water is expressed in parts per thousand or as a ratio to a reference solution. When measured versus a reference, units are expressed as a practical salinity scale, PSS, where 1 PSS unit is equal to approximately 1 g sea salt per kg of seawater. There are numerous other cases where some standard protocol is adopted so that measurements made by different analysts in different locations can be compared directly. Air sampling can require different approaches. The EPA limits for particulate matter in indoor air, PM 2.5, are 15.0 μg/m 3 for the annual average limit and 35 μg/m 3 for the 24-h limit. 6 As this example illustrates, quoting a single simple quantity is not always sufficient to describe the concentration of an analyte in a real sample or situation. Example 1.1 Unit Conversion. The EPA maximum contaminant limit (MCL) for benzene, C 6 H 6, in drinking water is mg/l. What is this limit written in units of ppm, ppb, M, and wt%? The question does not specify the density of water, so we will take it to be 1.0 g/ml or 1.0 kg/l. As we have only one significant figure in mg/l, this 5 U.S. Environmental Protection Agency, 6 Solid and liquid particles less than 2.5 μm in diameter suspended in air. U.S. Environmental Protection Agency, National Ambient Air Quality Standards, /air/criteria.html.

23 INTRODUCTION 9 assumption does not affect this calculation. 7 Given a density of 1.0 kg/l, the units mg/l and ppm are the same mg C 6 H l water ( ) 1.0l = ppm C 1.0 kg 6 H 6 Converting ppm to ppb is accomplished by multiplying by 1000: ( ) 1000 ppb ppm C 6 H 6 = 5 ppb C 1 ppm 6 H 6 To convert to molarity, M, we use the formula weight of benzene, which is g/mol. Converting mg to mol is gc 6 H g/mol = mol C 6 H 6 Now dividing by 1.0 l gives c C6 H 6 = mol 1.0l = M To find the weight percent, wt%, we need grams of benzene per 100 g of solution. The MCL of mg/l for a water density of 1.0 g/ml is mg/1000 g. I multiply both numerator and denominator by 0.1 to get the units that I want in the denominator: gc 6 H g water ( ) 0.1 = gc 6 H g water gc 6 H g water 100% = %C 6 H 6 As you can see, mg/l or 5 ppb is simply more convenient for describing this limit than other concentration units. The following spreadsheet calculation is the first You-Try-It exercise in the text. An Excel spreadsheet file containing this exercise and a step-by-step guide are available on the course web site. I have placed these exercises at appropriate locations in the chapter, and I recommend that you practice on the just-completed topics before moving to new material. If you are new to or want more practice using spreadsheets, work through the spreadsheet-help.pdf document that is also available on the course web site, 7 Pure water at 25 C has a density of g/ml.

24 10 MAKING MEASUREMENTS You-Try-It 1.A The conversions worksheet in you-try-it-01.xlsx contains two tables of measurement results. The exercise is to convert these results to other common units. 1.2 GLP AND A FEW OTHER IMPORTANT ACRONYMS GLP (Good Laboratory Practice) refers to specific regulations by which laboratories must conduct, verify, and maintain their procedures, results, and records. These regulations, effective in the United States in 1979, were a response to the unreliability of data that were submitted to government agencies to certify that agricultural chemicals, food additives, drugs, and cosmetics were safe and effective. The current GLP regulations are found in the Code of Federal Regulations as 8 Title 21, Part 58: U.S. Food and Drug Administration, Title 40, Part 160: U.S. Environmental Protection Agency pertaining to the Federal Insecticide, Fungicide, and Rodenticide Act (FIFRA), Title 40, Part 792: U.S. Environmental Protection Agency pertaining to the Toxic Substances Control Act (TSCA). The different regulations are similar in their overall structure and purpose, but tailored to specific types of chemicals and laboratories. The details of the Code of Federal Regulations are not important to us, but they are useful to recognize the origin of regulations that we will discuss. Reference to sections of the federal code use an abbreviation based on title, part, and section number. The following paragraph illustrates the coding with a small excerpt of the Code of Federal Regulations, 21CFR58.83, where the.83 in the title refers to this specific section [Code of Federal Regulations] [Title 21, Volume 1] [Revised as of April 1, 2006] From the U.S. Government Printing Office via GPO Access [CITE: 21CFR58] [Page 308] TITLE 21--FOOD AND DRUGS CHAPTER I--FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES 8 Available from U.S. Government Printing Office:

25 GLP AND A FEW OTHER IMPORTANT ACRONYMS 11 PART 58_GOOD LABORATORY PRACTICE FOR NONCLINICAL LABORATORY STUDIES --Table of Contents Subpart E_Testing Facilities Operation Sec Reagents and solutions. All reagents and solutions in laboratory areas shall be labeled to indicate identity, titer or concentration, storage requirements, and expiration date. Deteriorated or outdated reagents and solutions shall not be used Besides codifying good science and common sense as federal law, the GLP regulations stipulate a framework for personnel responsibilities, analytical methods, and record keeping. These tasks include 1. Personnel and management responsibilities for individual laboratory workers, Study Director, and a Quality Assurance unit (QAU). 2. Written protocols, study plans, and standard operating procedures (SOP) for individual steps or instruments. 3. Record keeping, final written reports, and retention of records. With the formalized GLP regulations, quality assurance (QA) now indicates an auditing role, while quality control (QC) refers to instrument calibration and method validation (discussed in Section 1.5). My point in this discussion is not that we must memorize government regulations but that all workers in an analytical laboratory have certain roles. Laboratory workers must follow documented and reproducible procedures and create an audit trail of all work. Following GLP ensures the reliability of analytical measurements and maintains the credibility of reported results. An analyst should develop GLP habitsof-mind to become adept at measurement science and to work safely in the laboratory. Standard Operating Procedure (SOP) An SOP is a document containing the instructions for a specific analytical procedure or instrument. SOPs can serve as a user s guide, reference source, and safety manual. As per GLP regulations, they are reviewed and approved by other members of the laboratory management team. The following example shows a simple SOP. Other SOPs might be the size of a novella, depending on the complexity of a task, an instrument, or a hazard.

26 12 MAKING MEASUREMENTS ========== Standard Operating Procedure ========== Title: Use of Ocean Optics FL-400 Flame-Resistant Fiber Probe SOP No: AC-21 Author: B. Tissue version: 05/27/06 1. Purpose To ensure correct and safe usage of the FL-400 Flame-Resistant Fiber Probe when recording flame emission spectra. 2. Scope This SOP provides operating procedures for the Ocean Optics FL-400 Flame-Resistant Fiber Probe for recording flame emission spectra. Consult other SOPs or your instructor for sample handling and use of the flame source and spectrometer. 3. Precautions Do not touch the tip when hot. Handle the probe with care. It contains a glass fiber and should not be dropped or bent. Let tip cool for at least 10 seconds before placing in sample or cleaning solutions. Do not place a hot probe tip in a cool solution as the glass fiber could crack. 4. Specific Procedures 4.A. Set-up: The flame-resistant fiber probe should be connected to a 1-m optical patch cable using the stainless steel SMA splice bushing stored with the probe. Do not attempt to use the probe connected directly to the USB2000 R spectrometer. 4.B. Use: To load a test portion onto the flame loop (1) dip into a solution and allow to dry or (2) wet the loop with dilute HCl and dip into solid sample. Insert only the loop into a flame and not the whole tip. A separate sample loop or splint may also be used. 4.C. Cleaning: The flame loop and fiber tip should be cleaned with distilled water. A mild detergent and ultrasound is acceptable if necessary. 5. Related SOPs and References a. SOP No. AC-20 Use of Ocean Optics USB2000 R spectrometer. b. Ocean Optics web site: c. Chemistry Hypermedia Project: chem-ed/index.html. Reviewed: J.R. Morris 5/29/06 Approved: M. Anderson 6/7/06

27 GLP AND A FEW OTHER IMPORTANT ACRONYMS 13 SOPs provide a handy reference to ensure proper use of a procedure or instrument to obtain reproducible results. They, or other procedural documentation, also serve as traceable legal documents in patent and criminal legal proceedings. For example see the Toxicology Procedures Manual at the Virginia Department of Forensic Science. 9 Failure of an analyst to follow the approved procedures can result in measurements being inadmissible in court cases. Still More Acronyms There are a number of other regulatory infrastructures that are similar to GLP. Some examples are Good Clinical Practice (GCP). Regulations governing clinical trials. Good Manufacturing Practice (GMP). Regulations governing the pharmaceutical industry. Also cgmp denotes current Good Manufacturing Practice. 10 Organisation for Economic Co-operation and Development (OECD). An international organization concerned chiefly with economic and social issues. Also a publisher of guidelines on science and technology including chemical safety, chemical testing, and GLP. 11 International Union of Pure and Applied Chemistry (IUPAC). A nongovernmental agency that recommends standardization of chemical nomenclature, terminology, and chemical and physical data. The point of this list is not that you should memorize acronyms but that you can determine the purpose and origin of regulations when you see them in your work context. Many of these regulatory structures are available online, and a search will often find all that you need to know about the details of the regulations. Chances are that you will deal with scientific regulations sometime during your work life, possibly in all roles in a laboratory as analyst, supervisor, or auditor. Material Safety Data Sheet I finish this section with a very important reference document for performing laboratory work called the material safety data sheet (MSDS). The purpose of an MSDS is to provide sufficient information for a laboratory worker to select facilities, equipment, and precautions to work safely and to prevent exposing 9 VA DFS, Forensic Toxicology, Also note the Training manual at the bottom of the page for maintaining GLP. 10 GCP and GMP are both administered by the Food and Drug Administration in the United States. 11 OECD Principles on Good Laboratory Practice and Guidelines for Testing of Chemicals can be found by searching at

28 14 MAKING MEASUREMENTS people or the environment to hazardous chemicals. The information includes what you should know before using a substance, that is, before spilling, inhaling, or igniting anything. Some of the more useful sections of an MSDS are Section 3: Hazards Identification, Including Emergency Overview Section 4: First Aid Measures Section 5: Fire Fighting Measures Section 6: Accidental Release Measures Section 7: Handling and Storage Section 8: Exposure Controls & Personal Protection The following section of an MSDS illustrates the type of information that they contain. The most important aspect about the information in an MSDS is that it is very easy to read before starting work with an unfamiliar chemical. It is very difficult to read an MSDS when you are dizzy, burnt, or unconscious. Section 4 - First Aid Measures 134 HYDROCHLORIC ACID ACS GRADE First Aid: EYES/SKIN: IMMEDATELY FLUSH W/WATER FOR 15 MINS. INGESTION: DO NOT INDUCE VOMITING. GIVE 1 2 GLASSES OF WATER. NEVER GIVE ANYTHING BY MOUTH TO AN UNCONSCIOUS PERSON. INHALATION: MOVE TO FRESH AIR. GIVE CPR IF NEEDED. OBTAIN MEDICAL ATTENTION IN ALL CASES If the point of this discussion on MSDS is not clear, let me spell it out: You must know the location of relevant MSDS s before beginning work. If you are working with an unfamiliar substance, you must read the MSDS before beginning work. MSDS sheets must be easily available, as medical personnel can refuse to treat a case of chemical exposure without an MSDS for the substance involved. If you do not have an MSDS, most chemical suppliers provide them online, and there are a variety of other resources: Where to find MSDS on the Internet, 12 For many common chemicals the NIOSH Pocket Guide to Chemical Hazards is online at Interactive Learning Paradigms, Inc., copyright by Rob Toreki. This company also provides a free online MSDS HyperGlossary for technical terms, 13 National Institute for Occupational Safety and Health, a part of the U.S. CDC (Centers for Disease Control and Prevention).

29 PRECISION AND RANDOM ERROR PRECISION AND RANDOM ERROR A GLP to improve the credibility of any type of measurement is to simply repeat a measurement more than once. We call measurements of multiple test portions from the same sample replicate measurements. Placing a test portion in an instrument and recording the signal multiple times is signal averaging, which is useful if the signal fluctuates, but signal averaging is not the same as making replicate measurements. After dividing a sample into several test portions, each test portion is treated and measured using the identical procedures. If an analytical procedure requires 10 steps, the 10 steps are done for each test portion. Doing replicate measurements can identify gross errors such as omitting one step in a procedure, one-time instrument glitches, or recording a value incorrectly. Analytical methods will often specify measurement of duplicate samples at some frequency: once daily, a duplicate set every 10 samples, and so on. A duplicate sample is a sample that is collected and split into two portions. These are then treated as two distinct samples and are used to monitor variability in the analytical methods. The repeatability in making replicate measurements is called precision. 14 We use the term repeatability when we are talking about replicate measurements made on the same sample and performed under identical conditions. When we compare measurements of the same sample by different analysts or different methods, we use the term reproducibility. The calculations of precision are the same for repeatability and reproducibility, the difference is the source of the measurements being described. Quantitative measures of precision include standard deviation, standard error, and confidence limits (CL). These measures quantitate the variation or spread in the individual measurements due to random fluctuations, which we call random errors. The distribution of random fluctuations follows a Gaussian-shape bell curve, and precision is a measure of the width of this distribution. Graphically, we display the precision by placing error bars about a data point. The accuracy of a measurement is how close an experimental result comes to the true value. The difference between a measurement and the true value is called the systematic error or bias. We determine the bias in a measurement by measuring samples of known composition, which we call standards. We will discuss accuracy and systematic errors fully in Section 1.5 on calibration, which is the process of measuring standards to be able to obtain accurate results of unknowns. 15 We can never know with 100% certainty if we have determined the true concentration of an analyte in a sample. The accuracy of a measurement depends 14 Imprecision, or the lack of precision, is probably a better term to describe the repeatability of measurements, but precision is the more common term. 15 The source of the sample is usually known. Calling a sample an unknown is common usage to indicate that the analyte concentration in the sample is unknown.

30 16 MAKING MEASUREMENTS on the care in validating sample processing procedures, calibrating the measurement, and making sure that standards match the samples. The best that we can do, by following GLP, is to use reproducible methods; maintain equipment, reagents, and instruments in good working condition; verify the accuracy of our analytical abilities with standards of known concentrations; use a calibration procedure that is appropriate for the unknown sample. As an example, the chemical treatment of a municipal water supply is based on the measurement of a pollutant being less than the EPA action level. Analysis of randomly collected water samples shows that it contains 14 ppb Pb, below the EPA action level of 15 ppb. 16 If you drink water from this system, you will probably have a few questions about the result. First, you might ask the analyst how confident she is of the accuracy of the result. Did the analyst follow GLP and check the accuracy of the analytical methods by measuring a known standard? Next, what is the spread in the measurement, that is, what is the precision of the reported result? Is the range of measured values 14.0 ± 0.1 ppb or is it 14 ± 5 ppb? You might be more concerned if the upper value of the error bars exceeds the EPA action level. The terms accuracy and precision are often used interchangeably, but they are not the same. Precision does not tell us anything about the accuracy of a result owing to systematic errors in the measurement procedure. Figure 1.1 illustrates the difference between accuracy and precision using the results of arrows shot at targets by three different archers. The archer in (a) was very steady, she had high precision, but she hit high on the target with every shot. She possibly aligned her sight incorrectly, estimated the distance to the target incorrectly, or made some other systematic error. Unfortunately, it is not always Precise but not accurate Figure 1.1 Accurate and precise Accurate but not precise The results from three archers. 16 The EPA action level is the concentration of a contaminant that requires a response such as public notification, exposure monitoring, or remediation.