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Multiresidue Methods for the Analysis of Pesticide Residues in Food 1st Edition Heinzen
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Library of Congress Cataloging–in–Publication Data
Names: Franca, Adriana S., editor. | Nollet, Leo M. L., 1948- editor. Title: Spectroscopic methods in food analysis / [edited by] Adriana S. Franca and Leo M.L. Nollet.
Description: Boca Raton, FL : CRC Press, Taylor & Francis Group, 2017. Identifiers: LCCN 2017008762 | ISBN 9781498754613 (978-1-4987-5461-3)
Subjects: LCSH: Food–Analysis. | Food–Quality. | Spectrum analysis. Classification: LCC TX547 .S638 2017 | DDC 664/.07–dc23 LC record available at https://lccn.loc.gov/2017008762
Visit the Taylor & Francis Web site at http://www.taylorandfrancis.com
and the CRC Press Web site at http://www.crcpress.com
David Lee Nelson Chapter 2
Suzana Lucy Nixdorf
Chapter
Ouissam Abbas and Vincent Baeten
Chapter 4 Fourier Transform Spectroscopy
Daniel Cozzolino
Chapter 5 Raman Spectroscopy
Sagar Dhakal, Jianwei Qin, Moon S. Kim, and Kuanglin Chao
Chapter 6
Laura R. Cagliani, Paola Scano, and Roberto Consonni
Chapter 7 Fluorescence Spectroscopy
Jana Sádecká, Veronika Urícˇková, and Michaela Jakubíková
Chapter 8 Ultrasound Spectroscopy in Food Analysis
Semih Otles and Vasfiye Hazal Ozyurt
Chapter 9 Instrumentation
Didem P. Aykas and Luis E. Rodriguez-Saona
Chapter 10 Multivariate
Marcelo M. Sena, Mariana R. Almeida, Jez W. B. Braga, and Ronei J. Poppi
PART II APPLICATIONS
Chapter 11 Food Composition 317
Semih Otles and Vasfiye Hazal Ozyurt
Chapter 12 Food Authentication
Cristina Alamprese
Chapter 13 Food Adulteration
Daniel Cozzolino
Chapter 14 Food Quality 363
Nikolaos Nenadis, Anna Androulaki, and Maria Z. Tsimidou
PART III FOOD PRODUCTS
Chapter 15 Spectroscopy Analysis of Beverages
Daniel Cozzolino and Jessica Roberts
Chapter 16 Nonalcoholic Beverages
Basil K. Munjanja and Anna T.D. Gowera
Chapter 17 Novel and Conventional Spectroscopic Study of Cereals and Cereal Products
Molecular Structure, Chemistry, Imaging, and Nutrition 451
Peiqiang Yu
Chapter 18 Structural Responses of Chemical Functional Groups in Different Types of Cereal Grains to Heat-Related Processing Methods Revealed with Advanced Synchrotron and GlobarSourced Molecular (Micro) Spectroscopy 463
Yuguang Ying and Peiqiang Yu
Chapter 19 Coffee 485
Adriana S. Franca and Leandro S. Oliveira
Chapter 20 Spectroscopic Methods for Analysis of Edible Oils
Xiuzhu Yu
Chapter 21 Dairy Products
Basil K. Munjanja and Anna T.D. Gowera
Chapter 22 Fish and Meat
María José Ayora-Cañada and Ana Domínguez-Vidal
Chapter 23 Fruits and Vegetables 601
Adriana S. Franca
Chapter 24 Other Food Products 619
Ana Paula Craig and Joseph Irudayaraj
Index
Preface
The determination of product quality and authenticity and the detection of adulteration are the major issues in the food industry, causing concern among consumers and special attention among food manufacturers. The concepts of food quality and authenticity are quite broad, given the different demands of the manufacturer, consumer, oversight, and legislative bodies that will ultimately provide healthy and safe products, taking into account both economic and environmental issues. Given the inherent complexity of food products, most instrumental techniques (e.g., chromatographic methods) employed for quality and authenticity evaluation are time consuming, expensive, and labor intensive. Therefore, there has been an increasing interest in simpler, faster, and more reliable analytical methods for assessing food quality attributes.
Spectroscopic methods have been extensively employed in the analysis of food products because they often require minimal or no sample preparation, provide rapid and online analysis, and have the potential to run multiple tests on a single sample (i.e., nondestructive). Therefore, this book is dedicated to spectroscopic techniques that are of relevance to food analysis.
This book is divided into three parts: Part I—Fundamentals and instrumentation, Part II—Applications, and Part III—Food products. Part I begins with a comprehensive and historic overview of spectroscopic methods (Chapter 1) followed by an extensive discussion on fundamental and theoretical aspects as well as new trends on each technique (Chapters 2 through 8), instrumentation (Chapter 9), and statistical analysis (Chapter 10). Part II focuses on the specific application of these techniques in food analysis, dealing with important issues such as composition (Chapter 11), authentication (Chapter 12), adulteration (Chapter 13), and food quality (Chapter 14). Part III presents the recent advances in spectroscopy for the analysis of specific food products including beverages (Chapters 15 and 16), cereals (Chapters 17 and 18), coffee (Chapter 19), edible oils (Chapter 20), dairy products (Chapter 21), fish and meat (Chapter 22), fruits and vegetables (Chapter 23), and other food products (Chapter 24).
All chapters have been written by renowned scientists who are experts in their research fields. We would like to thank all contributing authors and colleagues for their effort in producing this excellent book. They are the ones who made this project possible.
“As coisas tangíveis tornam-se insensíveis à palma da mão. Mas as coisas findas muito mais que lindas, essas ficarão.” (Tangible things become insensible at the palm of the hand. But finished things, more than beautiful, these will stay)
Carlos
Drummond de Andrade
Editors
Adriana S. Franca, PhD, received her BSc in chemical engineering in 1988 and MSc in mechanical engineering in 1991 from the Universidade Federal de Minas Gerais, Belo Horizonte, Brazil. She completed her PhD in agricultural and biological engineering from Purdue University in 1995.
She is currently a professor in the Department of Mechanical Engineering, the Universidade Federal de Minas Gerais, Belo Horizonte, Brazil, and also teaches a graduate course on food sciences. She has published 94 articles in international journals, 21 book chapters, and has presented more than 200 research papers at various international conferences. Her research areas include food science, sustainable uses of agricultural residues, coffee chemistry, heat transfer, microwaves, and spectroscopic methods. For more information refer http://lattes.cnpq.br/1719405448685259.
Leo M.L. Nollet, PhD, received his MS (1973) and PhD (1978) in biology from the University of Leuven, Belgium. He is an editor and associate editor of numerous books. He edited for M. Dekker, New York—now CRC Press of Taylor & Francis—the first, second, and third editions of the books entitled Food Analysis by HPLC and Handbook of Food Analysis. The last edition is a two-volume book. He also edited the Handbook of Water Analysis (first, second, and third editions) and Chromatographic Analysis of the Environment, third edition (CRC Press).
With F. Toldrá, he coedited two books published in 2006 and 2007: Advanced Technologies for Meat Processing (CRC Press) and Advances in Food Diagnostics (Blackwell Publishing—now Wiley). With M. Poschl, he coedited the book Radionuclide Concentrations in Foods and the Environment also published in 2006 (CRC Press).
Dr. Nollet has also coedited with Y.H. Hui and other colleagues, several books: Handbook of Food Product Manufacturing (Wiley, 2007), Handbook of Food Science, Technology and Engineering (CRC Press, 2005), Food Biochemistry and Food Processing (first and second editions; Blackwell Publishing—now Wiley—2006 and 2012), and the Handbook of Fruits and Vegetable Flavors (Wiley, 2010).
In addition, he edited the Handbook of Meat, Poultry and Seafood Quality, first and second editions, (Blackwell Publishing—now Wiley—2007 and 2012). From 2008 to 2011, he published with F. Toldrá five volumes on animal product-related books, namely, the Handbook of Muscle Foods Analysis, Handbook of Processed Meats and Poultry Analysis, Handbook of Seafood and Seafood Products Analysis, Handbook of Dairy Foods Analysis, and Handbook of Analysis of Edible Animal By-Products. Also in 2011 with F. Toldrá, he coedited for CRC Press two volumes: Safety Analysis of Foods of Animal Origin and Sensory Analysis of Foods of Animal Origin. In 2012, they both published the Handbook of Analysis of Active Compounds in Functional Foods.
In a coedition with Hamir Rathore, the book Handbook of Pesticides: Methods of Pesticides Residues Analysis was marketed in 2009, Pesticides: Evaluation of Environmental Pollution in 2012, and the Biopesticides Handbook in 2015. Other finished book projects include Food Allergens: Analysis, Instrumentation, and Methods (with A. van Hengel; CRC Press, 2011) and Analysis of Endocrine Compounds in Food (Wiley-Blackwell, 2011).
Dr. Nollet's recent projects include Proteomics in Foods with F. Toldrá (Springer, 2013) and Transformation Products of Emerging Contaminants in the Environment: Analysis, Processes, Occurrence, Effects and Risks with D. Lambropoulou (Wiley, 2014). In this series, CRC Food Analysis & Properties, he edited with C. Ruiz-Capillas, Flow Injection Analysis of Food Additives (CRC Press, 2015) and Marine Microorganisms: Extraction and Analysis of Bioactive Compounds (CRC Press, 2016).
Ouissam Abbas
Food and Feed Quality Unit
Contributors
Quality Department of Agricultural Products
Walloon Agricultural Research Centre (CRA-W)
Gembloux, Belgium
Cristina Alamprese
Department of Food, Environmental and Nutritional Sciences (DeFENS) Università degli Studi di Milano Milan, Italy
Mariana R. Almeida
Departamento de Química
Instituto de Ciências Exatas Universidade Federal de Minas Gerais (UFMG)
Jez W.B. Braga Instituto de Química Universidade de Brasília Brasília, Brazil
Laura R. Cagliani
NMR Laboratory, National Research Council Institute for Macromolecular Studies (ISMAC) Milan, Italy
Kuanglin Chao
USDA/ARS Environmental Microbial and Food Safety Laboratory
Beltsville Agricultural Research Center
Beltsville, Maryland
Roberto Consonni
NMR Laboratory, National Research Council
Institute for Macromolecular Studies (ISMAC)
Milan, Italy
Daniel Cozzolino
School of Medical and Applied Sciences
Central Queensland Innovation and Research Precinct (CQIRP)
Central Queensland University (CQU)
North Rockhampton, Australia
Ana Paula Craig
Department of Agricultural and Biological Engineering
Bindley Bioscience Center and Birck
Nanotechnology Center
Purdue University
West Lafayette, Indiana
Sagar Dhakal
USDA/ARS Environmental Microbial and Food Safety Laboratory
Beltsville Agricultural Research Center
Beltsville, Maryland
Ana Domínguez-Vidal
Department of Physical and Analytical Chemistry
Universidad de Jaén
Jaén, Spain
Anna T.D. Gowera
Certification Services Department
Standards Association of Zimbabwe
Harare, Zimbabwe
Joseph Irudayaraj
Department of Agricultural and Biological Engineering
Bindley Bioscience Center and Birck
Nanotechnology Center
Purdue University
West Lafayette, Indiana
Michaela Jakubíková
Faculty of Chemical and Food Technology
Institute of Analytical Chemistry
Slovak University of Technology in Bratislava
Bratislava, Slovak Republic
Moon S. Kim
USDA/ARS Environmental Microbial and Food Safety Laboratory
Beltsville Agricultural Research Center
Beltsville, Maryland
Basil K. Munjanja
Department of Chemistry
Faculty of Natural and Agricultural Sciences
University of Pretoria Pretoria, South Africa
David Lee Nelson
Pro-Reitoria de Pesquisa e Pós-Graduação
Universidade Federal dos Vales de Jequitinhonha e Mucuri Diamantina, Minas Gerais, Brazil
Nikolaos Nenadis
Laboratory of Food Chemistry and Technology (LFCT)
School of Chemistry
Aristotle University of Thessaloniki
Thessaloniki, Greece
Suzana Lucy Nixdorf
Departamento de Química
Universidade Estadual de Londrina (UEL) Londrina, Brazil
Leandro S. Oliveira
Departamento de Engenharia Mecânica (DEMEC)
Universidade Federal de Minas Gerais (UFMG)
Belo Horizonte, Brazil
Semih Otles
Food Engineering Department
Ege University
Izmir, Turkey
Vasfiye Hazal Ozyurt
Food Engineering Department
Ege University
Izmir, Turkey
Ronei J. Poppi
Instituto de Química
Universidade Estadual de Campinas
Campinas, Brazil
Jianwei Qin
USDA/ARS Environmental Microbial and Food Safety Laboratory
Beltsville Agricultural Research Center
Beltsville, Maryland
Jessica Roberts
School of Medical and Applied Sciences
Central Queensland Innovation and Research Precinct (CQIRP)
Central Queensland University (CQU) North Rockhampton, Australia
Luis E. Rodriguez-Saona
Department of Food Science and Technology
The Ohio State University Columbus, Ohio
Jana Sádecká
Faculty of Chemical and Food Technology
Institute of Analytical Chemistry
Slovak University of Technology in Bratislava Bratislava, Slovak Republic
Paola Scano
NMR Laboratory, National Research Council
Institute for Macromolecular Studies (ISMAC)
Milan, Italy
and Department of Chemical and Geological Sciences
University of Cagliari Cagliari, Italy
Marcelo M. Sena
Departamento de Química
Instituto de Ciências Exatas
Universidade Federal de Minas Gerais (UFMG)
Belo Horizonte, Brazil
Maria Z. Tsimidou
Laboratory of Food Chemistry and Technology (LFCT)
School of Chemistry
Aristotle University of Thessaloniki Thessaloniki, Greece
Veronika Uríc ˇ ková
Faculty of Chemical and Food Technology Institute of Analytical Chemistry
Slovak University of Technology in Bratislava
Bratislava, Slovak Republic
Yuguang Ying
College of Agriculture and Bioresources University of Saskatchewan Saskatoon, Saskatchewan, Canada
Peiqiang Yu
Department of Animal and Poultry Science
College of Agriculture and Bioresources University of Saskatchewan Saskatoon, Saskatchewan, Canada
Xiuzhu Yu
College of Food Science and Engineering
Northwest A&F University
Shaanxi, People's Republic of China
PART I
Fundamentals and Instrumentation
CHAPTER 1
Introduction to Spectroscopy
David Lee Nelson
CONTENTS
1.1 UV–Visible Spectroscopy
1.5
1.6
1.7
1.8
1.9
Spectroscopy has had an ever-increasing role in the determination of the composition and adulteration of foods and beverages. It is important for determining food safety, accompanying food and beverage production, and for the control of food, beverages, and packaging in general.
The study of spectroscopy is considered to have begun with Isaac Newton’s experiments with the dispersion of light into its components of various wavelengths with the aid of a prism (Thomas 1991; James 2007). However, nothing more was studied until the time of William Wollaston, who improved upon Newton’s experiment in 1802. The dark lines that appeared in the spectrum (Figure 1.1) were later studied by Joseph von Fraunhofer (Jackson 2000), followed by Anders J. Angstrom, who measured the wavelengths of these lines. Fraunhofer also constructed a grating that achieved greater resolution in the dispersion of light than the prism (Pasquini 2003). Sir John Herschel studied the spectrum of flames in 1822 and laid the foundation for spectral analyses. In 1859, Gustav Kirchhoff suggested that substances emitted and absorbed light at the same wavelength. These and other studies were the basis of Bohr’s theory of the atom, which specified that electrons existed in discrete energy levels in the atom (Thomas 1991). August Beer later proposed the linear relationship between absorbance and concentration, which has since been the basis for the quantitative determination of substances by measurements of absorbance or transmittance in the visible and ultraviolet (UV) regions (Thomas 1991).
Electromagnetic radiation consists of electromagnetic waves, which are synchronized oscillations of electric and magnetic fields that propagate at the speed of light through a vacuum. The oscillations of the two fields are perpendicular to each other and perpendicular to
FIGURE 1.1 Solar spectrum with Fraunhofer lines. (From Gebruiker, M. V. 2005. Spectrum-sRGB.svg https://en.wikipedia.org/wiki/Fraunhofer_lines.)
FIGURE 1.2 The electromagnetic spectrum. (From Ronan, P. and Gringer. 2013. File: EM spectrum.svg and File: Linear visible spectrum.svg. https://en.wikipedia.org/wiki/ Electromagnetic_radiation.)
the direction of energy and wave propagation, forming a transverse wave. Electromagnetic waves can be characterized by either the frequency or wavelength of their oscillations, which determines their position in the electromagnetic spectrum (Crowell 2013). Electromagnetic radiation involves a wide range of wavelengths, as is shown in Figure 1.2. The electromagnetic spectrum refers to all the known frequencies and their linked wavelengths of the known photons. The electromagnetic spectrum extends from above the long wavelengths (high frequencies) used for modern radio communication to gamma radiation at the shortwavelength (high-frequency) end, thereby covering wavelengths from thousands of kilometers down to a fraction of the size of an atom (Mehta 2011). The range of energies involved in this range of wavelengths varies from 12.4 feV to 1.24 Mev. In principle, the upper limit for the possible wavelengths of electromagnetic radiation is the dimension of the universe. The theoretical lower limit is thought to be the Planck length (1.616199(97) × 10 −35 m) (Bakshi and Godse 2009). Although all the wavelengths shown in Figure 1.2 can be used for analysis, the range of wavelengths usually employed in spectroscopy is relatively narrow and includes mainly the UV, visible, infrared (IR), ultrasound, and FM radio (nuclear magnetic resonance [NMR]) regions.
1.1 UV–VISIBLE SPECTROSCOPY
UV light was discovered by J. W. Ritter in 1801 (Thomas 1991). However, there remained no method by which to measure UV radiation until the development of the photodetector in the 1930s. The first commercial UV–visible (UV–Vis) spectrophotometer was introduced by Beckman in 1941 (Buie 2011).
The absorbance of light in the UV–Vis region occurs when an electron is excited and passes from a bonding or nonbonding orbital to an antibonding orbital. The amount of energy required to excite an electron depends on the difference in energy between the ground state and the excited state. Transitions of the σ – σ*, σ –π*, π– σ*, or simple π–π* type require light in the vacuum UV region. However, conjugated π systems exhibit π–π* transitions that absorb in the region between 200 and 800 nm. In conjugated systems, the excited state is more greatly stabilized by resonance than the ground state, so the difference in energy between the two states is smaller than in nonconjugated systems (Silverstein et al. 1974). The smaller the difference in energy between the ground state and the excited state, the greater is the probability that a transition between the ground state and the excited state will occur. The intensity of the absorbance is a function of this probability. For a given concentration, the intensity of absorbance will be greater when the difference in energy between the ground and excited state is small. And, this difference will be smaller when there is a greater degree of conjugation in the molecule.
The nonbonding (n) electrons normally have a higher energy than the ground state pi electrons. The nonbonding electrons are held less strongly. Therefore, the difference in energy between the nonbonding orbitals and the antibonding (π*) orbital is small, and the absorbance corresponding to this n–π* transition occurs at a longer wavelength than the π–π* transition. However, the nonbonding orbitals are perpendicular to the π* orbitals, and there is very little overlap between the two. Therefore, the probability that a nonbonding electron will be excited to a π* orbital is extremely small, and the intensity of the corresponding absorbance will also be very low. This transition is said to be “forbidden.”
The absorption bands in the UV–Vis region are very wide. The electronic state is made up of several vibrational energy sublevels. These different vibrational energy levels are each composed of several rotational energy levels. The energy differences between the vibrational levels are much smaller than the differences between electronic energy states and the differences between rotational energy levels are even smaller. The electronic excited state also has several vibrational and rotational energy sublevels. Excitation of the electron involves a transition from any one of the vibrational and rotational levels in the ground state to any of the vibrational and rotational levels in the electronic excited state, resulting in several absorptions with small differences in wavelength that form very wide bands.
UV–Vis spectroscopy has been widely used for the quantitative analysis of substances that absorb in this region because of the high sensitivity of the method. The UV–Vis detector has long been employed in chromatography, especially of proteins, and has been especially useful in high-performance liquid chromatography (HPLC) analysis of many classes of compounds. It has also proved useful for the qualitative identification of pure substances, although the quantity of information provided by the UV–Vis spectrum is much more limited than that of some other spectroscopic techniques. The proposal by R. B. Woodward (1941, 1942a,b) of a set of empirical rules for calculating the λ max of the absorbance of unsaturated compounds was an important tool in the identification of such compounds.
A development that increased the usefulness of UV–Vis spectroscopy was the use of derivatives of the original spectra (Griffiths et al. 1982; Rojas et al. 1988). The first to fourth derivative of the spectra can be obtained, and this mathematical technique has been incorporated into the instrumentation. This technique permits the identification and quantification of mixtures of substances, whereas this identification is much more difficult or impossible in the original spectrum. This technique has been used in many studies, such as the determination of tyrosine in proteins (Ragone et al. 1984), the detection of toxic substances (Gill et al. 1982), the study of the fractions obtained from the partial hydrolysis of casein and other proteins (Silvestre et al. 1993), and the determination of phenylalanine in wheat flour (Carreira et al. 2009).
The development of the diode-array detector was especially useful because it became possible to simultaneously measure the absorbance at several wavelengths, and the spectrum could be registered while determining the concentration of the substance eluted from a column. Another more recent development is the use of diffuse reflectance spectroscopy for the analysis of substances, including food and nanostructures (Gao and Wachs 2000; Morales et al. 2007; Rossel et al. 2006; Liu 2016), although this spectroscopic technique has been more extensively used in the mid- and near-infrared (NIR) regions. Reflectance spectroscopy does not require modification of the sample and can be applied for quality control on the production line. This technique is one of the advancements in spectroscopy that has been made possible by the development of multivariate analysis and other chemometric techniques, without which the spectra obtained would make no sense. Instrumental modifications necessary for the recording of reflectance spectra have also played a major role in this type of analysis and in the use of microspectroscopy, where the spectrometer is adapted to a microscope and spectra can be obtained from minute particles or cells (Les 2010).
When the substance does not absorb significantly in the UV–Vis region, derivatization can be performed to obtain a product that does absorb in this region. An example is the preparation of phenylthiohydantoin (PTH) derivatives of amino acids that do not have aromatic rings (Nollet and Toldrá 2012). Of course, it is essential that the reaction be 100% complete.
1.2 FLUORESCENCE SPECTROSCOPY
Fluorescence spectroscopy is a very valuable, highly sensitive technique for determining the quantity of certain kinds of substances that possess the capability to fluoresce. In 1565, Nicolás Monardes, a Spanish physician and botanist, reported a bluish opalescence from a water infusion of the wood from a Mexican tree. A Franciscan missionary named Bernardino de Sahagún observed a similar phenomenon in a wood named “coatli.” Both woods were reported to have medicinal benefits for the kidney. This type of luminescence has since been reported in chlorophyll, barium sulfate, quinine, acridine, fluorosceine, and rhodamine. In 1845, Sir John Frederick William Herschel observed the fluorescence from a solution of quinine sulfate and termed this phenomenon as “epipolic dispersion.” In 1852, G. G. Stokes invented the term fluorescence from the mineral fluorspar. He was also the first person to propose the use of fluorescence as an analytical tool (Chakraborty 2013).
The first fluorimetric analysis was performed by F. Goppelsröder in 1867 for the quantitative determination of Al(III) from the fluorescence of its morin chelate. Otto Heimstaedt and Heinrich Lehmann (1911–1913) first developed the fluorescence microscope to investigate the autofluorescence of biosamples such as bacteria, protozoa,
plant, and animal tissues. Later, the American Instrument Company collaborated with Dr. Robert Bowman who designed and marketed the first spectrophotofluorimeter (SPF) in 1956 (National Institutes of Health 2016). Antimalarial research actually initiated the invention of the SPF as an analytical instrument that can determine the presence of analytes that fluoresce. The story dated back to 1940, during World War II, when scientists in the United States were required to determine the amount of drug that reached the malarial parasites in a patient’s blood for a clinical trial of antimalarial drugs. Bernard Brodie and Sidney Udenfriend of Goldwater Memorial Hospital in New York City designed a new test using an instrument called a fluorometer that could determine the amount of the drugs in the blood plasma from the intensity of the fluorescence emitted from the drug, because many of the drugs used in the trial fluoresce. This observation helped them to come up with a critical dose of a drug to minimize adverse side effects (National Institutes of Health 2016).
Normally, only aromatic or highly unsaturated organic compounds fluoresce. One of the advantages of fluorescence spectroscopy over absorption spectroscopy is the fact that these compounds can be detected in the presence of substances that do not fluoresce. Therefore, they can be detected in mixtures without the competition of nonfluorescing compounds. The technique has several advantages and some limitations. If the compound to be analyzed does not fluoresce, it must be derivatized or tagged to form a product that does fluoresce. An example is the detection of polyamines and biogenic amines when they are separated by HPLC (Ubaldo et al. 2015; Custódio et al. 2015; Kalac and Glória 2009; Fernandes and Glória 2015). These amines do not fluoresce, so they are treated with o-phthalaldehyde to produce a fluorescent derivative that can be detected by the fluorescence detector. A 100% conversion to the derivative is necessary. o-Phthalaldehyde, 4-dimethylaminobenzenesulfonyl chloride, 1-dimethylaminonaphthalene-5-sulfonyl chloride, and 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate have also been used to prepare fluorescent derivatives of amino acids and peptides (Nollet and Toldrá 2012).
In fluorescence spectroscopy, the substance absorbs light in the UV, visible, or NIR region of the electromagnetic radiation. The electrons are excited from a singlet ground state to one of several singlet excited states (Figure 1.3). The electron then decays to the
FIGURE 1.3 Jablonski diagram showing the intersystem decay and fluorescence after absorption of light. (From Jacobhed. 2012. Jablonski diagram. https://en.wikipedia.org/ wiki/Fluorescence.)
lowest singlet excited state via vibrational relaxation and internal conversion and then returns to the ground state with the emission of light. Therefore, the wavelength of the light emitted is longer than the wavelength of the absorbed light. This shift to a longer wavelength is known as the Stokes shift. The λ max of the light emitted does not shift with the wavelength of the light being absorbed; only the intensity of the light emitted is affected because the intensity of the light emitted depends on the number of excited molecules, and the number of molecules that become excited depends on the wavelength of the light being absorbed. The spectral range for most fluorescence measurements is 200–1000 nm (Wehry 1997). As with absorption spectroscopy, there is a transition from the lowest singlet state to a variety of vibrational levels in the ground state, so the emission band is relatively wide.
Fluorescence spectroscopy is orders of magnitude more sensitive than most other methods of detection of organic compounds. One of the reasons for the greater sensitivity when compared to UV–Vis absorption spectroscopy is the fact that any light emitted is compared to a black background. In absorption spectroscopy, if the concentration of the sample is very dilute, the difference between the absorbance of the sample and that of the reference cell will be minimal. That is, the amount of light transmitted will be nearly the same, and two large values with only a slight difference between them will be compared. In fluorescence spectroscopy, there is zero light emitted by the reference, so any light emitted by the sample can be more easily detected. Detection limits down to 10 −10 mol L −1 or lower can be reached, and extremely small samples can be used. The sensitivity depends on the quantum yield, which represents the efficiency of the fluorescence process. It is defined as the ratio of the number of photons emitted to the number of photons absorbed. Another important factor is the lifetime of the excited state because it represents the time available for the excited electron to interact with its environment (Lakowicz 1999).
An advantage of fluorescence spectroscopy over the UV–Vis absorption technique is the fact that two frequencies (absorption and emission maxima) are available for identification of a compound rather than only one. If two sample constituents with similar absorption spectra fluoresce at different wavelengths, they may be distinguished from one another by the appropriate choice of emission wavelength. Or, if two compounds have similar fluorescence spectra but absorb strongly at different wavelengths, they may be distinguished by proper choice of excitation wavelength (Wehry 1997). When the fluorescent spectrum is composed of contributions from a mixture of compounds, synchronized scanning spectroscopy can be used to distinguish between the components of the mixture (Sikorska et al. 2005). In synchronized fluorometry, the absorption spectrum and the emission spectrum are recorded simultaneously with a constant difference in wavelength between the two.
Another advantage is that the technique is fast and simple and the equipment is relatively robust so that it can be used in the field for preliminary analyses. It is also possible to detect emissions remotely if fiber optics or excitation with lasers is employed. This advantage means that the technique can be used for environmental studies and for the control of food products during production without the necessity for extraction or other types of modification of the product. In these cases, filter fluorometers can be used because the intensity of fluorescence at single excitation and emission wavelengths can be measured to detect specific analytes without the need for high resolution or array detectors. Portability, low cost, and small size are most important. A large number of photons can be transmitted by filters, and this characteristic makes them useful for trace analyses. Laboratory fluorometers usually employ grating monochromators.
One of the limitations of the use of fluorescence spectroscopy is the fact that the glassware and solvents utilized must be very clean so that no fluorescent impurities will interfere with the fluorescence from the sample. Extensive cleanup of mixtures, such as chromatography, may be required, and this process can be time consuming. Solvents that absorb in the UV region cannot be used. Another possible interference is the presence of substances that absorb in the same region in which the analyte emits light, thereby reducing the quantum yield.
Photobleaching can sometimes occur, especially in the case of fluorescent probes that might be submitted to radiation over a prolonged period. Photobleaching occurs when a photolytic reaction causes a rupture in one or more bonds and results in the loss of fluorescence (ThermoFisher Scientific 2016). The last type of interference involves quenching, which can occur when some substance interacts with the excited state of the molecule and inhibits fluorescence. The possibility of quenching in mixtures means that care should be exercised during calibration. Quenching can be caused by collision with another molecule, such as iodide, oxygen, and acrylamide, or by the formation of a nonfluorescing complex. This latter type of quenching occurs in the ground state. Quenching can also occur because of attenuation of the incident light by either the fluorophore or some other species (Lakowicz 1999).
The lifetime of the excited state can furnish some information regarding its interactions with other molecules in the environment. The lifetime of the excited state might be sufficiently long so that the solvent molecules can reorient themselves around the molecule. This relaxation of the solvent is responsible for the Stokes shift caused by the solvent (Lakowicz 1999). The Stokes shift can indicate whether a protein is folded or if the fluorophore (tryptophan) is exposed to the solvent water. The shift for a tryptophan residue buried within the protein molecule will be different from that of a residue on the surface. Labeling with extrinsic probes can also be used to determine the environment within a macromolecule.
Fluorescence anisotropy measurements can furnish information regarding the size and shape of proteins. Fluorescence anisotropy involves the photoselective excitation by polarized light. Those fluorophores whose absorption transition dipole is parallel to the electronic vector of the excitation will be preferentially excited. The population of molecules will be partially oriented, and the light emitted will be partially polarized. Fluorescence anisotropy can be reduced by rotational diffusion. If the fluorophore is bound to a large molecule such as a protein or a membrane, rotational diffusion becomes very limited and anisotropy is more easily observed (Lakowicz 1999).
If the emission spectrum of the fluorophore overlaps with the absorption spectrum of another molecule, the energy of the excited state can be transferred by resonance without the emission of light (Lakowicz 1999). The effect depends on whether the two species are free in solution, covalently linked, or trapped within a membrane or nucleic acid molecule. Resonance energy transfer can be used to measure distances between sites on a macromolecule.
There are two types of fluorescence measurements: steady-state and time-resolved measurements. The steady-state measurement is the more commonly used. The timeresolved measurement measures the rate of decay of the intensity of the emitted light or the anisotropy. The anisotropic decay furnishes information regarding the molecular shape and flexibility. The decay in intensity also furnishes information regarding the conformation of the molecule or the presence of more than one conformation.
Fluorimetric detectors are widely used in chromatography, especially HPLC, and in electrophoresis. Capillary electrophoresis is a relatively new technique that achieves
a high degree of resolution of the components in the sample. Fluorescence detection is a very valuable tool for detecting these components.
By employing front-face fluorescence spectroscopy, food samples can be analyzed without destruction of the sample (Karoui et al. 2006; Veberg et al. 2006). It is also a valuable technique for the analysis of proteins and peptides (Ladokhin 2000), as well as nucleic acids (Lakowicz 1999; Ono et al. 2012; Suzuki et al. 1997; Behlke et al. 2005; Xiao and Kwok 2003) and for applications such as determining bitterness in beer (Christensen et al. 2005). Other important subjects include phase-sensitive and phase-modulation resolution of emission spectra, DNA sequencing, fluorescence sensing, time-resolved protein fluorescence, excited state reactions, and energy transfer to multiple acceptors (Lakowicz 1999).
1.3 FOURIER TRANSFORM IR SPECTROSCOPY
The existence of IR radiation was discovered by Herschel in 1800 (Herschel 1800a,b). However, no more interest in this region of the electromagnetic spectrum existed until absorption spectra were obtained in 1882 by Abney and Festing (1886). They also correlated the absorption bands with some functional groups. In 1903, Coblenz (1906) correlated the absorbances in the mid-IR region with the vibrations of functional groups. The recording of IR spectra was difficult until Perkin-Elmer and Beckman developed the first commercial instruments (Thomas 1991).
Midrange IR spectroscopy was the most extensively employed because of its usefulness for the identification of the vibrations of functional groups in pure compounds. In addition, the supports for liquids and solids were inexpensive and easily prepared. The technique was highly valuable as a tool for use in the identification of compounds. It could be used for the identification of liquids using films supported between sodium chloride plates. Spectra of solids could be obtained in solution, in the form of a mull or as a suspension in a KBr pellet. The technique measured the IR light transmitted through the sample. The resulting spectra were not exactly reproducible because the concentration of the sample and conditions were difficult to reproduce exactly. In addition, for aqueous solutions or humid samples, the supports used needed to be insoluble in water. Also, the IR bands corresponding to water interfered with some of those obtained with organic compounds such as alcohols, amines, and amides. The use of midrange IR transmission spectroscopy for the study of pure compounds is very useful, but for the study of foods, it is somewhat limited because of the complexity of the matrices. The energy levels involved in these vibrations are low (2.5–25 μm wavelength). For the vibration to result in an absorption band in the spectrum, the vibration must cause a change in the dipole moment of the molecule.
There are basically two methods by which one can obtain the IR absorption spectrum. The original IR instruments were of the dispersive type. These instruments separated the individual frequencies of energy emitted from the IR source by the use of a prism or grating, and a few wavelengths at a time passed through a slit and then through the sample. The grating is a more modern dispersive element that furnishes a better resolution of the frequencies of IR energy. The detector measures the amount of energy at each frequency that has passed through the sample. This results in a spectrum, which is a plot of intensity versus frequency or wavelength. This method is slow. Because of the necessity for the light to pass through a slit, part of the energy transmitted by the sample is lost.
The second method is called Fourier transform infrared (FTIR) spectroscopy. In this method, the light that impinges on the sample is composed of all of the wavelengths, and
the computer transforms the signals of all of the transmitted wavelengths into a spectrum using a mathematical technique, the Fourier transformation. Instead of a prism or grating, an interferometer is used. The spectrum is obtained in a few seconds instead of a few minutes. After passing through the interferometer, the beam passes through the sample and impinges on the detector. The signal that is measured is digitized and sent to the computer where the transformation takes place. FTIR still involves the transmission of light through the sample and, thus, suffers from some of the same limitations that dispersive instruments possess (Thomas 1991; Blum and John 2012).
This technique has been used for the study of proteins and protein–ligand interactions (Jung 2000). It has also been used for the characterization of spoilage fungi (Shapaval et al. 2013) and for the study of phospholipids in edible oils (Meng et al. 2014). Other examples of the use of FTIR for the analysis of foods include its use for the determination of thermoxidized olive oil (Tena et al. 2013), the study of defective and normal coffees (Craig et al. 2012), the detection of H 2O2 in food (Şansal and Somer 1999), the study of the secondary structure and conformation changes in polyphenol oxidase (Baltacioğ lu et al. 2015), and the detection of adulteration in food (Rodriguez-Saona and Allendorf 2011).
1.3.1 Attenuated Total Reflectance
Although transmission measurements are used, the diffuse reflectance measurements have proved to be much more useful for the analysis of food. New techniques of surface analysis (Chabal 1988) and reflectance analysis (NUANCE 2016) have been employed. These techniques facilitate the monitoring of foods during the production process without the destruction of the sample. Thus, the detection of adulteration and the determination of the quality of the product can frequently be accomplished more rapidly. The technique is called attenuated total reflectance (ATR). It is a sampling technique used in conjunction with IR spectroscopy that enables samples to be examined directly in the solid or liquid state without further separation and purification (Carter et al. 2010). In this aspect, it is similar to Raman spectroscopy (RS) (Mauricio-Iglesias et al. 2009).
A beam of IR light is passed through an ATR crystal so that it reflects at least once off the internal surface in contact with the sample. This reflection forms an evanescent wave, which extends into the sample to a depth of 0.5–2 μm with each reflection along the top surface (NUANCE 2016). The exact value is dependent on the wavelength of the light, the angle of incidence, and the indices of refraction for the ATR crystal and the medium being probed (Minnich et al. 2010), the efficiency of sample contact, the area of contact with the sample, and the crystal material (SpectraTech 2000).
The number of reflections can be varied by varying the angle of incidence. The beam is collected by a detector as it exits the crystal. Most modern IR spectrometers can be converted to ATR by mounting the ATR accessory in the spectrometer’s sample compartment (WOW 2016; Sawant 2016).
The sampling surface is pressed into intimate contact with the top surface of a crystal such as KRS-5, ZnSe, or germanium. To obtain internal reflectance, the angle of incidence must exceed the critical angle. This angle is a function of the refractive indices of both the sample and the ATR crystal. The evanescent wave decays into the sample exponentially with distance from the surface of the crystal over a distance on the order of micrometers. The depth of penetration of the evanescent wave is defined as the distance from the crystal–sample interface at which the intensity of the evanescence decays to 1/e
(37%) of its original value. Different crystals have different refractive indices because of the crystal material. Different crystals are applied to different transmission ranges (ZnSe for 20,000–650 cm−1 and Ge for 5500–800 cm−1) (NUANCE 2016; Sawant 2016). In a spectrum obtained by transmission, the path length is the thickness of the sample. In ATR, the effective path length (EPL) can be calculated as
=× EPL penetrationdepthnumberofreflections.
The EPL is directly related to the absorbance intensity. An increase in either the depth of penetration or in the number of reflections will increase the absorbance intensity of the spectrum. The penetration depth of the IR energy into the sample is proportional to the wavelength. In other words, the depth of penetration decreases when the wavenumber increases. Thus, the relative band intensities in the ATR spectrum decrease with increasing wavenumbers when compared to a transmission spectrum of the same sample (SpectraTech 2000; Suraj 2013).
Examples of the use of ATR include the study of the structure, orientation, and tertiary structure changes in peptides and membrane proteins (Vigano et al. 2000), the study of the structure of potato starch (van Soest et al. 1995), the drying process of sodium alginate films (Xiao et al. 2014), and the determination of linoleic acid in potato chips (Kadamne et al. 2011). Micro-ATR IR spectroscopy has also been investigated (Suraj 2013). ATR-IR has been applied to the microfluidic flow of aqueous solutions in microreactors (Greener et al. 2010) or in flow cells (Carter et al. 2010; Minnich et al. 2010).
Diffuse reflectance infrared Fourier transform spectroscopy (DRIFTS) is a technique that collects and analyzes scattered IR energy. It is used for measurement of fine particles and powders, as well as rough surfaces (NUANCE 2016). When an IR beam enters the sample, it can either be reflected off the surface of a particle or be transmitted through a particle. The IR energy reflecting off the surface is usually lost. The IR beam that passes through a particle can either reflect off the next particle or be transmitted through the next particle. This transmission–reflectance event can occur many times in the sample. The scattered IR energy is focused onto the detector. The IR light is partially absorbed by the sample particles, and this absorption yields information regarding the sample. In the case of colloids and particles in suspension in a volatile solvent, the solvent can be evaporated and the spectrum obtained on the residue (NUANCE 2016).
1.4 NIR SPECTROSCOPY
The NIR and far-IR regions of the spectrum originally furnished much less information about the composition of material because of the wide, overlapping peaks and weak absorbance. NIR began to be employed in 1938, but its use only took hold in the 1980s. The first work on the analytical exploitation of the NIR spectral region involved the determination of the amount of water in gelatin by employing its absorption in the NIR region (Ellis and Bath 1938). Barchewitz was the first to apply NIR spectroscopy for the analysis of fuel (Barchewitz 1943). Other studies were performed in the 1950s (Evans
et al. 1951; White and Barrett 1956; Whetsel et al. 1958) on the use of NIR for the study of mixtures and its obedience to Beer’s law (Pasquini 2003). NIR spectroscopy is a type of vibrational spectroscopy that employs photon energy (h ν) in the energy range from 2.65 × 1019 to 7.96 × 1020 J, which corresponds to the wavelength range from 750 to 2500 nm (Pasquini 2003).
The technique has various advantages: It is fast, nondestructive, and noninvasive, and requires very limited sample preparation. The radiation is highly penetrating and can be used for analyses on the production line. Nearly any molecule containing CH, NH, SH, or OH bonds can be detected. Because of its high penetrability, which allows the detection of substances within the upper layers of tissue, NIR has been employed for the study of the brain and muscle physiology (Ferrari and Quaresima 2004). Several constituents can be measured simultaneously (Osborne 2006). However, because of the large number of wavelengths and absorbances that must normally be computed, the technique requires the use of chemometric techniques and computer control and analysis. It also requires good standards that are adequate for defining the data points that should be measured. It is a secondary method of analysis. A primary method is required to furnish the analytical results necessary for modeling of the NIR spectral data. The model may be very complicated and may need to be updated frequently because of changes in the sample matrix. Robust models might require that many samples be analyzed by the primary method. Also, the technique is not very sensitive; the limit of determination is approximately 0.1% (Pasquini 2003).
Although absorption measurements are used, the diffuse reflectance measurements have proved to be much more useful for the analysis of food. New techniques of surface analysis (Chabal 1988) have been employed. These techniques facilitate the monitoring of foods during the production process without the destruction of the sample. Thus, the detection of adulteration and the determination of the quality of products can frequently be accomplished more rapidly. The NIR region is composed principally of overtones and combination bands corresponding to absorptions in the mid-IR region, as well as the NIR region. The bands observed in the NIR region are weak and generally poorly resolved. Each successive overtone band is approximately an order of magnitude less intense than the preceding one. Thus, several choices of absorptions of different intensity containing the same chemical information are available. Because water absorbs weakly in this region, high-moisture foods can be analyzed (Osborne 2006).
The first type of instrument employed was the dispersive type, which used diffraction gratings. These instruments were used in the early days of NIR spectroscopy and are still being used. They are of relatively low cost compared with other NIR instruments. The main disadvantages of dispersive instruments are the slow scan speed and a lack of wavelength precision, which deteriorates during long-term operation because of mechanical fatigue. Also, the presence of moving parts limits the use of dispersive instruments in the field. However, the development of linear sensor arrays allows the entire spectrum to be scanned in a few seconds, and the lack of moving parts in such instruments means that the dispersive optics have a longer lifetime.
The NIR instruments may use filters to determine the wavelengths to be detected. These instruments are more limited with respect to the range of wavelengths available, but are less expensive and are used mostly for measuring the quantities of specific substances such as water, proteins, and fats. They are usually more robust and have found use in the field or in the online control of production (Morimoto et al. 2001). NIR instruments that use filters are more robust because the optical parts are not harmed by environmental humidity. The detectors for the NIR spectral region are usually based on silicon, PbS, and
InGa. The last has a high sensitivity and response speed. When high-powered radiation sources are used, these detectors can impart a very high signal-to-noise ratio.
Light-emitting diode (LED)-based instruments can produce NIR radiation with a bandwidth of about 30–50 nm, centered in any wavelength of the spectral region. The instruments can employ a set of LEDs as sources of narrow NIR bands (Ellekker et al. 1993; Evans et al. 1993) or use them to produce a polychromatic, highly stable source whose radiation is dispersed by using common monochromator devices such as those based on gratings or filter optics (Goto 1989). The LED and filter instruments are of lower cost, they are smaller, and they can be more adequate for use in the field (Pasquini 2003).
The fourth type of instrument is based on acousto-optic tunable filters (AOTFs) (Abe et al. 1996). Such instruments utilize a technology that involves no moving parts. They are capable of very high speeds over a wide range of the spectral region. The scan speed is very fast, and random access to a large number of frequencies is possible.
The type of spectrophotometer of choice for research utilizes the interferometer and Fourier transform to convert the intensities of the individual frequencies. These instruments possess the highest precision and accuracy of wavelength, a high signal-to-noise ratio, and a high scan speed, although they are slower than AOTF-based instruments (Kays and Barton 2003). These instruments do not have entrance or exit slits that can limit the intensity of the radiation reaching the detector. The wavelength accuracy is better than 0.05 nm and the resolution can reach values below 1 nm in the NIR region. However, the instruments are more expensive than the dispersive or filter types, and they are less robust than the filter or AOTF-based instruments.
Some of the applications of NIR spectroscopy include agricultural products, industrial food products, precision agricultural or soil analyses, polymer processing, polymer quality characteristics, fuel quality control, fuel production processes, petroleum, environmental analyses, textiles, biomedical or clinical analyses, pharmacy and cosmetics, and NIR imaging (Williams and Norris 1987; Pasquini 2003). NIR spectroscopy is used routinely for the compositional, functional, and sensory analysis of food ingredients, process intermediates, and the final products (Pasquini 2003).
NIR spectroscopy has been used to determine protein content (Kays et al. 2000) and soluble and insoluble dietary fiber (Kays 1998; Kays and Barton 2002) in cereal foods and to predict gross energy and usable energy (Kays and Barton 2003). Evans et al. (1993) utilized NIR spectroscopy to determine the authenticity of orange juice. It has also been employed to determine the protein and lactose contents of goat’s milk (Diaz-Carrillo et al. 1993) and the protein, casein, and fat contents in cow’s milk (Laporte and Paquin 1999). Studies of starch and water in cereal food products have been performed (Osborne 1996). The control of the variation in water content of foods (Wahlby and Skjoldebrand 2001) is another application.
1.5 RAMAN SPECTROSCOPY
Chandrasekhara Venkata Raman discovered Raman radiation in 1928 using sunlight as the source, a telescope as a collector, and his eye as the detector. Later, lamps using helium, bismuth, lead, zinc, and mercury were tested, and the mercury lamp was adopted, followed by the mercury burner and the mercury arc. Other types of lamps were studied, but in 1962, the laser source was developed (Ferraro et al. 2003). Photographic plates were first used as detectors until the photomultiplier tube was developed after World War II. Double and triple monochromators were introduced to reduce stray light. Fourier
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A W O R D to an
U N H A P P Y W O M A N.
1.W
HITHER are you going? To heaven or hell? Do you not know? Do you never think about it? Why do you not? Are you never to die? Nay, it is appointed for all men to die. And what comes after? Only heaven or hell.――Will the not thinking of death, put it farther off? No; not a day: not one hour. Or will your not thinking of hell, save you from it? O no: you know better. And you know that every moment you are nearer hell, whether you are thinking of it or no: that is, if you are not nearer heaven. You must be nearer one or the other.
2. I intreat you, think a little on that plain question, Are you going toward heaven or hell? To which of the two does this way lead? Is it possible you should be ignorant? Did you never hear, that neither adulterers nor fornicators, shall inherit the kingdom? That fornicators and adulterers God will judge? And how dreadful will be their sentence, “Depart ye cursed into everlasting fire, prepared for the devil and his angels!”
3. Surely you do not mock at the word of God! You are not yet sunk so low as this. Consider then that awful word, know ye not, that ye are the temples of God? Was not you designed for the Spirit of God to dwell in? Was not you devoted to God in baptism? But if any man defile the temple of God, him shall God destroy. O do not provoke him to it any longer. Tremble before the great, the holy God!
4. Know you not, that your body is, or ought to be, the temple of the Holy Ghost which is in you? Know you not, that you are not your own? For you are bought with a price. And, O how great a price! You are not redeemed with corruptible things, as silver and gold: but with the precious blood of Christ, as of a lamb without blemish and without spot. O when will you glorify God, with your body and your spirit, which are God’s!
5. Ah poor wretch! How far are you from this? How low are you fallen? You yourself are ashamed of what you do. Are you not? Conscience, speak in the sight of God? Does not your own heart condemn you at this very hour? Do not you shudder at the condition you are in?――Dare, for once, to lay your hand upon your breast, and ask, “What am I doing? And what must the end of these things be?” Destruction both of body and soul.
6. Destruction of body as well as of soul! Can it be otherwise? Are you not plunging into misery in this world, as well as in the world to come? What have you brought upon yourself already! What infamy? What contempt? How could you now appear, among those relations or friends, that were once so loved, and so loving to you? What pangs have you given them? How do some of them still weep for you in secret places? And will you not weep for yourself? When you see nothing before you, but want, pain, diseases, death? O spare yourself! Have pity upon your body, if not your soul. Stop! Before you rot above ground and perish!
7. Do you ask, what shall I do? First, Sin no more. First of all, secure this point. Now, this instant now, escape for your life. Stay not. Look not behind you. Whatever you do, sin no more: starve, die, rather than sin. Be more careful for your soul than your body. Take care of that too: but of your poor soul first.
8. “But you have no friend: none, at least, that is able to help you.” Indeed you have: one that is a present help in time of trouble. You have a friend that has all power in heaven and earth, even Jesus Christ the righteous. He loved sinners of old: and he does so still. He then suffered the publicans and harlots to come unto him. And one of them washed his feet with her tears, and wiped them with the hairs of her head. I would to God you were in her place! Say, Amen! Lift up your heart, and it shall be done. How soon will he say, “Woman be of good chear! Thy sins which are many, are forgiven thee.—Go in peace. Sin no more. Love much; for thou hast much forgiven.”
9. Do you still ask, but what shall I do for ♦bread? For food to eat, and raiment to put on? I answer, in the name of the Lord God, (and mark well! His promise shall not fail) seek thou first the kingdom of God and his righteousness, and all these things shall be added unto thee.
♦ “read” replaced with “bread”
Settle it first in your heart, whatever I have or have not, I will not have everlasting burnings. I will not sell my soul and body for bread: better even starve on earth than burn in hell. Then ask help of God. He is not slow to hear. He hath never failed them that seek him. He who feeds the young ravens that call upon him, will not let you perish for lack of sustenance. He will provide, in a way you thought not of, if you seek him with your whole heart. O let your heart be toward him: seek him from the heart. Fear sin, more than want, more than death. And cry mightily to him who bore your sins, till you have bread to eat, that the world knoweth not of; till you have angels food, even the love of God, shed abroad in your heart: till you can say, now I know that my Redeemer liveth, that he hath loved me and given himself for me: and though after my skin worms destroy this body, yet in my flesh shall I see God!
A W O R D to a
S M U G G L E R.
I. “W
HAT is smuggling?” It is the importing, selling, or buying of run goods: that is, those which have not paid the duty appointed by law to be paid to the king.
1. Importing run goods. All smuggling vessels do this with an high hand. It is the chief, if not the whole business of these, to bring goods which have not paid duty.
2. Next to these are all sea captains, officers, sailors, or passengers, who import any thing without paying the duty which the law requires.
3. A third sort of smugglers are all those, who sell any thing which has not paid the duty.
4. A fourth sort, those who buy tea, liquors, linen, handkerchiefs, or any thing else which has not paid duty.
II. “But why should they not? What harm is there in it?”
1. I answer, open smuggling (such as was common a few years ago, on the southern coasts especially) is robbing on the highway: and as much harm as there is in this, just so much there is in smuggling. A smuggler of this kind is no honester than an highwayman. They may shake hands together.
2. Private smuggling is just the same with picking of pockets. There is full as much harm in this as in that. A smuggler of this kind is no honester than a pickpocket. These may shake hands together.
3. But open smugglers are worse than common highwaymen, and private smugglers are worse than common pickpockets. For it is undoubtedly worse to rob our father, than one we have no obligation to. And it is worse still, far worse, to rob a good father, one who sincerely loves us, and is at that very time doing all he can, to provide for us, and to make us happy. Now this is exactly the present case. King George is the father of all his subjects: and not only so, but he is a good father. He shews his love to them on all occasions: and is continually doing all that is in his power, to make his subjects happy
4. An honest man therefore would be ashamed to ask, where is the harm in robbing such a father? His own reason, if he had any at all, would give him a speedy answer. But you are a Christian: are you not? You say, you believe the bible. Then I say to you, in the name of God, and in the name of Christ, Thou shalt not steal. Thou shalt not take what is not thine own, what is the right of another man. But the duties appointed by law are the King’s right, as much as your coat is your right. He has as good a right to them, as you have to this: these are his property, as much as this is yours. Therefore you are as much a thief if you take his duties, as a man is that takes your coat.
5. If you believe the bible, I say to you, as our Saviour said to them of old time, Render unto Cæsar the things that are Cæsar’s, and unto God the things that are God’s. If then you mind our Saviour’s words, be as careful to honour the King, as to fear God. Be as exact in giving the king, what is due to the king, as in giving God what is due to God. Upon no account whatever rob or defraud him of the least thing which is his lawful property.
6. If you believe the bible, I say to you, as St. Paul said to the ancient Christians, Render unto all their dues: in particular, Custom to whom custom is due, tribute to whom tribute. Now custom is by the laws of England due to the king. Therefore every one in England is bound to pay it him. So that robbing the king herein, is abundantly worse than common stealing, or common robbing on the highway.
7. And so it is, on another account also: for it is a general robbery: it is, in effect, not only robbing the king, but robbing every honest man in the nation. For the more the king’s duties are diminished, the more the taxes must be increased. And these lie upon us all: they are the burden not of some, but of all the people of England. Therefore every smuggler is a thief-general, who picks the pockets both of the king, and all his fellow-subjects. He wrongs them all; and above all, the honest traders: many of whom he deprives of their maintenance: constraining them either not to sell their goods at all, or to sell them to no profit. Some of them are tempted hereby, finding they cannot get bread for their families, to turn thieves too. And then you are accountable for their sin as well as your own: you bring their blood upon your own head. Calmly consider this, and you will never more ask, “What harm there is in smuggling?”
III. 1. But for all this, cannot men find excuses for it? Yes, abundance; such as they are. “I would not do this, says one; I would not sell uncustomed goods: but I am under a necessity: I can’t live without it.” I answer, may not the man who stops you on the highway, say the very same? “I would not take your purse; but I am under a necessity: I cannot live without it.” Suppose the case to be your own; and will you accept of this excuse? Would not you tell him, “Let the worst come to the worst, you had better be honest, though you should starve.” But that need not be neither. Others who had no more than you to begin with, yet find a way to live honestly. And certainly so may you: however, settle it in your heart, “Live or die, I will be an honest man.”
2. “Nay, says another, we do not wrong the king: for he loses nothing by us. Yea, on the contrary, the king is rather a gainer, namely by the seizures that are made.”
So you plunder the king, out of stark love and kindness! You rob him, to make him rich! It is true, you take away his purse: but you put an heavier in its place! Are you serious? Do you mean what you say? Look me in the face and tell me so. You cannot. You know in your own conscience, that what comes to the king, out of all seizures made the year round, does not amount to the tenth, no not to the hundredth part of what he is defrauded of.
But if he really gained more than he lost, that would not excuse you. You are not to commit robbery, though the person robbed were afterwards to gain by it. You are not to do evil, that good may come If you do, your damnation is just.
“But certainly, say some, the king is a gainer by it, or he might easily suppress it.” Wilt you tell him, which way? By Custom-house officers? But many of them have no desire to suppress it. They find their account in its continuance: they come in for a share of the plunder. But what if they had a desire to suppress it? They have not the power. Some of them have lately made the experiment: and what was the consequence? Why they lost a great part of their bread, and were in danger of losing their lives.
♦8. Can the king suppress smuggling, by parties of soldiers? That he cannot do. For all the soldiers he has are not enough, to watch every port and every creek in Great-Britain. Besides, the soldiers that are employed, will do little more than the Custom-house officers. For there are ways and means to take off their edge too, and making them as quiet as lambs.
♦ Paragraph number out of sequence.
“But many courtiers and great men, who know the king’s mind, not only connive at smuggling, but practise it.” And what can we infer from this? Only that those great men are great villains. They are great highwaymen and pickpockets: and their greatness does not excuse, but makes their crime tenfold more inexcusable.
But besides. Suppose the king were willing to be cheated, how would this excuse your cheating his subjects? All your fellowsubjects, every honest man, and in particular, every honest trader? How would it excuse, your making it impossible for him to live, unless he will turn knave as well as yourself?
3. “Well, but I am not convinced it is a sin: My conscience does not condemn me for it.” No! Are you not convinced, that robbery is a sin? Then I am sorry for you. And does not your conscience condemn you for stealing? Then your conscience is asleep. I pray God to smite you to the heart, and awaken it this day!
4. “Nay, but my soul is quite happy in the love of God: therefore I cannot think it is wrong.” I answer, wrong it must be, if the bible is right. Therefore either that love is a mere delusion, a fire of your own kindling; or God may have hitherto winked at the times of ignorance. But now you have the means of knowing better. Now light is offered to you. And if you shut your eyes against the light, the love of God cannot possibly continue.
5. “But I only buy a little brandy or tea now and then, just for my own use.” That is, I only steal a little. God says, steal not at all.
6. “Nay, I do not buy any at all myself: I only send my child or servant for it.” You receive it of them: Do you not? And the receiver is as bad as the thief.
7. “Why I would not meddle with it, but I am forced, by my parent, husband, or master.” If you are forced by your father or mother to rob, you will be hanged nevertheless. This may lessen, but does not take away the fault: for you ought to suffer rather than sin.
8. “But I do not know, that it was run.” No! Did not he that sold it, tell you it was? If he sold it under the common price, he did. The naming the price, was telling you, “This is run.”
9. “But I don’t know where to get tea which is not run.” I will tell you where to get it. You may have it from those whose tea is duly entered, and who make a conscience of it. But were it otherwise, if I could get no wine, but what I knew to be stolen, I would drink water: yea, though not only my health, but my life depended upon it: for it is better to die, than to live by thieving.
10. “But if I could get what has paid duty, I am not able to pay the price of it. And I can’t live without it.” I answer, 1. You can live without it, as well as your grandmother did. But 2. If you could not live without it, you ought to die, rather than steal. For death is a less evil than sin.
11. “But my husband will buy it, whether I do or no. And I must use what he provides, or have none.” Undoubtedly to have none is a less evil, than to be partaker with a thief.
IV. Upon the whole then, I exhort all of you that fear God, and desire to save your souls, without regarding what others do, resolve at all hazards, to keep yourselves pure. Let your eye be fixed on the word of God, not the examples of men. Our Lord says to every one of you, What is that to thee? Follow thou me! Let no convenience, no gain, no pleasure, no friend, draw you from following him. In spite of all the persuasions, all the reasonings of men, keep to the word of God. If all on the right-hand and the left will be knaves, be you an honest man. Probably God will repay you (he certainly will, if this be best for you) even with temporal blessings: there have not been wanting remarkable instances of this. But if not, he will repay you with what is far better: with the testimony of a good conscience towards God; with joy in the Holy Ghost; with an hope full of immortality; with the love of God shed abroad in your hearts. And the peace of God, which passeth all understanding, shall keep your hearts and minds in Christ Jesus!
L , January 30, 1767.
A
WHAT a condition are you in? The sentence is past: you are condemned to die: and this sentence is to be executed shortly. You have no way to escape; these fetters, these walls, these gates and bars, these keepers cut off all hope. Therefore die you must: but must you die like a beast, without thinking what it is to die?
You need not: you will not: you will think a little first: you will consider, what is death? It is leaving this world, these houses, lands, and all things under the sun; leaving all these things, never to return; your place will know you no more. It is leaving these pleasures; for there is no eating, drinking, gaming, no merriment in the grave. It is leaving your acquaintance, companions, friends: your father, mother, wife, children. You cannot stay with them, nor can they go with you: you must part; perhaps for ever. It is leaving a part of yourself; leaving this body which has accompanied you so long. Your soul must now drop its old companion, to rot and moulder into dust. It must enter upon a new, strange, unbodied state. It must stand naked before God!
2. But O! how will you stand before God? The great, the holy, the just, the terrible God? Is it not his own word, Without holiness no man shall see the Lord? No man shall see him with joy: rather he will call for the mountains to fall upon him and the rocks to cover him. And what do you think holiness is? It is purity both of heart and life. It is the mind that was in Christ, enabling us to walk as he also walked. It is the loving God with all our heart, the loving our neighbour, every man as ourselves, and the doing to all men, in every point, as we would they should do unto us. The least part of holiness is, to do good to all men, and to do no evil either in word or work. This is only the outside of it. But this is more than you have. You are from it; far as darkness from light. You have not the mind that was in Christ: there was no pride, no malice in him: no hatred, no revenge, no furious anger, no foolish or worldly desire. You have not walked as Christ walked: no; rather as the devil would have walked, had he been in a body; the works of the devil you have done, not the works of God. You have not loved God with all your heart. You have not loved him at all. You have not thought about him. You hardly knew or cared, whether there was any God in the world. You have not done to others as you would they should do to you; far, very far from it. Have you done all the good you could to all men? If so, you had never come to this place. You have done evil exceedingly: your sins against God and man are more than the hairs of your head. Insomuch that even the world cannot bear you; the world itself spues you out. Even the men that know not God declare, you are not fit to live upon the earth.
3. O repent, repent! Know yourself: see and feel what a sinner you are. Think of the innumerable sins you have committed, even from your youth up. How many wicked words have you spoken? How many wicked actions have you done? Think of your inward sins! Your pride, malice, hatred, anger, revenge, lust. Think of your sinful nature, totally alienated from the life of God. How is your whole soul prone to evil, void of good, corrupt, full of all abominations! Feel, that your carnal mind is enmity against God. Well may the wrath of God abide upon you. He is of purer eyes than to behold iniquity: he hath said, The soul that sinneth, it shall die It shall die eternally, shall be punished with everlasting destruction, from the presence of the Lord and from the glory of his power.
4. How then can you escape the damnation of hell? The lake of fire burning with brimstone? Where the worm dieth not, and the fire is not quenched? You can never redeem your own soul. You cannot atone for the sins that are past. If you could leave off sin now, and live unblamable for the time to come, that would be no atonement for what is past. Nay, if you could live like an angel for a thousand years, that would not atone for one sin. But neither can you do this: you cannot leave off sin: it has the dominion over you. If all your past sins were now to be forgiven, you would immediately sin again: that is, unless your heart were cleansed; unless it were created anew. And who can do this? Who can bring a clean thing out of an unclean? Surely none but God. So you are utterly sinful, guilty, helpless! What can you do to be saved?
5. One thing is needful: believe in the Lord Jesus Christ, and thou shalt be saved! Believe (not as the devils only, but) with that faith which is the gift of God, which is wrought in a poor, guilty, helpless sinner, by the power of the Holy Ghost. See all thy sins on Jesus laid. God laid on him the iniquities of us all. He suffered once the just for the unjust. He bore our sins in his own body on the tree. He was wounded for thy sins; he was bruised for thy iniquities. Behold the Lamb of God, taking away the sin of the world! Taking away thy sins, even thine, and reconciling thee unto God the Father! Look unto him and be thou saved! If thou look unto him by faith, if thou cleave to him with thy whole heart, if thou receive him both to atone, to teach and to govern thee in all things, thou shalt be saved, thou art saved, both from the guilt, the punishment, and all the power of sin. Thou shalt have peace with God, and a peace in thy own soul, that passeth all understanding. Thy soul shall magnify the Lord, and thy Spirit rejoice in God thy Saviour. The love of God shall be shed abroad in thy heart, enabling thee to trample sin under thy feet. And thou wilt then have an hope full of immortality. Thou wilt no longer be afraid to die, but rather long for the hour having a desire to depart, and to be with Christ.
6. This is the faith that worketh by love, the way that leadeth to the kingdom. Do you earnestly desire to walk therein? Then put away all hindrances. Beware of company: At the peril of your soul, keep from those who neither know nor seek God. Your old acquaintance are no acquaintance for you, unless they too acquaint themselves with God. Let them laugh at you, or say, you are running mad. It is enough, if you have praise of God. Beware of strong drink. Touch it not, lest you should not know when to stop. You have no need of this to chear your spirits; but of the peace and the love of God: beware of men that pretend to shew you the way to heaven, and know it not themselves. There is no other name whereby you can be saved, but the name of our Lord Jesus Christ. And there is no other way whereby you can find the virtue of his name but by faith. Beware of Satan transformed into an angel of light, and telling you, it is presumption to believe in Christ, as your Lord and your God, your wisdom and righteousness, sanctification and redemption. Believe in him with your whole heart. Cast your whole soul upon his love. Trust him alone: love him alone: fear him alone: and cleave to him alone: Till he shall say to you (as to the dying malefactor of old,) This day shalt thou be with me in paradise.
Advice to an ENGLISHMAN. ¹
¹ This was published at the beginning of the late rebellion.
O you ever think? Do you ever consider? If not, ’tis high time you should. Think a little, before it is too late. Consider what a state you are in. And not you alone, but our whole nation. We would have war. And we have it. And what is the fruit? Our armies broken in pieces: And thousands of our men either killed on the spot or made prisoners in one day. Nor is this all. We have now war at our own doors: our own countrymen turning their swords against their brethren. And have any hitherto been able to stand before them? Have they not already seized upon one whole kingdom? Friend, either think now, or sleep on and take your rest, till you drop into the pit where you will sleep no more?
2. Think, what is likely to follow, if an army of French also, should blow the trumpet in our land! What desolation may we not then expect? What a wide-spread field of blood? And what can the end of these things be? If they prevail, what but Popery and Slavery? Do you know what the spirit of Popery is? Did you never hear of that in queen Mary’s reign? And of the holy men who were then burnt alive by the Papists, because they did not dare to do as they did? To worship angels and saints; to pray to the virgin Mary; to bow down to images, and the like. If we had a king of this spirit, whose life would be safe? At least, what honest man’s? A knave indeed might turn with the times. But what a dreadful thing would this be to a man of conscience? “Either turn, or burn. Either go into that fire: or into the fire that never shall be quenched.”
3. And can you dream that your property would be any safer than your conscience? Nay, how should that be? Nothing is plainer than that the Pretender cannot be king of England, unless it be by conquest. But every conqueror may do what he will. The laws of the land are no laws to him. And who can doubt, but one who should conquer England by the assistance of France, would copy after the French rules of government?
4. How dreadful then is the condition wherein we stand? On the very brink of utter destruction! But why are we thus? I am afraid the answer is too plain, to every considerate man. Because of our sins: because we have well-nigh filled up the measure of our iniquities. For, what wickedness is there under heaven, which is not found among us at this day? Not to insist on the sabbath-breaking in every corner of our land, the thefts, cheating, fraud, extortion; the injustice, violence, oppression; the lying and dissimulating; the robberies, sodomies and murders (which, with a thousand unnamed ♦villainies are common to us and our neighbour Christians of Holland, France, and Germany:) consider over and above, what a plentiful harvest we have of wickedness almost peculiar to ourselves? For who can vie with us, in the direction of courts of justice? In the management of public charities? Or, in the accomplished, barefaced wickedness, which so abounds in our prisons, and fleets, and armies? Who in Europe can compare with the sloth, laziness, luxury and effeminacy of the English gentry? Or with the drunkenness, and stupid, senseless cursing and swearing, which are daily seen and heard in our streets? One great inlet, no doubt, to that flood of perjury, which so increases among us day by day: the like whereunto is not to be found, in any other part of the habitable earth.
♦ “villanies” replaced with “villainies”
5. Add to all these (what is indeed the source as well as completion of all) that open and profess’d D and rejection of the G , that public, avowed apostacy from the Christian faith, which reigns among the rich and great, and hath spread from them to all ranks and orders of men (the vulgar themselves not excepted) and made us a people fitted for the destroyer of the Gentiles.
6. Because of these sins is this evil come upon us. For (whether you are aware of it, or no) there is a God: a God, who tho’ he sits upon the circle of the heavens, sees and knows all that is done upon earth. And this God is holy; he does not love sin: he is just, rendering to all their due. And he is strong; there is none able to withstand him: he hath all power in heaven and in earth. He is patient indeed, and suffers long; but he will at last repay the wicked to his face. He often does so in this world; especially when a whole nation is openly and insolently wicked. Then doth God arise and maintain his own cause; then doth he terribly shew both his justice and power: that if these will not repent, yet others may fear, and flee from the wrath to come.
7. There hath been among them that feared God, a general expectation for many years, that the time was coming, when God would thus arise, to be avenged on this sinful nation. At length the time is come. The patience of God, long provoked, gives place to justice. The windows of heaven begin to be opened, to rain down judgments on the earth. And yet, with what tenderness does he proceed? In the midst of wrath remembring mercy. By how slow degrees does his vengeance move! Nor does his whole displeasure yet arise.
8. Brethren, countrymen, Englishmen, What shall we do? To-day! While it is called to-day! Before the season of mercy is quite expired, and our destruction cometh as a whirlwind? Which way can we remove the evils we feel? Which way prevent those we fear? Is there any better way, than the making God our friend? The securing his help against our enemies? Other helps are little worth. We see armies may be destroyed, or even flee away from old men and children. Fleets may be dashed to pieces in an hour, and sunk in the depth of the sea. Allies may be treacherous, or slow, or foolish, or weak, or cowardly But God is a friend who cannot betray, and whom none can either bribe or terrify. And who is wise, or swift, or strong like him? Therefore, whatever we do, let us make God our friend. Let us with all speed remove the cause of his anger. Let us cast away our sins. Then shall his love have free course, and he will send us help, sufficient help, against all our enemies.
9. Come; will you begin? Will you, by the grace of God, amend one, and that without delay? First then, own those sins which have long cried for vengeance in the ears of God. Confess, that we and all (and you in particular) deserve for our inward and outward abominations, not only to be swept from the face of the earth, but to suffer the vengeance of eternal fire. Never aim at excusing either yourself or others: Let your mouth be stopt. Plead guilty before God. Above all, own that impudence of wickedness, that utter ♦carelessness, that pert stupidity, which is hardly to be found in any part of the earth, (at least, not in such a degree) except in England. Do you not know what I mean? You was not long since praying to God for “damnation upon your own soul.” One who has heard you, said, is that right? Does not God hear? “What if he takes you at your word?” You replied, with equal impudence and ignorance, “What, Are you a Methodist?”――What, if he is a Turk? Must thou therefore be a Heathen?――God humble thy brutish, devilish spirit.
♦ “carlessness” replaced with “carelessness”
10. Lay thee in the dust, for this and for all thy sins. Let thy laughter be turned into heaviness; thy joy into mourning; thy senseless jollity and mirth, into sorrow and brokenness of heart. This is no time to eat and drink and rise up to play; but to afflict thy soul before the Lord. Desire of God a deep piercing sense of the enormous sins of the nation, and of thy own. Remember that great example: how when the king of Nineveh was warned of the near approaching vengeance of God, he caused it to be proclaimed, Let none taste any thing, let them not feed nor drink water. But let them be covered with sackcloth, and cry mightily to God; yea let them turn every one from his evil way; who can tell, if God will turn and repent, and turn away from his fierce anger that we perish not. Jonah iii.
11. Let them turn every one from his evil way. Cease to do evil. Learn to do well. And see that this reformation be universal: for there is no serving God by halves. Avoid all evil, and do all good unto all men; else you only deceive your own soul. See also, that it be from the heart: lay the axe to the root of the tree. Cut up, by the grace of God, evil desire, pride, anger, unbelief. Let this be your continual prayer to God, the prayer of your heart, (as well as lips) “Lord, I would believe: help thou mine unbelief! Give me the faith that worketh by love. The life which I now live, let me live by faith in the Son of God. Let me so believe, that I may love thee, with all my heart, and mind, and soul, and strength! and that I may love every child of man, even as thou hast loved us! Let me daily add to my faith courage, knowledge, temperance, patience, brotherly kindness, charity: that so an entrance may be ministered to me abundantly, into the everlasting kingdom of our Lord and Saviour Jesus Christ.”
REGARD, thou righteous God and true, Regard thy weeping people’s prayer, Before the sword our land go through, Before thy latest plague we bear, Let all to thee their smiter turn, Let all beneath thine anger mourn. The sword, which first bereav’d abroad, We now within our borders see: We see, but slight thy nearer rod, So oft so kindly warn’d by thee: We still thy warning love despise, And dare thine utmost wrath to rise Yet for the faithful remnants sake Thine utmost wrath awhile defer, If haply we at last may wake, And trembling at destruction near The cause of all our evils own, And leave the sins for which we groan. Or if the wicked will not mourn, And ’scape the long-suspended blow, Yet shall it to thy glory turn, Yet shall they all thy patience know, Thy slighted love and mercy clear, And vindicate thy justice here.
