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Food Science Text Series
S. Suzanne Nielsen
Food Analysis
Laboratory Manual
Third Edition
Food Science
Text Series
Third Edition
For other titles published in this series, go to

www.springer.com/series/5999
Series editor:
Dennis R. Heldman
Heldman Associates
Mason, Ohio, USA
The Food Science Text Series provides faculty with the leading teaching tools. The Editorial Board has
outlined the most appropriate and complete content for each food science course in a typical food science
program and has identified textbooks of the highest quality, written by the leading food science educators.
Series Editor Dennis R. Heldman, Professor, Department of Food, Agricultural, and Biological Engineering,
The Ohio State University. Editorial Board; John Coupland, Professor of Food Science, Department of Food
Science, Penn State University, David A. Golden, Ph.D., Professor of Food Microbiology, Department of Food
Science and Technology, University of Tennessee, Mario Ferruzzi, Professor, Food, Bioprocessing and
Nutrition Sciences, North Carolina State University, Richard W. Hartel, Professor of Food Engineering,
Department of Food Science, University of Wisconsin, Joseph H. Hotchkiss, Professor and Director of the
School of Packaging and Center for Packaging Innovation and Sustainability, Michigan State University,
S. Suzanne Nielsen, Professor, Department of Food Science, Purdue University, Juan L. Silva, Professor,
Department of Food Science, Nutrition and Health Promotion, Mississippi State University, Martin
Wiedmann, Professor, Department of Food Science, Cornell University, Kit Keith L. Yam, Professor of Food
Science, Department of Food Science, Rutgers University
Food Analysis
Laboratory Manual
Third Edition
edited by
S. Suzanne Nielsen
Purdue University
West Lafayette, IN, USA
S. Suzanne Nielsen
Department of Food Science
Purdue University
West Lafayette
Indiana
USA
ISSN 1572-0330 ISSN 2214-7799 (electronic)

Food Science Text Series
ISBN 978-3-319-44125-2 ISBN 978-3-319-44127-6 (eBook)
DOI 10.1007/978-3-319-44127-6
Library of Congress Control Number: 2017942968
© Springer International Publishing 2017

This work is subject to copyright. All rights are reserved by the Publisher, whether the whole or part of the material is concerned,
specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfilms or in any other
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The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication does not imply, even in the
absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for
general use.
The publisher, the authors and the editors are safe to assume that the advice and information in this book are believed to be true and
accurate at the date of publication. Neither the publisher nor the authors or the editors give a warranty, express or implied, with respect
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Printed on acid-free paper
This Springer imprint is published by Springer Nature

The registered company is Springer International Publishing AG
The registered company address is: Gewerbestrasse 11, 6330 Cham, Switzerland
Preface and Acknowledgments
This laboratory manual was written to accompany able for teaching food analysis laboratory
the textbook, Food Analysis, fifth edition. The labora- sessions vary considerably between schools,
tory exercises are tied closely to the text and cover 21 as do student numbers and their level in
of the 35 chapters in the textbook. Compared to the school. Therefore, instructors may need to
second edition of this laboratory manual, this third modify the laboratory procedures (e.g., num-
edition contains four introductory chapters with ber of samples analyzed, replicates) to fit their
basic information that compliments both the text- needs and situation. Some experiments
book chapters and the laboratory exercises (as include numerous parts/methods, and it is
described below). Three of the introductory chapters not assumed that an instructor uses all parts
include example problems and their solutions, plus of the experiment as written. It may be logical
additional practice problems at the end of the chap- to have students work in pairs to make things
ter (with answers at the end of the laboratory man- go faster. Also, it may be logical to have some
ual). This third edition also contains three new students do one part of the experiment/one
laboratory exercises, and previous experiments have type of sample and other students to another
been updated and corrected as appropriate. Most of part of the experiment/type of sample.
the laboratory exercises include the following: back- 4. Use of Chemicals: The information on hazards
ground, reading assignment, objective, principle of and precautions in the use of the chemicals for
method, chemicals (with CAS number and hazards), each experiment is not comprehensive but
reagents, precautions and waste disposal, supplies, should make students and a laboratory assis-
equipment, procedure, data and calculations, ques- tant aware of major concerns in handling and
tions, and resource materials. disposing of the chemicals.
Instructors using these laboratory exercises 5. Reagent Preparation: It is recommended in the
should note the following: text of the experiments that a laboratory assis-
tant prepare many of the reagents, because of
1. Use of Introductory Chapters: the time limitations for students in a laboratory
• Chap. 1, “Laboratory Standard Operating session. The lists of supplies and equipment for
Procedures” – recommended for students experiments do not necessarily include those
prior to starting any food analysis labora- needed by the laboratory assistant in preparing
tory exercises reagents for the laboratory session.
• Chap. 2, “Preparation of Reagents and 6. Data and Calculations: The laboratory exer-
Buffers” – includes definition of units of cises provide details on recording data and
concentrations, to assist in making chemi- doing calculations. In requesting laboratory
cal solutions reports from students, instructors will need to
• Chap. 3, “Dilution and Concentration specify if they require just sample calculations
Calculations” – relevant for calculations in or all calculations.
many laboratory exercises
• Chap. 4, “Use of Statistics in Food Even though this is the third edition of this labo-
Analysis” – relevant to data analysis ratory manual, there are sure to be inadvertent omis-
2. Order of Laboratory Exercises: The order of sions and mistakes. I will very much appreciate
laboratory exercises has been changed to be receiving suggestions for revisions from instructors,
fairly consistent with the reordering of chap- including input from lab assistants and students.
ters in the textbook, Food Analysis, fifth edition I maintain a website with additional teaching
(i.e., chromatography and spectroscopy near materials related to both the Food Analysis textbook
the front of the book). However, each labora- and laboratory manual. Instructors are welcome to
tory exercise stands alone, so they can be cov- contact me for access to this website. To compliment
ered in any order. the laboratory manual, the website contains more
3. Customizing Laboratory Procedures: It is rec- detailed versions of select introductory chapters and
ognized that the time and equipment avail- Excel sheets related to numerous laboratory exercises.
v
vi Preface and Acknowledgments
I am grateful to the food analysis instructors much appreciated. Special thanks go to Baraem
identified in the text who provided complete labora- (Pam) Ismail and Andrew Neilson for their input
tory experiments or the materials to develop the and major contributions toward this edition of the
experiments. For this edition, I especially want to laboratory manual. My last acknowledgment goes to
thank the authors of the new introductory chapters my former graduate students, with thanks for their
who used their experience from teaching food analy- help in working out and testing all experimental pro-
sis to develop what I hope will be very valuable cedures written for the initial edition of the labora-
chapters for students and instructors alike. The input tory manual.
I received from other food analysis instructors, their
students, and mine who reviewed these new intro- West Lafayette, IN, USA S. Suzanne Nielsen
ductory chapters was extremely valuable and very
Contents
Preface and Acknowledgments v 4.6 t-Scores 58
4.7 t-Tests 59
4.8 Practical Considerations 61
Part 1 Introductory Chapters 4.9 Practice Problems 62
4.10 Terms and Symbols 62
1 Laboratory Standard Operating Procedures 3
1.1 Introduction 5
1.2 Precision and Accuracy 5 Part 2 Laboratory Exercises
1.3 Balances 6
1.4 Mechanical Pipettes 7 5 Nutrition Labeling Using a Computer
1.5 Glassware 9 Program 65
1.6 Reagents 16 5.1 Introduction 67
1.7 Data Handling and Reporting 18 5.2 Preparing Nutrition Labels for Sample
1.8 Basic Laboratory Safety 19 Yogurt Formulas 67
5.3 Adding New Ingredients to a Formula
2 Preparation of Reagents and Buffers 21 and Determining How They Influence
2.1 Preparation of Reagents of Specified the Nutrition Label 68
Concentrations 22 5.4 An Example of Reverse Engineering
2.2 Use of Titration to Determine in Product Development 69
Concentration of Analytes 24 5.5 Questions 70
2.3 Preparation of Buffers 25
2.4 Notes on Buffers 30 6 Accuracy and Precision Assessment 71
2.5 Practice Problems 31 6.1 Introduction 72
6.2 Procedure 73
3 Dilutions and Concentrations 33 6.3 Data and Calculations 74
3.1 Introduction 34 6.4 Questions 74
3.2 Reasons for Dilutions
and Concentrations 34 7 High-Performance Liquid
3.3 Using Volumetric Glassware Chromatography 77
to Perform Dilutions 7.1 Introduction 79
and Concentrations 34 7.2 Determination of Caffeine in Beverages
3.4 Calculations for Dilutions By HPLC 79
and Concentrations 34 7.3 Solid-Phase Extraction and HPLC Analysis
3.5 Special Cases 40 of Anthocyanidins from Fruits
3.6 Standard Curves 41 and Vegetables 81
3.7 Unit Conversions 44
3.8 Avoiding Common Errors 45 8 Gas Chromatography 87
3.9 Practice Problems 46 8.1 Introduction 89
8.2 Determination of Methanol and Higher
4 Statistics for Food Analysis 49 Alcohols in Wine by Gas
4.1 Introduction 50 Chromatography 89
4.2 Population Distributions 50 8.3 Preparation of Fatty Acid Methyl
4.3 Z-Scores 51 Esters (FAMEs) and Determination
4.4 Sample Distributions 54 of Fatty Acid Profile of Oils by Gas
4.5 Confidence Intervals 55 Chromatography 91
vii
viii Contents
9 Mass Spectrometry with High-Performance 16 Water Hardness Testing by Complexometric

Liquid Chromatography 97 Determination of Calcium 147
9.1 Introduction 98 16.1 Introduction 149
9.2 Procedure 100 16.2 EDTA Titrimetric Method for Testing
9.3 Data and Calculations 101 Hardness of Water 149
9.4 Questions 102 16.3 Test Strips for Water Hardness 151
9.5 Case Study 102
17 Phosphorus Determination by Murphy-Riley
10 Moisture Content Determination 105 Method 153
10.2 Forced Draft Oven 107 17.2 Procedure 155
10.3 Vacuum Oven 109 17.3 Data and Calculations 155
10.4 Microwave Drying Oven 110 17.4 Questions 155
10.5 Rapid Moisture Analyzer 111
10.6 Toluene Distillation 111
10.7 Karl Fischer Method 112 18 Iron Determination by Ferrozine Method 157
10.8 Near-Infrared Analyzer 114 18.1 Introduction 158
10.9 Questions 114 18.2 Procedure 158
18.3 Data and Calculations 159
18.4 Question 159
11 Ash Content Determination 117
11.1 Introduction 118
11.2 Procedure 118 19 Sodium Determination Using Ion-Selective
11.3 Data and Calculations 118 Electrodes, Mohr Titration, and Test Strips 161
11.4 Questions 119 19.1 Introduction 163
19.2 Ion-Selective Electrodes 163
19.3 Mohr Titration 165
12 Fat Content Determination 121 19.4 Quantab® Test Strips 167
12.1 Introduction 123 19.5 Summary of Results 169
12.2 Soxhlet Method 123 19.6 Questions 170
12.3 Goldfish Method 125
12.4 Mojonnier Method 125
12.5 Babcock Method 127 20 Sodium and Potassium Determinations
by Atomic Absorption Spectroscopy
and Inductively Coupled Plasma-Optical
13 Protein Nitrogen Determination 131 Emission Spectroscopy 171
13.2 Kjeldahl Nitrogen Method 132 20.2 Procedure 174
13.3 Nitrogen Combustion Method 135 20.3 Data and Calculations 176
20.4 Questions 177
14 Total Carbohydrate by Phenol-Sulfuric
Acid Method 137 21 Standard Solutions and Titratable Acidity 179
14.2 Procedure 139 21.2 Preparation and Standardization of Base
14.3 Data and Calculations 140 and Acid Solutions 180
14.4 Questions 141 21.3 Titratable Acidity and pH 182
15 Vitamin C Determination by Indophenol 22 Fat Characterization 185

Method 143 22.1 Introduction 187
15.1 Introduction 144 22.2 Saponification Value 187
15.2 Procedure 145 22.3 Iodine Value 188
15.3 Data and Calculations 145 22.4 Free Fatty Acid Value 190
15.4 Questions 146 22.5 Peroxide Value 191
22.6 Thin-Layer Chromatography Separation
of Simple Lipids 193
Contents ix
23 Proteins: Extraction, Quantitation, 27 CIE Color Specifications Calculated

and Electrophoresis 195 from Reflectance or Transmittance Spectra 219
23.2 Reagents 197 27.2 Procedure 222
23.3 Supplies 198 27.3 Questions 224
23.4 Procedure 198
23.6 Questions 200 28 Extraneous Matter Examination 225
28.2 Extraneous Matter in Soft Cheese 227
24 Glucose Determination by Enzyme 28.3 Extraneous Matter in Jam 228
Analysis 203 28.4 Extraneous Matter in Infant Food 229
24.1 Introduction 204 28.5 Extraneous Matter in Potato Chips 229
24.2 Procedure 205 28.6 Extraneous Matter in Citrus Juice 230
24.3 Data and Calculations 205 28.7 Questions 230
24.4 Questions 205
Part 3 Answers to Practice Problems
25 Gliadin Detection by Immunoassay 207
29 Answers to Practice Problems in Chap. 2,
Preparation of Reagents and Buffers 233
25.2 Procedure 209
25.4 Questions 211 30 Answers to Practice Problems in Chap. 3,
Dilutions and Concentrations 239
26 Viscosity Measurements of Fluid Food
Products 213 31 Answers to Practice Problems in Chap. 4,
26.1 Introduction 214 Use of Statistics in Food Analysis 247
26.2 Procedure 214
26.3 Data 216
26.4 Calculations 216
26.5 Questions 217
Contributors
Charles E. Carpenter Department of Nutrition, Oscar A. Pike Department of Nutrition, Dietetics,
Dietetics and Food Sciences, Utah State University, and Food Science, Brigham Young University, Provo,
Logan, UT, USA UT, USA
Young-Hee Cho Department of Food Science, Michael C. Qian Department of Food Science and
Purdue University, West Lafayette, IN, USA Technology, Oregon State University, Corvallis, OR,
USA
M. Monica Giusti Department of Food Science and
Technology, The Ohio State University, Columbus, Qinchun Rao Department of Nutrition, Food and
OH, USA Exercise Sciences, Florida State University,
Tallahassee, FL, USA
Y.H. Peggy Hsieh Department of Nutrition, Food
and Exercise Sciences, Florida State University, Ann M. Roland Owl Software, Columbia, MO, USA
Tallahassee, FL, USA
Daniel E. Smith Department of Food Science and
Baraem P. Ismail Department of Food Science and Technology, Oregon State University, Corvallis, OR,
Nutrition, University of Minnesota, St. Paul, MN, USA
USA
Denise M. Smith School of Food Science,
Helen S. Joyner School of Food Science, University Washington State University, Pullman, WA, USA
of Idaho, Moscow, ID, USA
Stephen T. Talcott Department of Nutrition and
Dennis A. Lonergan The Vista Institute, Eden Food Science, Texas A&M University, College
Prairie, MN, USA Station, TX, USA
Lloyd E. Metzger Department of Dairy Science, Catrin Tyl Department of Food Science and
University of South Dakota, Brookings, SD, USA Nutrition, University of Minnesota, St. Paul, MN,
USA
Andrew P. Neilson Department of Food Science
and Technology, Virginia Polytechnic Institute and Robert E. Ward Department of Nutrition, Dietetics
State University, Blacksburg, VA, USA and Food Sciences, Utah State University, Logan, UT,
USA
S. Suzanne Nielsen Department of Food Science,
Purdue University, West Lafayette, IN, USA Ronald E. Wrolstad Department of Food Science
and Technology, Oregon State University, Corvallis,
Sean F. O’Keefe Department of Food Science and
OR, USA
Technology, Virginia Tech, Blacksburg, VA, USA
xi
1
part
Introductory Chapters
1
chapter
Laboratory Standard
Operating Procedures
Andrew P. Neilson (*)
Department of Food Science and Technology,
Virginia Polytechnic Institute and State University,
Blacksburg, VA, USA
e-mail: andrewn@vt.edu
Dennis A. Lonergan
The Vista Institute,
Eden Prairie, MN, USA
e-mail: dennis@thevistainstitute.com
S. Suzanne Nielsen
Department of Food Science, Purdue University,
West Lafayette, IN, USA
e-mail: nielsens@purdue.edu
S.S. Nielsen, Food Analysis Laboratory Manual, Food Science Text Series, 3
DOI 10.1007/978-3-319-44127-6_1, © Springer International Publishing 2017
1.1 Introduction 1.6 Reagents
1.2 Precision and Accuracy 1.6.1 Acids
1.3 Balances 1.6.2 Distilled Water
1.3.1 Types of Balances 1.6.3 Water Purity
1.3.2 Choice of Balance 1.6.4 Carbon Dioxide-Free Water
1.3.3 Use of Top Loading Balances 1.6.5 Preparing Solutions and Reagents
1.3.4 Use of Analytical Balances 1.7 Data Handling and Reporting
1.3.5 Additional Information 1.7.1 Significant Figures
1.4 Mechanical Pipettes 1.7.2 Rounding Off Numbers
1.4.1 Operation 1.7.3 Rounding Off Single Arithmetic
1.4.2 Pre-rinsing Operations
1.4.3 Pipetting Solutions of Varying Density or 1.7.4 Rounding Off the Results of a Series
Viscosity of Arithmetic Operations
1.4.4 Performance Specifications 1.8 Basic Laboratory Safety
1.4.5 Selecting the Correct Pipette 1.8.1 Safety Data Sheets
1.5 Glassware 1.8.2 Hazardous Chemicals
1.5.1 Types of Glassware/Plasticware 1.8.3 Personal Protective
1.5.2 Choosing Glassware/Plasticware Equipment and Safety Equipment
1.5.3 Volumetric Glassware 1.8.4 Eating, Drinking, Etc.
1.5.4 Using Volumetric Glassware to 1.8.5 Miscellaneous Information
Perform Dilutions and Concentrations
1.5.5 Conventions and Terminology
1.5.6 Burets
1.5.7 Cleaning of Glass and Porcelain
Chapter 1 • Laboratory Standard Operating Procedures 5
1.1 INTRODUCTION cal relationship between the analyte concentration and

the analytical response). There are a number of differ-
This chapter is designed to cover “standard operating ent methods available for the determination of preci-
procedures” (SOPs), or best practices, for a general sion. One method follows:
food analysis laboratory. The topics covered in this
chapter include balances, mechanical pipettes, glass- 1. Three separate concentration levels should be
ware, reagents, precision and accuracy, data handling, studied, including a low concentration near the
data reporting, and safety. These procedures apply to sensitivity level of the method, an intermediate
all the laboratory experiments in this manual, and concentration, and a concentration near the
therefore a thorough review of general procedures will upper limit of application of the method.
be invaluable for successful completion of these labo- 2. Seven replicate determinations should be made
ratory exercises. at each of the concentrations tested.
This manual covers many of the basic skills and 3. To allow for changes in instrument conditions,
information that are necessary for one to be a good the precision study should cover at least 2 h of
analytical food chemist. Much of this material is the normal laboratory operation.
type that one “picks up” from experience. Nothing can 4. To permit the maximum interferences in sequen-
replace actual lab experience as a learning tool, but tial operation, it is suggested that the samples be
hopefully this manual will help students learn proper run in the following order: high, low, and inter-
lab techniques early rather than having to correct mediate. This series is then repeated seven times
improper habits later. When one reads this manual, to obtain the desired replication.
your reaction may be “is all of this attention to detail 5. The precision statement should include a range
necessary?” Admittedly, the answer is “not always.” of standard deviations over the tested range of
This brings to mind an old Irish proverb that “the best concentration. Thus, three standard deviations
person for a job is the one that knows what to ignore.” will be obtained over a range of three
There is much truth to this proverb, but a necessary concentrations.
corollary is that one must know what they are ignor-
ing. The decision to use something other than the Accuracy refers to the degree (absolute or relative)
“best” technique must be conscious decision and not of difference between observed and “actual” values.
one made from ignorance. This decision must be based The “actual” value is often difficult to ascertain. It may
not only upon knowledge of the analytical method be the value obtained by a standard reference method
being used but also on how the resulting data will be (the accepted manner of performing a measurement).
used. Much of the information in this manual has been Another means of evaluating accuracy is by the addi-
obtained from an excellent publication by the US tion of a known amount of the material being analyzed
Environmental Protection Agency entitled Handbook for the food sample and then calculation of % recov-
for Analytical Quality Control in Water and Wastewater ery. This latter approach entails the following steps:
Laboratories. 1. Known amounts of the particular constituent
are added to actual samples at concentrations
for which the precision of the method is satis-
1.2 PRECISION AND ACCURACY factory. It is suggested that amounts be added
to the low-concentration sample, sufficient to
To understand many of the concepts in this chapter, a double that concentration, and that an amount
rigorous definition of the terms “precision” and “accu- be added to the intermediate concentration, suf-
racy” is required here. Precision refers to the ficient to bring the final concentration in the
reproducibility of replicate observations, typically sample to approximately 75 % of the upper limit
measured as standard deviation (SD), standard error of application of the method.
(SE), or coefficient of variation (CV). Refer to Chap. 4 2. Seven replicate determinations at each concen-
in this laboratory manual and Smith, 2017, for a more tration are made.
complete discussion of precision and accuracy. The 3. Accuracy is reported as the percent recovery at
smaller these values are, the more reproducible or pre- the final concentration of the spiked sample.
cise the measurement is. Precision is determined not on Percent recovery at each concentration is the
reference standards, but by the use of actual food sam- mean of the seven replicate results.
ples, which cover a range of concentrations and a vari-
ety of interfering materials usually encountered by the A fast, less rigorous means to evaluate precision
analyst. Obviously, such data should not be collected and accuracy is to analyze a food sample and replicate
until the analyst is familiar with the method and has a spiked food sample, and then calculate the recovery
obtained a reproducible standard curve (a mathemati- of the amount spiked. An example is shown in Table 1.1.
6 A.P. Neilson et al.
alances are usually sensitive to 0.001–0.00001 g,

b
1. 1
Measured calcium content (g/L) of milk and
depending on the specific model. It should be remem-
table bered, however, that sensitivity (ability to detect small
spiked milk
differences in mass) is not necessarily equal to accuracy
Replicate Milk Milk + 0.75 g Ca/L (the degree to which the balances correctly report the
actual mass). The fact that a balance can be read to
1 1.29 2.15
0.01 mg does not necessarily mean it is accurate to
2 1.40 2.12
0.01 mg. What this means is that the balance can distin-
3 1.33 2.20
4 1.24 2.27 guish between masses that differ by 0.01 mg, but may
5 1.23 2.07 not accurately measure those masses to within 0.01 mg
6 1.40 2.10 of the actual masses (because the last digit is often
7 1.24 2.20 rounded). The accuracy of a balance is independent of
8 1.27 2.07 its sensitivity.
9 1.24 1.74
10 1.28 2.01 1.3.2 Choice of Balance
11 1.33 2.12
Mean 1.2955 2.0955 Which type of balance to use depends on “how much
SD 0.062 0.138 accuracy” is needed in a given measurement. One way
%CV 4.8 6.6 to determine this is by calculating how much relative
(%) error would be introduced by a given type of bal-
ance. For instance, if 0.1 g of a reagent was needed,
The accuracy can then be measured by calculating weighing it on a top loading balance accurate to within
the % of the spike (0.75 g/L) detected by comparing only ± 0.02 g of the actual mass would introduce
the measured values from the unspiked and spiked approximately 20 % error:
samples:
% error in measured mass =
accuracy ≈ % recovery = (1.1)
absolute error in measured mass
measured spiked sample ´ 100% (1.2)
× 100% measured mass
measured sample + amount of spike
0.02 g
2.0955 g / L % error in measured mass = ´ 100% = 20%
accuracy » % recovery = 0.1 g
1.2955 g / L + 0.75 g / L
´ 100% = 102.44% This would clearly be unacceptable in most situations.
The method measured the spike to within 2.44 %. By Therefore, a more accurate balance would be needed.
adding 0.75 g/L Ca to a sample that was measured to However, the same balance (with accuracy to
have 1.2955 g/L Ca, a perfectly accurate method within ± 0.02 g) would probably be acceptable for
would result in a spiked sample concentration of 1.295 weighing out 100 g of reagent, as the error would be
5 g/L + 0.75 g/L = 2.0455 g/L. The method actually approximately 0.02 %:
measured the spiked sample at 2.0955 g/L, which is 0.02 g
2.44 % greater than it should be. Therefore, the accu- % error in measured mass = ´ 100% = 0.02%
100 g
racy is estimated at ~2.44 % relative error.
The decision on “how much accuracy” is needed can
only be answered when one knows the function of the
1.3 BALANCES reagent in the analytical method. This is one reason
why it is necessary to understand the chemistry
1.3.1 Types of Balances
involved in an analytical method, and not to simply
Two general types of balances are used in most laborato- approach an analytical method in a cookbook fashion.
ries. These are top loading balances and analytical Therefore, a general guideline regarding which bal-
balances. Top loading balances usually are sensitive to ance to use is hard to define.
0.1–0.001 g, depending on the specific model in use (this Another situation in which care must be exercised
means that they can measure differences in the mass of a in determining what type of balance to use is when a
sample to within 0.1–0.001 g). In, general, as the capacity difference in masses is to be calculated. For instance, a
(largest mass that can be measured) increases, the sensi- dried crucible to be used in a total ash determination
tivity decreases. In other words, balances that can mea- may weigh 20.05 g on a top loading balance, crucible
sure larger masses generally measure differences in plus sample = 25.05 g, and the ashed crucible 20.25 g. It
those masses to fewer decimal places. Analytical may appear that the use of the top loading balance
with its accuracy of ± 0.02 g would introduce approxi- with the vessel. The mass of the vessel must be
mately 0.1 % error, which would often be acceptable. known so that it can be subtracted from the
Actually, since a difference in weight (0.20 g) is being final mass to get the mass of the dried sample or
determined, the error would be approximately 10 % ash. Therefore, make sure to obtain the mass of
and thus unacceptable. In this case, an analytical bal- the vessel before the analysis. This can be done
ance is definitely required because sensitivity is by either weighing the vessel before taring the
required in addition to accuracy. balance and then adding the sample or obtain-
ing the mass of the vessel and then the mass of
1.3.3 Use of Top Loading Balances the vessel plus the sample.
2. The accumulation of moisture from the air or
These instructions are generalized but apply to the use
fingerprints on the surface of a vessel will add a
of most models of top loading balances:
small mass to the sample. This can introduce
1. Level the balance using the bubble level and the errors in mass that affect analytical results, par-
adjustable feet (leveling is required so that the ticularly when using analytical balances.
balance performs correctly). Therefore, beakers, weigh boats, and other
2. Either zero the balance (so the balance reads 0 weighing vessels should be handled with tongs
with nothing on the pan) or tare the balance so or with gloved hands. For precise measure-
that the balance reads 0 with a container that ments (moisture, ash, and other measurements),
will hold the sample (empty beaker, weighing weighing vessels should be pre-dried and
boat, etc.) on the weighing pan. The tare func- stored in a desiccator before use, and then
tion is conveniently used for “subtracting” the stored in a desiccator after drying, ashing, etc.
weight of the beaker or weighing boat into prior to weighing the cooled sample.
which the sample is added. 3. Air currents or leaning on the bench can cause
3. Weigh the sample. appreciable error in analytical balances. It is
best to take the reading after closing the side
1.3.4 Use of Analytical Balances doors of an analytical balance.
4. Most balances in modern laboratories are elec-
It is always wise to consult the specific instruction
tric balances. Older lever-type balances are no
manual for an analytical balance before using it.
longer in wide use, but they are extremely
Speed and accuracy are both dependent on one being
reliable.
familiar with the operation of an analytical balance. If
it has been a while since you have used a specific type
of analytical balance, it may be helpful to “practice”
1.4 MECHANICAL PIPETTES
before actually weighing a sample by weighing a
spatula or other convenient article. The following Mechanical pipettes (i.e., automatic pipettors) are
general rules apply to most analytical balances and standard equipment in many analytical laboratories.
should be followed to ensure that accurate results are This is due to their convenience, precision, and accept-
obtained and that the balance is not damaged by able accuracy when used properly and when calibrated.
improper use: Although these pipettes may be viewed by many as
1. Analytical balances are expensive precision being easier to use than conventional glass volumetric
instruments; treat them as such. pipettes, this does not mean that the necessary accuracy
2. Make sure that the balance is level and is on a and precision can be obtained without attention to
sturdy table or bench free of vibrations. proper pipetting technique. Just the opposite is the
3. Once these conditions are met, the same proce- case; if mechanical pipettes are used incorrectly, this
dure specified above for top loading balances is will usually cause greater error than the misuse of glass
used to weigh the sample on an analytical volumetric pipettes. The proper use of glass volumetric
balance. pipettes is discussed in the section on glassware. The
4. Always leave the balance clean. PIPETMAN mechanical pipette (Rainin Instrument
Co., Inc.) is an example of a continuously adjustable
1.3.5 Additional Information design. The proper use of this type of pipette, as recom-
mended by the manufacturer, will be described here.
Other points to be aware of regarding the use of bal- Other brands of mechanical pipettes are available, and
ances are the following: although their specific instructions should be followed,
1. Many analyses (moisture, ash, etc.) require their proper operation is usually very similar to that
weighing of the final dried or ashed sample described here.
1.4.1 Operation 10. With plunger fully depressed, withdraw

mechanical pipette from vessel carefully with
1. Set the desired volume on the digital microme-
tip sliding along wall of vessel.
ter/volumeter. For improved precision, always
11. Allow plunger to return to top position.
approach the desired volume by dialing down-
12. Discard tip by depressing tip-ejector button
ward from a larger volume setting. Make sure
smartly.
not to wind it up beyond its maximum capacity;
13. A fresh tip should be used for the next measure-
this will break it beyond repair.
ment if:
2. Attach a disposable tip to the shaft of the pipette
and press on firmly with a slight twisting (a) A different solution or volume is to be
motion to ensure a positive, airtight seal. pipetted.
3. Depress the plunger to the first positive stop. (b) A significant residue exists in the tip (not to
This part of the stroke is the calibrated volume be confused with the visible “film” left by
displayed. Going past the first positive stop will some viscous or organic solutions).
cause inaccurate measurement.
4. Holding the mechanical pipette vertically,
1.4.2 Pre-rinsing
immerse the disposable tip into sample liquid
to a depth indicated (Table 1.2), specific to the Pipetting very viscous solutions or organic solvents
maximum volume of the pipette (P-20, 100, 200, will result in a significant film being retained on the
500, 1000, 5000, correspond to maximum vol- inside wall of the tip. This will result in an error that
umes of 20, 100, 200, 500, 1000, and 5000 μL, will be larger than the tolerance specified if the tip is
respectively). only filled once. Since this film remains relatively con-
5. Allow plunger to slowly return to the “up” posi- stant in successive pipettings with the same tip, accu-
tion. Never permit it to snap up (this will suck liquid racy may be improved by filling the tip, dispensing
up into the pipette mechanism, causing inaccu- the volume into a waste container, refilling the tip a
rate measurement and damaging the pipette). second time, and using this quantity as the sample.
6. Wait 1–2 s to ensure that full volume of sample This procedure is recommended in all pipetting oper-
is drawn into the tip. If the solution is viscous ations when critical reproducibility is required,
such as glycerol, you need to allow more time. whether or not tips are reused (same solution) or
7. Withdraw tip from sample liquid. Should any changed (different solutions/different volumes).
liquid remain on outside of the tip, wipe care- Note that the “non-wettability” of the polypropylene
fully with a lint-free cloth, taking care not to tip is not absolute and that pre-rinsing will improve
touch the tip opening. the precision and accuracy when pipetting any
8. To dispense sample, place tip end against side solution.
wall of vessel and depress plunger slowly past
the first stop until the second stop (fully 1.4.3 ipetting Solutions of Varying
P
depressed position) is reached. Density or Viscosity
9. Wait (Table 1.3).
Compensation for solutions of varying viscosity or
density is possible with any adjustable pipette by
setting the digital micrometer slightly higher or
1. 2
Appropriate pipette depth for automatic lower than the required volume. The amount of
table pipettors compensation is determined empirically. Also, when
dispensing viscous liquids, it will help to wait 1 s
Pipette Depth (mm) longer at the first stop before depressing to the sec-
P-20D, P-100D, P-200D 1–2
ond stop.
P-500D, P-1000D 2–4
P-5000D 3–6 1.4.4 Performance Specifications
The manufacturer of PIPETMAN mechanical pipettes
provides the information in Table 1.4, on the precision
1. 3
Appropriate dispense wait time for auto-
and accuracy of their mechanical pipettes.
table matic pipettors
1.4.5 Selecting the Correct Pipette
Pipette Time (s)
Although automatic pipettes can dispense a wide
P-20D, P-100D, P-200D 1 range of volumes, you may often have to choose the
P-500D, P-1000D 1–2 “best” pipette with the most accuracy/precision from
P-5000D 2–3 among several choices. For example, a P5000
1. 4
Accuracy and precision of PIPETMAN 1. 5
Recommended volume ranges for mechani-
table mechanical pipettes table cal pipettors
Reproducibilitya Maximum volume Lowest recommended volume

Model Accuracy a
(standard deviation)
5 mL (5000 μL) 1 mL (1000 μL)
P-2OD <0.l μL @ 1–10 μL <0.04 μL @ 2 μL 1 mL (1000 μL) 0.1–0.2 mL (100–200 μL)
<1 % @ 10–20 μL <0.05 μL @ 10 μL 0.2 mL (200 μL) 0.02–0.04 mL (20–40 μL)
P-200D <0.5 μL @ 20–60 μL <0.15 μL @ 25 μL 0.1 mL (100 μL) 0.01–0.02 mL (10–20 μL)
<0.8 % @ 60–200 μL <0.25 μL @ 100 μL 0.05 mL (50 μL) 0.005–0.01 mL (5–10 μL)
<0.3 μL @ 200 μL 0.02 mL (20 μL) 0.002–0.004 mL (2–4 μL)
P-1000D <3 μL @ 100–375 μL <0.6 μL @ 250 μL 0.01 mL (10 μL) 0.001–0.002 mL (1–2 μL)
<0.8 % @ 375–l000 μL <1.0 μL @ 500 μL
<1.3 μL @ 1000 μL
P-5000D <12 μL @ 0.5–2 mL <3 μL @ 1.0 mL s tudent grade to others possessing specific properties
<0.6 % @ 2.0–5.0 mL <5 μL @ 2.5 mL such as resistance to thermal shock or alkali, low boron
<8 μL @ 5.0 mL content, and super strength. The most common type is
a
Aqueous solutions, tips prerinsed once a highly resistant borosilicate glass, such as that
manufactured by Corning Glass Works under the

name “Pyrex” or by Kimble Glass Co. as “Kimax.”
(i.e., 5 mL) automatic pipettor could theoretically
Brown/amber actinic glassware is available, which
pipette anywhere between 0 and 5 mL. However, there
blocks UV and IR light to protect light-sensitive solu-
are several limitations that dictate which pipettes to
tions and samples. The use of vessels, containers, and
use. The first is a practical limitation: mechanical
other apparatus made of Teflon, polyethylene, poly-
pipettes are limited by the graduations (the incre-
styrene, and polypropylene is common. Teflon stop-
ments) of the pipette. The P5000 and P1000 are typi-
cock plugs have practically replaced glass plugs in
cally adjustable in increments of 0.01 mL (10 μL).
burets, separatory funnels, etc., because lubrication to
Therefore, these pipettes cannot dispense volumes of
avoid sticking (called “freezing”) is not required.
<10 μL, nor can they dispense volumes with more pre-
Polypropylene, a methylpentene polymer, is available
cision that of 10 μL. However, just because these
as laboratory bottles, graduated cylinders, beakers,
pipettes can technically be adjusted to 10 μL does not
and even volumetric flasks. It is crystal clear, shatter-
mean that they should be used to measure volumes
proof, autoclavable, chemically resistant, but relatively
anywhere near this small. Most pipettes are labeled
expensive as compared to glass. Teflon (polytetrafluo-
with a working range that lists the minimum and max-
roethylene, PTFE) vessels are available, although they
imum volume, but this is not the range for ideal per-
are very expensive. Finally, most glassware has a polar
formance. Mechanical pipettes should be operated
surface. Glassware can be treated to derivatize the sur-
from 100 % down to 10–20 % of their maximum capac-
face (typically, tetramethylsilane, or TMS) to make it
ity (Table 1.5). Below 10–20 % of their maximum capac-
nonpolar, which is required for some assays. However,
ity, performance (accuracy and precision) suffers. A
acid washing will remove this nonpolar layer.
good way of thinking of this is to use the largest pipette
capable of dispensing the volume in a single aliquot.
Mechanical pipettes are invaluable pieces of labo- 1.5.2 Choosing Glassware/Plasticware
ratory equipment. If properly treated and maintained, Some points to consider in choosing glassware and/or
they can last for decades. However, improper use can plasticware are the following:
destroy them in seconds. Mechanical pipettes should
1. Generally, special types of glass are not required
be calibrated, lubricated, and maintained at least
to perform most analyses.
yearly by a knowledgeable pipette technician.
2. Reagents and standard solutions should be
Weighing dispensed water is often a good check to see
stored in borosilicate or polyethylene bottles.
if the pipette needs calibration.
3. Certain dilute metal solutions may plate out on
glass container walls over long periods of stor-
1.5 GLASSWARE age. Thus, dilute metal standard solutions
should be prepared fresh at the time of
1.5.1 Types of Glassware/Plasticware analysis.
4. Strong mineral acids (such as sulfuric acid) and
Glass is the most widely used material for construc- organic solvents will readily attack polyethyl-
tion of laboratory vessels. There are many grades and ene; these are best stored in glass or a resistant
types of glassware to choose from, ranging from plastic.
5. Borosilicate glassware is not completely inert, meniscus should be tangent to the calibration mark.
particularly to alkalis; therefore, standard solu- There are other sources of error, however, such as
tions of silica, boron, and the alkali metals (such changes in temperature, which result in changes in the
as NaOH) are usually stored in polyethylene actual capacity of glass apparatus and in the volume of
bottles. the solutions. The volume capacity of an ordinary
6. Certain solvents dissolve some plastics, includ- 100 mL glass flask increases by 0.025 mL for each 1°
ing plastics used for pipette tips, serological rise in temperature, but if made of borosilicate glass,
pipettes, etc. This is especially true for acetone the increase is much less. One thousand mL of water
and chloroform. When using solvents, check (and of most solutions that are ≤ 0.1 N) increases in
the compatibility with the plastics you are volume by approximately 0.20 mL per 1 °C increase at
using. Plastics dissolved in solvents can cause room temperature. Thus, solutions must be measured
various problems, including binding/precipi- at the temperature at which the apparatus was cali-
tating the analyte of interest, interfering with brated. This temperature (usually 20 °C) will be indi-
the assay, clogging instruments, etc. cated on all volumetric ware. There may also be errors
7. Ground-glass stoppers require care. Avoid of calibration of the adjustable measurement appara-
using bases with any ground glass because the tus (e.g., measuring pipettes), that is, the volume
base can cause them to “freeze” (i.e., get stuck). marked on the apparatus may not be the true volume.
Glassware with ground-glass connections Such errors can be eliminated only by recalibrating the
(burets, volumetric flasks, separatory funnels, apparatus (if possible) or by replacing it.
etc.) are very expensive and should be handled A volumetric apparatus is calibrated “to contain”
with extreme care. or “to deliver” a definite volume of liquid. This will be
indicated on the apparatus with the letters “TC” (to
For additional information, the reader is referred contain) or “TD” (to deliver). Volumetric flasks are cali-
to the catalogs of the various glass and plastic manu- brated to contain a given volume, which means that the
facturers. These catalogs contain a wealth of informa- flask contains the specified volume ± a defined toler-
tion as to specific properties, uses, sizes, etc. ance (error). The certified TC volume only applies to
the volume c ontained by the flask and it does not take
1.5.3 Volumetric Glassware into account the volume of solution that will stick to
the walls of the flask if the liquid is poured out.
Accurately calibrated glassware for accurate and pre- Therefore, for example, a TC 250 mL volumetric flask
cise measurements of volume has become known as will hold 250 mL ± a defined tolerance; if the liquid is
volumetric glassware. This group includes volumet- poured out, slightly less than 250 mL will be dispensed
ric flasks, volumetric pipettes, and accurately cali- due to solution retained on the walls of the flask (this is
brated burets. Less accurate types of glassware, the opposite of “to deliver” or TD, glassware discussed
including graduated cylinders, serological pipettes, below). They are available in various shapes and sizes
and measuring pipettes, also have specific uses in the ranging from 1 to 2000 mL capacity. Graduated cylin-
analytical laboratory when exact volumes are unnec- ders, on the other hand, can be either TC or TD. For
essary. Volumetric flasks are to be used in preparing accurate work the difference may be important.
standard solutions, but not for storing reagents. The Volumetric pipettes are typically calibrated to
precision of an analytical method depends in part deliver a fixed volume. The usual capacities are
upon the accuracy with which volumes of solutions 1–100 mL, although micro-volumetric pipettes are also
can be measured, due to the inherent parameters of the available. The proper technique for using volumetric
measurement instrument. For example, a 10 mL volu- pipettes is as follows (this technique is for TD pipettes,
metric flask will typically be more precise (i.e., have which are much more common than TC pipettes):
smaller variations between repeated measurements)
than a 1000 mL volumetric flask, because the neck on 1. Draw the liquid to be delivered into the pipette
which the “fill to” line is located is narrower, and above the line on the pipette. Always use a
therefore smaller errors in liquid height above or pipette bulb or pipette aid to draw the liquid
below the neck result in smaller volume differences into the pipette. Never pipette by mouth.
compared to the same errors in liquid height for the 2. Remove the bulb (when using the pipette aid,
larger flask. However, accuracy and precision are often or bulbs with pressure release valves, you can
independent of each other for measurements on simi- deliver without having to remove it) and replace
lar orders of magnitude. In other words, it is possible it with your index finger.
to have precise results that are relatively inaccurate 3. Withdraw the pipette from the liquid and wipe
and vice versa. There are certain sources of error, off the tip with tissue paper. Touch the tip of the
which must be carefully considered. The volumetric pipette against the wall of the container from
apparatus must be read correctly; the bottom of the which the liquid was withdrawn (or a spare
beaker). Slowly release the pressure of your fin- standard for laboratory glassware. Class A glassware
ger (or turn the scroll wheel to dispense) on the has the tightest tolerances and therefore the best
top of the pipette and allow the liquid level in accuracy and precision. These flasks are rated
the pipette to drop so that the bottom of the TC. Therefore, volumetric flasks are used to bring
meniscus is even with the line on the pipette. samples and solutions up to a defined volume. They
4. Move the pipette to the beaker or flask into are not used to quantitatively deliver or transfer sam-
which you wish to deliver the liquid. Do not ples because the delivery volume is not known. Other
wipe off the tip of the pipette at this time. Allow types of glassware (non-Class A flasks, graduated
the pipette tip to touch the side of the beaker or cylinders, Erlenmeyer flasks, round-bottomed flasks,
flask. Holding the pipette in a vertical position, beakers, bottles, etc., Fig. 1.1b) are less accurate and
allow the liquid to drain from the pipette. less precise. They should not be used for quantitative
5. Allow the tip of the pipette to remain in contact volume dilutions or concentrations if Class A volu-
with the side of the beaker or flask for several metric flasks are available.
seconds. Remove the pipette. There will be a For transferring a known volume of a liquid sam-
small amount of liquid remaining in the tip of ple for a dilution or concentration, the “gold standard”
the pipette. Do not blow out this liquid with the providing maximal accuracy and precision is a Class A
bulb, as TD pipettes are calibrated to account glass volumetric pipette (Fig. 1.2a). These pipettes are
for this liquid that remains. rated “to deliver” (TD), which means that the pipette
will deliver the specified volume ± a defined tolerance
Note that some volumetric pipettes have calibra- (error). The certified TD volume takes into account the
tion markings for both TC and TD measurements. volume of solution that will stick to the walls of the
Make sure to be aware which marking refers to which pipette as well as the volume of the drop of solution
measurement (for transfers, use the TD marking). The that typically remains in the tip of the pipette after
TC marking will be closer to the dispensing end of the delivery (again, you should not attempt to get this
pipette (TC does not need to account for the volume drop out, as it is already accounted for). Therefore, for
retained on the glass surface, whereas TD does account example, a TD 5 mL pipette will hold slightly more
for this). than 5 mL but will deliver (dispense) 5 mL ± a defined
Measuring and serological pipettes should also be tolerance (the opposite of TC glassware). It is impor-
held in a vertical position for dispensing liquids; how- tant to note that volumetric pipettes are used only to
ever, the tip of the pipette is only touched to the wet deliver a known amount of solution. Typically they
surface of the receiving vessel after the outflow has should not be used to determine the final volume of
ceased. Some pipettes are designed to have the small the solution unless the liquids dispensed are the only
amount of liquid remaining in the tip blown out and components of the final solution. For example, if a
added to the receiving container; such pipettes have a sample is dried down and then liquid from a volumet-
frosted band near the top. If there is no frosted band ric pipette is used to resolubilize the solutes, it is
near the top of the pipette, do not blow out any remain- unknown if the solutes significantly affect the volume
ing liquid. of the resulting solution, unless the final volume is
measured, which may be difficult to do. Although the
effect is usually negligible, it is best to use volumetric
1.5.4 Using Volumetric Glassware
glassware to assure that the final volume of the result-
to Perform Dilutions
ing solution is known (the dried solutes could be dis-
and Concentrations
solved in a few mL of solvent and then transferred to a
Typically, dilutions are performed by adding a liq- volumetric flask for final dilution). However, it is
uid (water or a solvent) to a sample or solution. acceptable to add several solutions together using vol-
Concentrations may be performed by a variety of umetric pipettes and then add the individual volumes
methods, including rotary evaporation, shaking together to calculate the final volume. However, using
vacuum evaporation, vacuum centrifugation, boil- a single volumetric flask to dilute to a final volume is
ing, oven drying, drying under N2 gas, or freeze still the favored approach, as using one measurement
drying. for the final volume reduces the uncertainty. (The
For bringing samples or solutions up to a known errors, or tolerances, of the amounts added are also
volume, the “gold standard” providing maximal added together; therefore, using fewer pieces of glass-
accuracy and precision is a Class A glass volumetric ware lowers the uncertainty of the measurement even
flask (Fig. 1.1a). During manufacture, glassware to be if the tolerances of the glassware are the same.) For
certified as Class A is calibrated and tested to comply example, suppose you need to measure out 50 mL of
with tolerance specifications established by the solution. You have access to a 50 mL volumetric flask
American Society for Testing and Materials (ASTM, and a 25 mL volumetric pipette, both of which have
West Conshohocken, PA). These specifications are the tolerances of ± 0.06 mL. If you obtain 50 mL by filling
a b c d e
1. 1
Class A volumetric flask (a) and other types of non-Class A volume measuring glassware: graduated cylinder
figure
(b), Erlenmeyer flask (c), beaker (d), and bottle (e)
the volumetric flask, the measured volume is Information typically printed on the side of the
50 mL ± 0.06 mL (or somewhere between 49.94 and pipette or flask includes the class of the pipette or
50.06 mL). If you pipette 25 mL twice into a beaker, the flask, whether the glassware is TD or TC, the TC or TD
tolerance of each measurement is 25 mL ± 0.06 mL, and volume, and the defined tolerance (error) (Fig. 1.3).
the tolerance of the combined volume is the sum of the Note that the specifications are typically valid at a
means and the errors: specified temperature, typically 20 °C. Although it is
rare that scientists equilibrate solutions to exactly
( 25 mL ± 0.06 mL ) + ( 25 mL ± 0.06 mL ) = 20 °C before volume measurement, this temperature is
50 mL ± 0.12 mL = 49.88 − 50.12 mL assumed to be approximate room temperature. Be
aware that the greater the deviation from room tem-
This additive property of tolerances, or errors, com- perature, the greater the error in volume measure-
pounds further as more measurements are combined; ment. The specific gravity (density) of water at 4, 20,
conversely, when the solution is brought to volume 60, and 80 °C relative to 4 °C is 1.000, 0.998, 0.983, and
using a volumetric flask, only a single tolerance factors 0.972. This means that a given mass of water has lower
into the error of the measurement. density (greater volume for given mass) at tempera-
Other types of pipettes (non-Class A volumetric tures above 20 °C. This is sometimes seen when a volu-
glass pipettes, adjustable pipettors, automatic pipet- metric flask is brought exactly to volume at room
tors, reed pipettors, serological pipettes, etc., Fig. 1.2b) temperature and then is placed in an ultrasonic bath to
and other glassware (graduated cylinders, etc.) are less help dissolve the chemicals, warming the solution. A
accurate and less precise. They should not be used for solution that was exactly at the volume marker at
quantitative volume transfers. Pipettes are available room temperature will be above the volume when the
(but rare) that are marked with lines for both TC and solution is warmer. To minimize this error, volumes
TD. For these pipettes, the TD line would represent the should be measured at room temperature.
volume delivered when the drop at the tip is dispensed Volumetric glassware (flasks and pipettes) should
and TC when the drop remains in the pipette. be used for quantitative volume measurements during
a b c d e
1. 2
Class A volumetric pipette (a) and non-volumetric pipettes: adjustable pipettors (b), reed pipettor (c), serological
figure
pipettes (d)
a b
1. 3
Image of the label on a Class A volumetric flask pipette (a) and Class A volumetric pipette (b)
figure
volumetric measurements can result in significant

error being introduced into the measurement.
Typical tolerances for lab glassware are presented
in Tables 1.6 and 1.7. References for ASTM specifica-
tions are found at http://www.astm.org/.
A comparison of Tables 1.6 and 1.7 reveals some
important points. First, even for Class A glassware, the
tolerances for volumetric transfer pipettes (pipettes
with a single TD measurement) are much tighter than
for graduated measuring pipettes (pipettes with grad-
uations that can be used to measure a wide range of
volumes) of the same volume. Second, even for Class
A glassware, the tolerances for volumetric transfer
pipettes and volumetric flasks are much tighter than
1. 4
Image of a liquid meniscus at the line for a
figure
Class A volumetric flask
dilutions and concentrations whenever possible to 1. 6

Volume tolerances of Class A glassware
maximize the accuracy and precision of the procedure. table required by ASTM specifications
For both volumetric flasks and pipettes, the level of the Tolerance (± mL)
liquid providing the defined volume is indicated by a
Volumetric Measuring Volumetric
line (usually white or red) etched or printed on the Volume (transfer) (graduated) flask Graduated
neck of the glassware. To achieve the TD or TC vol- (mL) Buret pipette pipettes cylinder
ume, the bottom of the meniscus of the liquid should
0.5 0.006
be at the line as shown in Fig. 1.4.
1 0.006 0.010
For a volumetric flask, the proper technique for 2 0.006 0.01 0.015
achieving the correct volume is to pour the liquid into 3 0.01 0.02 0.015
the flask until the meniscus is close to the marking line, 4 0.01 0.03 0.020
and then add additional liquid dropwise (with a man- 5 0.01 0.05 0.020 0.05
ual pipette or Pasteur pipette) until the bottom (NOT 10 0.02 0.02 0.08 0.020 0.10
the top or middle) of the meniscus is at the line with 25 0.03 0.03 0.10 0.030 0.17
your eye level to the line. (If you do not look straight at 50 0.05 0.05 0.050 0.25
the line, occur, making it appear that so that your eye 100 0.10 0.08 0.080 0.50
and the line are at the same level, a phenomenon 250 0.012 1.00
500 0.013 2.00
known as “parallax” can occur, making it appear that
1000 0.015 3.00
the bottom of the meniscus is at the line when in fact it
is not, resulting in errors in volume measurement.) If
the level of the liquid is too high, liquid can be removed
using a clean pipette (or the liquid poured out and start
again). However, be aware that this cannot be done 1. 7
Volume tolerances of non-Class A glass-
table ware required by ASTM specifications
when preparing a reagent for which the solutes were
accurately measured into the flask and you are adding Tolerance (± mL)
liquid to make up to volume. In this case, you must Volume Volumetric Volumetric Graduated
start over. For this reason, the best practice is to add (mL) Buret (transfer) pipette flask cylinder
liquid slowly, and then use a pipette to add liquid
0.5 0.012
dropwise when approaching the desired volume. 1 0.012
For a volumetric pipette, the proper technique for 2 0.012
achieving the correct volume is to draw liquid into the 3 0.02
pipette until the meniscus is above the line, and then 4 0.02
withdraw the pipette from the liquid and dispense the 5 0.02 0.10
excess liquid from the pipette until the bottom of the 10 0.04 0.04 0.04 0.20
meniscus is at the line. It is critical that the pipette be 25 0.06 0.06 0.06 0.34
withdrawn from the solution for this step. If the level 50 0.10 0.10 0.24 0.50
of the liquid goes below the line, additional liquid is 100 0.20 0.16 0.40 1.00
250 0.60 2.00
drawn up, and the process is repeated. Proper volu-
500 4.00
metric measurements require practice and should be
1000 6.00
repeated until they are performed correctly. Improper
for graduated cylinders of the same volume. Therefore, salt solution is concentrated tenfold (10X), the volume is
volumetric transfer pipettes and volumetric flasks are decreased to 9 mL (either by reducing to 9 mL or drying
preferred for dilutions and concentrations. For exam- completely and reconstituting to 9 mL, tenfold or 10X
ple, a 1000 mL Class A volumetric flask has a tolerance lower than 90 mL), and the final concentration is 3.1 ppm
of ±0.015 mL (the actual TC volume is somewhere salt (tenfold or 10X more than 0.31 ppm). Although ten-
between 999.985 and 1000.015 mL), while a 1000 mL fold or 10X was used for these examples, any value can
graduated cylinder has a tolerance of ± 3.00 mL (the be used. In microbiology, values of 10X, 100X, 1000X, etc.
actual TC volume is somewhere between 997 and are commonly used due to the log scale used in that
1003 mL). This is a 200-fold larger potential error in the field. However, less standard dilutions of any value are
measurement of 1000 mL! Finally, tolerances for non- routinely used in analytical chemistry.
Class A glassware are much broader than for Class A, The last terminology system for dilutions and
and thus Class A should be used if available. concentrations involves ratios. This system is some-
what ambiguous and is not used in the Food Analysis
1.5.5 Conventions and Terminology text or lab manual. This system refers to dilutions as
“X:Y,” where X and Y are the masses or volumes of the
To follow the analytical procedures described in this
initial and final solutions/samples. For example, it
manual and perform calculations correctly, common
may be stated that “the solution was diluted 1:8.” This
terminology and conventions (a convention is a stan-
system is ambiguous for the following reasons:
dard or generally accepted way of doing or naming
something) must be understood. A common phrase in 1. The first and last numbers typically refer to the
dilutions and concentrations is “diluted to” or “diluted initial and final samples, respectively (there-
to a final volume of.” This means that the sample or fore, a 1:8 dilution would mean 1 part initial
solution is placed in a volumetric flask, and the final sample and 8 parts final sample). However,
volume is adjusted to the specified value. In contrast, there is no standard convention. Therefore, an
the phrase “diluted with” means that the specified “X:Y” dilution could be interpreted either way.
amount is added to the sample or solution. In this latter 2. There is no standard convention as to whether
case, the final mass/volume must be calculated by add- this system describes the “diluted to” or
ing the sample mass/volume and the amount of liquid “diluted with” (as described above) approach.
added. For example, suppose you take a 1.7 mL volume Therefore, diluting a sample 1:5 could be inter-
and either (1) dilute to 5 mL with methanol or (2) dilute preted as either (1) diluting 1 mL sample with
with 5 mL methanol. In the first case, this means that the 4 mL for a final volume of 5 mL (“diluted to”) or
sample (1.7 mL) is placed in a volumetric flask and (2) diluting 1 mL sample with 5 mL for a final
methanol (~3.3 mL) is added so that the final volume is volume of 6 mL (“diluted with”).
5 mL total. In the second case, the sample (1.7 mL) is
combined with 5 mL methanol, and the final volume is Because of these ambiguities, the ratio system is
6.7 mL. As you can see, these are very different values. discouraged in favor of the “X-fold” terminology.
This will always be the case except when one of the vol- However, ratio dilutions still appear in some litera-
umes is much larger than the other. For example, if you ture. If possible, it is recommended that you investi-
were working with a 10 μL sample, diluting it “to 1 L” gate to clarify what is meant by this terminology.
or “with 1 L” would result in final volumes of 1 L and Another factor to consider is that liquid volumes
1.00001 L, respectively. It is important to understand the are often not strictly additive. For example, exactly
differences between these two conventions to perform 500 ml 95 % v/v ethanol aq. added to 500 ml distilled
procedures correctly and interpret data accurately. water will not equal 1000 ml; in fact, the new volume
Another common term in dilutions/concentrations will be closer to 970 ml. Where did the missing 30 ml
is the term “fold” or “X.” This refers to the ratio of the go? Polar molecules such as water undergo different
final and initial concentrations (or volumes and masses) three-dimensional intermolecular bonding in a pure
of the sample or solution during each step. An “X-fold solution versus in a mixture with other solute or chemi-
dilution” means that the concentration of a sample cals such as ethanol. The difference in bonding causes
decreases (and typically the volume increases) by a an apparent contraction in this case. As well, addition of
given factor. For example, if 5 mL of an 18.9 % NaCl solu- solute to an exact volume of water will change the vol-
tion is diluted tenfold (or 10X) with water, 45 mL water ume after dissolved. To account for this effect, volumet-
is added so that the final volume is 50 mL (tenfold or 10X ric glassware is used to bring mixed solutions up to a
greater than 5 mL) and the final concentration is 1.89 % final volume after initial mixing. When two liquids are
NaCl (tenfold or 10X less than 18.9 %). Conversely, an mixed, the first liquid is volumetrically transferred into
“X-fold concentration” means that the concentration of a a volumetric flask, and then the second liquid is added
sample increases (and typically the volume decreases) to volume, with intermittent swirling or vortexing to
by the stated factor. For example, if 90 mL of a 0.31 ppm mix the liquids as they are being combined. For mixing
solids into solvents, the chemicals are first placed in a “ultrapure” grades. The purity of these materials
volumetric flask, dissolved in a partial volume, and required in analytical chemistry varies with the type
then brought to exact volume with additional solvent. of analysis. The parameter being measured and the
sensitivity and specificity of the detection system are
1.5.6 Burets important factors in determining the purity of the
reagents required. Technical grade is useful for mak-
Burets are used to deliver definite volumes. The more
ing cleaning solutions, such as the nitric acid and
common types are usually of 25 or 50 ml capacity,
alcoholic potassium hydroxide solutions mentioned
graduated to tenths of a milliliter, and are provided
previously. For many analyses, analytical reagent
with stopcocks. For precise analytical methods in
grade is satisfactory. Other analyses, e.g., trace
microchemistry, microburets are also used. Microburets
organic and HPLC, frequently require special “ultra-
generally are of 5 or 10 ml capacity, graduated in hun-
pure” reagents and solvents. In methods for which
dredths of a milliliter division. General rules in regard
the purity of reagents is not specified, it is intended
to the manipulation of a buret are as follows:
that analytical reagent grade be used. Reagents of
1. Do not attempt to dry a buret that has been lesser purity than that specified by the method should
cleaned for use, but rather rinse it two or three not be used.
times with a small volume of the solution with There is some confusion as to the definition of the
which it is to be filled. terms analytical reagent grade, reagent grade, and
2. Do not allow alkaline solutions to stand in a buret, ACS analytical reagent grade. A review of the litera-
because the glass will be attacked, and the stop- ture and chemical supply catalogs indicates that the
cock, unless made of Teflon, will tend to freeze. three terms are synonymous. National Formulary
3. A 50 ml buret should not be emptied faster than (NF), US Pharmaceutical (USP), and Food Chemicals
0.7 ml per second; otherwise, too much liquid Codex (FCC) are grades of chemicals certified for use
will adhere to the walls; as the solution drains as food ingredients. It is important that only NF, USP,
down, the meniscus will gradually rise, giving a or FCC grades be used as food additives if the product
high false reading. is intended for consumption by humans, rather than
for chemical analysis.
It should be emphasized that improper use of
and/or reading of burets can result in serious calcula- 1.6.1 Acids
tion errors.
The concentration of common commercially available
acids is given in Table 1.8.
1.5.7 Cleaning of Glass and Porcelain
In the case of all apparatus for delivering liquids, 1.6.2 Distilled Water
the glass must be absolutely clean so that the film of
Distilled or demineralized water is used in the lab-
liquid never breaks at any point. Careful attention
oratory for dilution, preparation of reagent solu-
must be paid to this fact or the required amount of
tions, and final rinsing of washed glassware.
solution will not be delivered. The method of clean-
ing should be adapted to both the substances that
are to be removed and the determination to be per-
formed. Water-soluble substances are s imply 1. 8
Concentration of common commercial
washed out with hot or cold water, and the vessel is table strength acids
finally rinsed with successive small amounts of dis-
tilled water. Other substances more difficult to Molecular
remove, such as lipid residues or burned material, weight Concentration Specific
may require the use of a detergent, organic solvent, Acid (g/mol) (M) gravity
nitric acid, or aqua regia (25 % v/v conc. HNO3 in Acetic acid, glacial 60.05 17.4 1.05
conc. HCl). In all cases it is good practice to rinse a Formic acid 46.02 23.4 1.20
vessel with tap water as soon as possible after use. Hydriodic acid 127.9 7.57 1.70
Material allowed to dry on glassware is much more Hydrochloric acid 36.5 11.6 1.18
difficult to remove. Hydrofluoric acid 20.01 32.1 1.167
Hypophosphorous acid 66.0 9.47 1.25
Lactic acid 90.1 11.3 1.2
1.6 REAGENTS Nitric acid 63.02 15.99 1.42
Perchloric acid 100.5 11.65 1.67
Chemical reagents, solvents, and gases are available Phosphoric acid 98.0 14.7 1.70
Sulfuric acid 98.0 18.0 1.84
in a variety of grades of purity, including technical
Sulfurous acid 82.1 0.74 1.02
grade, analytical reagent grade, and various
Ordinary distilled water is usually not pure. It may 1.6.4 Carbon Dioxide-Free Water
be contaminated by dissolved gases and by materi-
Carbon dioxide (CO2) dissolved in water can interfere
als leached from the container in which it has been
with many chemical measurements. Thus, CO2-free
stored. Volatile organics distilled over from the orig-
water may need to be produced. CO2-free water may
inal source feed water may be present, and nonvola-
be prepared by boiling distilled water for 15 min and
tile impurities may occasionally be carried over by
cooling to room temperature. As an alternative, dis-
the steam, in the form of a spray. The concentration
tilled water may be vigorously aerated with a stream
of these contaminants is usually quite small, and
of inert gas (e.g., N2 or He2) for a period sufficient to
distilled water is used for many analyses without
achieve CO2 removal. The final pH of the water should
further purification. There are a variety of methods
lie between 6.2 and 7.2. It is not advisable to store CO2-
for purifying water, such as distillation, filtration,
free water for extended periods. To ensure that CO2-
and ion exchange. Distillation employs boiling of
free water remains that way, an ascarite trap should be
water and condensation of the resulting steam, to
fitted to the container such that air entering the con-
eliminate nonvolatile impurities (such as minerals).
tainer (as boiled water cools) is CO2-free. Ascarite is
Ion exchange employs cartridges packed with ionic
silica coated with NaOH, and it removes CO2 by the
residues (typically negatively charged) to remove
following reaction:
charged contaminants (typically positively charged
minerals) when water is passed through the car- 2NaOH + CO 2 ® Na 2 CO 3 + H 2 O
tridge. Finally, filtration and reverse osmosis remove
Ascarite should be sealed from air except when water
insoluble particulate matter above a specific size.
is being removed from the container.
1.6.3 Water Purity
1.6.5 Preparing Solutions and Reagents
Water purity has been defined in many different ways,
The accurate and reproducible preparation of labora-
but one generally accepted definition states that high
tory reagents is essential to good laboratory practice.
purity water is water that has been distilled and/or
Liquid reagents are prepared using volumetric glass-
deionized so that it will have a specific resistance of
ware (pipettes and flasks) as appropriate.
500,000 Ω (2.0 μΩ/cm conductivity) or greater. This defi-
To prepare solutions from solid reagents (such as
nition is satisfactory as a base to work from, but for more
sodium hydroxide):
critical requirements, the breakdown shown in Table 1.9
has been suggested to express degrees of purity. 1. Determine the amount of solid reagent needed.
Distilled water is usually produced in a steam- 2. Fill the TC volumetric flask ~ ¼–½ full with the
heated metal still. The feed water is (or should be) soft- solvent.
ened to remove calcium and magnesium to prevent 3. Add the solid reagent (it is best to pre-dissolve
scale (Ca or Mg carbonate) formation. Several compa- solids in a beaker with a small amount of liquid,
nies produce ion-exchange systems that use resin- and then add this to the flask; rinse the smaller
packed cartridges for producing “distilled water.” The beaker thoroughly and also put the rinses into
lifespan of an ion-exchange cartridge is very much a flask).
function of the mineral content of the feed water. Thus, 4. Swirl to mix until essentially dissolved.
the lifespan of the cartridge is greatly extended by using 5. Fill the flask to volume with the solvent.
distilled or reverse osmosis-treated water as the incom- 6. Cap and invert the flask ~10–20 times to com-
ing stream. This procedure can also be used for prepar- pletely mix the solution.
ing ultrapure water, especially if a low flow rate is used
and the ion-exchange cartridge is of “research” grade. Note that it is not appropriate to simply combine
the solid reagent with the final volume and assume
that the final volume does not change. This is particu-
larly true for high % concentrations. For example, 1 L
1. 9 of a 10 % aqueous NaOH solution is correctly made by
table
Classification of water purity filling a 1 L flask with ~25–500 mL water, adding 100 g
NaOH, mixing until dissolved, and diluting to 1 L. It
Maximum Approximate
conductivity concentration of
would be incorrect to simply combine 100 g NaOH
Degree of purity (μΩ /cm) electrolytes (mg/L) with 1 L water, as the dissolved solid will take up some
volume in solution. (Note that solid NaOH is difficult
Pure 10 2–5 to dissolve, requires a stir bar, and is exothermic,
Very pure 1 0.2–0.5 releasing heat upon dissolution; therefore, do not han-
Ultrapure 0.1 0.01–0.02
dle the glass with bare hands.) Additionally, if a stir
Theoretically pure 0.055 0.00
bar is used, make sure to remove this after the solution
is dissolved but BEFORE diluting to volume. Note that 2. Zeros before a decimal point with other preced-
sonication is preferred to using a stir bar in a volumet- ing digits are significant. With no preceding
ric flask. digit, a zero before the decimal point is not
The following similar procedures are used to pre- significant.
pare reagents from two or more liquids: 3. If there are no digits preceding a decimal point,
the zeros after the decimal point but preceding
1. Determine the total volume of the final reagent.
other digits are not significant. These zeros only
2. Obtain a TC volumetric flask (if possible) equal
indicate the position of the decimal point.
to the final volume.
4. Final zeros in a whole number may or may not
3. Use TD volumetric glassware to add the correct
be significant. In a conductivity measurement
amount of the liquids with the smallest
of 1000 μΩ/cm, there is no implication that the
volumes.
conductivity is 1000 ± 1 μΩ/cm. Rather, the
4. Dilute to volume with the liquid with the larg-
zeros only indicate the magnitude of the
est volume, gently swirling during addition.
number.
5. Cap and invert the flask ~10–20 times to com-
pletely mix the solution.
A good measure of the significance of one or more
zeros before or after another digit is to determine
Note that a TC volumetric flask should be used
whether the zeros can be dropped by expressing the
whenever possible to bring the solution to final vol-
number in exponential form. If they can, the zeros are
ume. For example, the correct way to prepare 1 L of a
not significant. For example, no zeros can be dropped
5 % ethanol in water solution is to use a 50 mL TD
when expressing a weight of 100.08 g is exponential
pipette to dispense 50 mL ethanol into a 1L TC flask
form; therefore the zeros are significant. However, a
and then fill the flask to volume with water. It would
weight of 0.0008 g can be expressed in exponential
be incorrect to simply combine 50 mL ethanol and
form as 8 × 10−4 g, and the zeros are not significant.
950 mL water, since complex physical properties gov-
Significant figures reflect the limits of the particular
ern the volume of a mixture of liquids, and it cannot be
method of analysis. If more significant figures are
assumed that two liquids of different densities and
needed, selection of another method will be required
polarities will combine to form a volume equal to the
to produce an increase in significant figures.
sum of their individual volumes. If the final volume is
Once the number of significant figures is estab-
not a commonly available TC flask size, then use TD
lished for a type of analysis, data resulting from such
glassware to deliver all reagents.
analyses are reduced according to the set rules for
The use of graduated cylinders and beakers
rounding off.
should be avoided for measuring volumes for reagent
preparation.
1.7.2 Rounding Off Numbers
Rounding off numbers is a necessary operation in all
1.7 DATA HANDLING AND REPORTING analytical areas. However, it is often applied in chemi-
cal calculations incorrectly by blind rule or prema-
1.7.1 Significant Figures turely and, in these instances, can seriously affect the
The term significant figure is used rather loosely to final results. Rounding off should normally be applied
describe some judgment of the number of reportable only as follows:
digits in a result. Often the judgment is not soundly 1. If the figure following those to be retained is
based and meaningful digits are lost or meaningless less than 5, the figure is dropped, and the
digits are accepted. Proper use of significant figures retained figures are kept unchanged. As an
gives an indication of the reliability of the analytical example, 11.443 is rounded off to 11.44.
method used. Thus, reported values should contain 2. If the figure following those to be retained is
only significant figures. A value is made up of signifi- greater than 5, the figure is dropped, and the
cant figures when it contains all digits known to be last retained figure is raised by 1. As an exam-
true and one last digit in doubt. For example, if a value ple, 11.446 is rounded off to 11.45.
is reported at 18.8 mg/l, the “18” must be a firm value, 3. When the figure following those to be retained
while the “0.8” is somewhat uncertain and may be is 5 and there are no figures other than zeros
between “0.7” or “0.9.” The number zero may or may beyond the 5, the figure is dropped, and the last
not be a significant figure: place figure retained is increased by 1 if it is an
1. Final zeros after a decimal point are always sig- odd number, or it is kept unchanged if an even
nificant figures. For example, 9.8 g to the near- number. As an example, 11.435 is rounded off to
est mg is reported as 9.800 g. 11.44, while 11.425 is rounded off to 11.42.
1.7.3 ounding Off Single Arithmetic

R reactivity, storage, disposal, protective equipment, and
Operations spill-handling procedures” (http://en.wikipedia.org/
wiki/Material_safety_data_sheet#United_States).
Addition: When adding a series of numbers, the sum
SDSs are available for all reagents, chemicals, sol-
should be rounded off to the same numbers of decimal
vents, gases, etc. used in your laboratory. You can con-
places as the addend with the smallest number of
sult these documents if you have questions regarding
places. However, the operation is completed with all
how to safely handle a material, the potential risks of
decimal places intact and rounding off is done after-
the material, how to properly clean up a spill, etc. They
ward. As an example:
should be available to you in a centralized location
11.1 + 11.12 + 11.13 = 33.35 (typically, a binder) in the lab. If not available, you
Thesum is rounded off to 33.4 may request these from your instructor or find them
online. Generally, the following information is avail-
able on a MSDS or SDS in a 16-section format:
Multiplication: When two numbers of unequal
digits are to be multiplied, all digits are carried 1. Identification of the substance/mixture
through the operation, and then the product is 2. Hazard identification
rounded off to the number of significant digits 3. Composition/information on ingredients
of the less accurate number. 4. First aid measures
5. Firefighting measures
Division: When two numbers of unequal digits 6. Accidental release measures
are to be divided, the division is carried out on 7. Handling and storage
the two numbers using all digits. Then the quo- 8. Exposure controls/personal protection
tient is rounded off to the lower number of sig- 9. Physical and chemical properties
nificant digits between the two values. 10. Stability and reactivity
Powers and roots: When a number contains n 11. Toxicological information
significant digits, its root can be relied on for n 12. Ecological information
digits, but its power can rarely be relied on for n 13. Disposal considerations
digits. 14. Transport information
15. Regulatory information
1.7.4 ounding Off the Results of a Series
R 16. Other information
of Arithmetic Operations
1.8.2 Hazardous Chemicals
The rules for rounding off are reasonable for simple
calculations. However, when dealing with two nearly Food analysis laboratories, like any chemical labora-
equal numbers, there is a danger of loss of all signifi- tory, often contain hazardous compounds, including:
cance when applied to a series of computations that 1. Acids (hydrochloric acid, sulfuric acid, etc.)
rely on a relatively small difference in two values. 2. Bases (e.g., sodium hydroxide)
Examples are calculation of variance and standard 3. Corrosives and oxidizers (sulfuric acid, nitric
deviation. The recommended procedure is to carry acid, perchloric acid, etc.)
several extra figures through the calculation and then 4. Flammables (organic solvents such as hexane,
to round off the final answer to the proper number of ether, alcohols)
significant figures. This operation is simplified by
using the memory function on calculators, which for 1.8.3 ersonal Protective Equipment
P
most calculators is a large number, often 10 or more, and Safety Equipment
digits.
It is important to understand the location and use of
lab safety equipment. The purpose of this is threefold:
1.8 BASIC LABORATORY SAFETY 1. To prevent accidents and/or injuries in the lab
2. To quickly and effectively respond to any acci-
1.8.1 Safety Data Sheets dent and/or injury in the lab
Safety Data Sheets (SDSs), formerly called Material 3. Be able to perform laboratory procedures with-
Safety Data Sheets (MSDSs), are informational packets out excessive worrying about lab hazards
that are “intended to provide workers and emergency
personnel with procedures for handling or working Your laboratory instructor should provide instruc-
with that substance in a safe manner and include infor- tion regarding basic laboratory safety equipment. You
mation such as physical data (melting point, boiling should be aware of these general rules and the exis-
point, flash point, etc.), toxicity, health effects, first aid, tence of this equipment.
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Cranesbill sports a crimson stem. The stalks of Poplar leaves are a
vivid yellow. To speak of “green leaves” is to speak in the most
general of terms. What is more exquisite than the silver gray to be
seen on the backs of many tree-leaves, notably the Alders, Willows,
and Poplars? Many leaves join the Wild Lettuce in having purple
backs. The reverse sides of Magnolias and Rhododendrons are red-
brown. In the autumn, nearly all leaves show brilliant patches of
colour.
In borrowing Nature’s colours to set forth our ideas, we have
become possessors of a mighty vehicle of expression. With yellow,
we can speak of life, light, cheer and vitality. Red tells of fire, heat,
blood, excitement and passion. Blue indicates coolness, quiet and
restraint. In choosing green for its most universal colour, Nature
harmonizes life and restraint, warmth and coolness, as represented
by the component blue and yellow. In the same way, when she
wants to concentrate the maximum colour power in a single fruit or
flower, she uses orange, a combination of light and heat, vitality and
excitement. Purple represents a neutralized idea. Red vitality is
tempered with blue restraint, which results in mysticism. Nature
clothes the Poppy in red to suggest power and strength. The royal
purple of the Aster and the Violet is purposely calculated to arouse a
feeling of mystery and awe.
Our man-made cloth designs often show various plant forms intact
in the weave. The same is true of lace, while one has only to look at
the miniature flower gardens which women wear on their heads to
realize the potent influence of plants in the domains of millinery. An
important plant element seems to run through many fields of applied
art.
In some ways, the beauties of form and structure are more
appealing than chromatic charms. Lines are more refined and
fundamental than colours. A feathery mass of tree-twigs seen
against a distant horizon is exquisitely beautiful. A symmetrically
shaped tree comes very close to presenting an idea of pure form.
One may argue that it is impossible to dissociate all idea of colour
from a natural object. This is theoretically true, but practically, while
we are impressed by the colour of the Rose, it is the structural
beauty of the Palm and Weeping Willow which attracts our eye.
Nature is the true and original sculptor. From her we learn our
rules of symmetry and design. All her plant creations are finished
with a faithfulness to artistic principles which is quite exact. Nor does
she build houses with false exteriors. Her structures show forth the
necessity of truth in real esthetic creation. Bartholdi’s exquisite
Statue of Liberty, viewed from the interior, is an ugly, hollow tube. A
stalk of corn not only has a pleasing exterior but is made up of
symmetrically formed and packed interior cells. From a giant
Redwood to a microscopic vegetable organism, every line and
structural unit in the plant world is perfect in its inception and
execution.
Each plant, viewed as a whole, has its own peculiar style of
structural beauty—the variation of line and form which stamps it with
charm. This differentiation extends to all parts of the plant and gives
character to leaves, stem, flowers and fruit. Marvellous is the art
worked out in the minute parts. The tendril of the Passion Flower, the
radicle of a Seedling Maple, the feathery hair on a stalk of Mullein—
all these are shaped according to the unknown law of beauty.
Probably every geometrical form exists in some seed pod or fruit.
The artistic little seeds of the Milkweed and the Dandelion are
packed into their containers with a skill which cannot be duplicated,
once they are dislodged. There are a million seeds in the capsules of
certain Orchids. Many seed vessels are tipped, balled, carved and
frescoed.
The same delicate touch is seen down to the last cell. Plant stems
range from the common tubular variety to four-sided, hexagonal and
octagonal forms. Trees exhibit exquisite mosaics in their rough bark.
Bell-shaped flowers and flowers which are tubes, rings, ovals,
trumpets, horns, and cones are only some of the pleasing shapes to
be found in this part of vegetable anatomy.
It is a significant thing that there are few straight lines in plantdom.
Everything is built in fascinating and alluring curves. There is a
definite idea of symmetry to be observed everywhere. The beautiful,
five-pointed, leaves of the Sweet Gum Tree are arranged so that
each one fits into an interstice between two others and so obtains a
maximum supply of air and light. In general, leaves nearest the
ground are largest, thus insuring each its supply of sunshine.
When we study ornamental design, ancient and modern, we see
plant forms on all hands. The Greeks and the Moors were the only
nations to be content with geometric shapes and lines—and they
were only content at times. All other peoples have given plants and
flowers a large place in their decorative conceptions. The Egyptians
and the Assyrians, who may be considered the first civilized artists,
used the Palm, Papyrus, Lotus and Lily. The Greeks and Romans
were partial to the Acanthus, Olive, Ivy, Vine, Fir and Oak. The
Gothic art of Germany, France and Spain featured the Lily, Rose,
Pomegranate, Oak, Maple, Iris, Buttercup, Passion Flower and
Trefoil. The modern Chinese are more conservative and seek
inspiration only from the Aster and the Peony. The Japanese use the
Almond, Cherry, Wistaria and the graceful Bamboo in their art work.
These various plant forms are sometimes quite conventionalized but
are readily recognizable, whether they occur in architecture,
carvings, paintings, illuminations, tapestries or cloth fabrics.
The plant world has been man’s most constant and readily
apprehended artistic model. Yet when we see the multitude of
attractive lines, curves and shapes in Nature’s great garden, we
wonder that he has so limited his imitation. One rarely sees the
Thorn-Apple, the Hawthorn, the Daisy or the Tulip in wood or stone,
yet they are all exquisitely beautiful.
Again, artists and artisans throughout the centuries have nearly
always confined themselves to but two phases of plant life—the
leaves and the matured fruit. Tendrils have been neglected or treated
with characterless mediocrity. Thorns, leaf stipules, buds, pods, and
leaf scars have been universally overlooked. Who has ever seen the
fruit of the Rose in ornamental art? Why is it no one has thought to
use the leaf scars of trees like the Horse Chestnut as decorative
units?
Grapes and Pomegranates are reproduced with some justice, but
the various small berries almost always appear as miscellaneous
spherical bodies, whereas they are really greatly varied. The
Snowberry, Privet, Laurel and Barberry have distinct characteristics
of form and shape.
There are chances for worlds of artistic expression in various seed
pods and fruit vessels. An open Pea Pod occurs in certain
Renaissance ornament. Why not (and this is not intended to be
humorous) a String Bean?
Even a lowly thing like the scarred stalk of an old Cabbage has a
pattern worthy of imitation. The shields or remains of leaves of
former seasons form an artistic detail of the growing Palm Tree. The
Romans occasionally reproduced them on their columns. Leaf
shields are also met with in Greek border ornament.
Why must our sculptors represent the various fruits as bursting
with mature mellowness? In many cases, the unripe fruit is
artistically more attractive than when in the later stages of
development.
We rarely think of disease or decay as being pleasing, yet some
plants are artistic even in their dissolution. Certain galls and cankers
draw beautiful designs on the bodies of their victims.
Everything in plantdom has its own peculiar style of structure and
beauty. All are worthy of imitation and reproduction, provided only it
is done in the right place and the right way. It must be remembered
that, in origin, ornament was first symbolic and then decorative. Real
ornament is never unduly prominent but subordinates itself to the
idea and structure of the whole.
Man has imitated the plants also in things of a lowlier nature.
Cups, vases, pitchers and other utensils were undoubtedly first
suggested by similar shapes in plantdom. It is not too fantastic to
imagine that the smoking pipe is modelled after the flower known as
the Dutchman’s Pipe. An electric wire running down the chain of a
suspended lighting fixture looks all the world like a climbing vine.
Human jewelry has its prototype among the flowers. Our garden
beauties powdered their faces long before their human sisters ever
thought of that method of self-adornment. It is said that Greek
dancers and athletes sometimes exercised before certain slender
plants in order to pattern their bodies after them.
We are not all artists or interior decorators, and yet we can all
make use of the artistic possibilities present and inherent in our plant
friends. We can cultivate and further the use of plants and flowers in
and about our homes. Europe is far ahead of us in this respect. In
England, a city house may be ever so frowsy and run-down but it will
be sure to have its well-kept window boxes. The suburban homes of
labourers and other lowly folk are often veritable bowers of
loveliness. The German must have a garden in which to drink his
beer. If there is none handy, he builds one, and cool and delightful he
makes it. In many European cities, all the houses come out to the
building line and even arch the sidewalks. Not a bit of greensward is
in sight. Yet shrubs, flowers and vines spring from every sill and
balcony and so make the streets to blossom as the Rose.
American cities are too inclined to be barren wastes of brick and
stone, with but scant provision for plant beauty. Even the rich, who
have their elaborate and beautiful country gardens, seem to forget
the plants and flowers when they come to the city. The self-tending
Ampelopsis and Wistaria vines are the only plants at all common.
Our short summer season and the fact that so many people do not
occupy their city homes in warm weather are a little discouraging,
but need not shake the enthusiasm of any one really interested in
plants. For a few dollars a season florists will assume all care of
exterior plants and vines.
The man who has a little plot of ground before his door is indeed
fortunate. Even a well-clipped grass lawn is a refreshing asset.
Sweet Peas train well against a wall. Pansies flourish in shady spots
and Nasturtiums wax beautiful where other plants fail.
A brown stone front, flushed to the sidewalk in the middle of a
block, need not go without floral decoration. Even a terra cotta box
on either side of the entrance is capable of holding much growing
joy. Evergreen shrubs fit well into such surroundings. A window box
has great possibilities. In early spring, Crocus, Narcissus and
Hyacinth flourish in it to advantage. Ivy-Geraniums of smooth waxy
leaves and graceful loose sprays will grow all summer. Vines of
various kinds can be trained so as to make very effective window
screens.
The subject of home plants is fascinating. It is well to note that it is
not always necessary to go in for the more elaborate varieties. It is
surprising what a delicate and pleasing decoration is made by so
humble a thing as a sprouting Carrot or a Sweet Potato Vine.
Outdoor and landscape gardening are whole sciences unto
themselves. In general, a Renaissance house looks best surrounded
by formal and well-clipt flower beds. Houses on the Gothic order
should have undulating lawns and irregular groups of shrubs and
trees about them.
Plants and flowers are the first and original artists. Their creations
are our best and most worthy models. We can use them both as
examples to be imitated and beautiful objects with which to surround
ourselves. They are one of our greatest esthetic inspirations.
CHAPTER VIII
Music in the Plant World
“Many voices there are in Nature’s choir, and none but were
good to hear
Had we mastered the laws of their music well, and could read
their meaning clear;
But we who can feel at Nature’s touch, cannot think as yet
with her thought;
And I only know that the sough of the pines with a spell of its
own is fraught.”
Music is a language—a species of soft, dreamy speech which
makes up for its lack of definiteness and precision by a beauty and
harmony which can best be described as divine. Indeed, the ancient
Greeks made music an all-inclusive term for the higher conceptions
of life. Dancing, poetry, and even science were supposed to be
under its sway, while the revolution of the heavenly bodies created
that “music of the spheres” which entertained the gods.
It would be better for mankind if this sentiment were more popular
today. It is a narrow notion which confines the idea of musical
harmony to the sounds produced by certain man-made instruments.
Art which is restricted to workings in oil may be very pleasing but it is
also very much limited. Music which is only interpreted on a violin or
a piano falls far short of its grandest possibilities. To certain minds,
the sighing of the wind through a Pine forest is more exquisitely
expressive than a hundred breath-blown symphonies. When men
cannot agree as to what is music among the sounds produced by
their self-created instruments, dare they lightly ignore the many
pleasing sounds which accompany the operations of Nature?
To an American ear, Chinese singing sounds like squealing and a
Fiji concert like a vociferous boiler factory. Yet a Chinaman or a Fiji
Islander will leave our grandest operatic efforts in disgust, though he
may be pleased with the preceding orchestral tunings. Where are we
to set the standard? Is it not safest to fall back on Nature for our
truest conceptions?
The real sublimity of Nature lies in her vocalism. A soundless
world would be greatly lacking in charm. The endearing noises of the
woods and the fields often become so familiar that we fail to notice
their individual merits. Yet they are there. Their sudden cessation
would leave a terrible and unbearable gap. The woods are filled with
gaily costumed feathered minstrels. The meadows are great emerald
stages of song and fancy. The very grass roots are filled with little
insect-fiddlers who chirp cheerfulness. Wind, water and rain all
furnish a grand and beautiful accompaniment.
Nature sings in the inharmonic scale, that is, a scale which takes
in all intervals. Between the piano notes “C” and “D” lies a great
space. They only represent halting points in the ascent of sound.
Just as in the spectrum there are a hundred variations of shade
between blue and green, so the cultivated human voice can hint at a
hundred intervals between “C” and “D”. Nature uses all the tiny
shades of sound there are, and certain humans have followed suit.
To the Arabians, water “lisps in a murmuring scale.”
Occasionally, Nature uses the diatonic scale familiar to our
western civilization. When the wind unites its vibrations into the long
shrill note we call the whistle, it is playing according to our musical
rules. Water, when falling perpendicularly from a great height also
gives forth a long, steady note. Even the rhythmical quality so
essential to good music is not lacking in such phenomena as rain
pattering on dry leaves. This sound has proved unusually appealing
to many people. The Mexicans sometimes attempt to imitate it by
means of clay rattles.
Not only does the countryside continually sing a great symphony,
but each region has its own acoustic properties. While large cities
maintain a discordant and incessant roar, the country is filled with
soft and pleasing voices. Birds, animals, water and wind give forth
quaint musings of the most soothing nature. Once in a while the
woods go on a musical jag and every instrument becomes
discordant. Under the influence of the bright moonlight, the
inhabitants of the South American jungles sometimes seem to go
mad. The hoarse roars of the Tiger mingle with the piercing shrieks
of Parrots and the shrill wailings of Monkeys, while the croaking of
Bull Frogs and the dismal hoot of Owls is deafening. Jaguars scream
as they chase Monkeys through the tree-tops.
The various members of the plant kingdom are the principal
instruments upon which the wind plays. Without the obstruction
offered by plants, trees, rocks, and houses, we should not hear the
wind at all. The trees, because of their size and exposed positions,
are most noted as plant-musicians, but the grasses and herbs are
also very susceptible to the caressings of the wind.
Who has not heard and gloried in the music of the Pines? The
sharp needles of these big conifers seem unusually fitted for esthetic
expression. They are the Aeolian harps of the woods. During a
storm, they sing in a mighty chorus of acclaim. At such a time, the
breaking of many small branches sounds like the snapping of
overstrained violin strings.
Almost any tree located on a cliff or on the edge of a mountain,
becomes a musician of the first order. It is apt to take on the
sorrowful tendencies of solitude. The weepings, wailings,
murmurings, groanings, sighs and whispers of the universe vibrate
through its branches. It would seem as if such a tree were trying to
express many mysterious wonders of which man has little
knowledge.
The trees are not altogether dependent upon their leaves for their
music. The barren branches of fall and winter sing in a most
attractive way. Their dry and discarded leaves litter the ground and
carry on crackly songs of their own, or sing as they play tag in whirls
of wind. The Elm is a pleasing autumn singer and the Willows, when
covered with ice, rattle their twigs like a minstrel’s bones. As the
winter wind hums around the Cottonwood Trees, it rocks the seed
balls in their natural cradles with a sighing, crooning sound. This is
the way the Tree sings to her babies! When the wind soughs through
a hollow tree, it produces a ghostly sound suggestive of a mourning
or dying person. A current of air rubbing two boughs together causes
a scrunching sound which sends the shivers up one’s back.
It is reasonable to believe that every tree and plant has its own
individual voice as set in motion by the wind. A Nature-lover does not
have much difficulty in distinguishing a great many. The desert Sage
whistles in the wind; the Cedar laughs in the storm; the air rustles
through a Wheat field; an agitated Sugar Cane or Corn field gives
forth a sound like tinkling glass. The noise produced by a high wind
in the Southern Smilax has been likened to a harp struck at random.
The bursting pods of the Witch Hazel pop gently and the seeds fall
among the dead leaves like so many buck shot; the Oxalis sends
forth its seed-babies with the crack of a pistol shot. Members of the
Bean family moan in the breeze like plaintive violins. The Squirting
Cucumber gurgles not unlike certain frogs. The Sunflower is a
professional drummer who rattles his seeds about in his pods. The
Rattlesnake Iris holds its seed-capsule in such a way that it gives an
excellent imitation of the warning noise of the reptile for which it is
named. Catalpa pods snap like horse-whips, but Cat-Tails sigh like
small reed instruments.
Early man gained more inspiration and pleasure from the music of
the plants than his wiser but more worldly successors. It is said that
the idea for the first flute was obtained by listening to the wind sigh
through the Reeds on the shore of a lake. The first stringed
instrument was probably a fibre accidentally stretched across a
hollow shell. The classic Aeolian harp consisted of a wooden frame
containing a thin sounding-board over which were stretched a
number of strips of cat-gut. If placed before a half-open window so
that an air current strikes it sideways, it gives forth a great volume of
harmonious notes in several octaves. This is a clear case of catching
the music of the wind. In a cruder, less harmonious way, the
Japanese glass tinklers of our day do the same thing. The humming
of telegraph wires and the strange chirping of a wireless instrument
are also a kind of singing.
All the plants are not expert musicians, which explains why they
often seek to make up for their own deficiencies by hiring numerous
birds and insects to make melody for them. These musicians are
employed in the truest sense of the word and receive their pay in
food, shelter and protection. In the air and on the ground, by day and
by night, they sing and fiddle for their hosts. The broad leaves of the
Water Lily (Victoria Regia) are veritable music schools of Frog
practice. Every voice from croaking bass to youthful tenor is heard!
Every tree has its Frogs and Birds—every bush and shrub
innumerable insect warblers.
The birds are the plants’ vocalists. Their songs and delightful
twitterings are among the most familiar things in Nature. The music
of the large body of insect-instrumentalists is carried on in such
obscure places, and often so far down among the very roots of the
plants, that a considerable investigation of their methods may not be
amiss. They are especially active after sundown.
The common Grasshoppers form a great corps of violinists. A
large vein on the inside of their thighs makes an ideal bow. It is
roughened not with resin but by a hundred minute spines. When this
vein is rubbed to and fro on the serrated veins of the insect’s wing-
cover, a shrill tone is produced. Sitting on its haunches, the
Grasshopper saws away with both hind legs at a great rate. The
interesting discovery has been made that the velocity of the strokes
increases with the temperature. Grasshoppers in large swarms emit
a low roar.
The Locust is a near relative of the Grasshopper. His music is
produced by scraping one wing across the other. The Cricket uses
the same method. When he is a house species, he fiddles in a
higher tone. The gold-green Muskback Beetle is an exquisite
violinist. His instrumental methods are most peculiar. His sharp
breast acts as a bow which he draws across a small group of veins
on his wing covers. The resulting music is so faint as to be almost
inaudible.
To Bees, Wasps, Hornets, Flies and Mosquitoes we may ascribe
reed instruments. They depend upon the rapid vibration of their tiny
wings to get their effects. The respiration openings distributed over
the body of a Bee, by giving resonance to the tone, aid in the
process and turn the whole insect’s body into a small clarionet. The
drowsy buzz of the honey-gatherer is only attained by swinging its
wings at the rate of four hundred vibrations a minute. People who
have good ears for music have observed that the ordinary Bee
drones his song out on G sharp. The House-Fly is credited with
singing at F with a preliminary grace note on E. Everyone is familiar
with the high thin plaint of the Mosquito.
There are many drummers in the insect orchestra. The Cicada
operates a small kettle drum. On the front of its body, a tough
membrane is stretched over a small cavity. When set in motion by a
special muscle, it gives out a surprisingly agreeable sound. The
Greeks enjoyed this music so well that they often caged the Cicada
much as they would a bird. In the hatching time of the seventeen-
year variety, the energetic drumming of thousands of the insects
rises into a scream which is far from melodious. Under such
conditions, the noise can be heard for half a mile. Travelers tell of a
giant South American species which produces a drumming which is
as loud as a locomotive whistle. An uncanny drummer is the “Death
Watch Beetle.” It uses its head for drumsticks and when in the wood
of furniture often plays a tattoo with considerable skill. Superstitious
people, for no apparent good reason, sometimes insist this is a
warning of impending death. Even the pretty little Butterfly on
occasion is a drummer. With hooks on its wings, it makes a sharp
crackle, not unlike one of the weird noises sometimes used by
human “traps.” Beetles play the bones.
The Bamboo Tree is sometimes the possessor of a whole corps of
intelligent and efficient drummers. They attach themselves to the
under side of the leaves, from which vantage-point they strike them
with their heads whenever their services are required. An Ant of the
Sumatran species keeps wonderful time. Though spread out over a
number of square yards of leaf space, a group of these tiny
creatures will start and stop tapping at the same instant.
Perhaps in some far-distant age, mankind will begin remotely to
understand the significance of the music of the plant world and its
allies. We have no right to say that the plants are not true musicians.
While we may only understand their system of harmony in part, we
can realize it contains hidden beauties just as the presence of
microscopic organisms in the world is indicated by their effects rather
than by actual perception.
CHAPTER IX
Science in the Plant World
“Weak with nice sense, the chaste Mimosa stands,

From each rude touch withdraws her timid hands.”
Plants are profound scientists. Their knowledge may not be as
broad and far-reaching as that of man, but they are more successful
workers than he. With all his wonderful discoveries in physics and
chemistry, man as a class has not yet learned to conduct his own
body so as to make it yield the highest efficiency. In fact, members of
the human race are today wearing out their frames at a faster rate
than ever before. Adept at running huge mechanisms of steel, they
are neglectful of those most delicate and wonderful machines which
are bound up with their own life processes.
Plants are not so prodigal. Whenever they are given a chance,
they develop and expand their powers in the most marvelous way.
They bring out the latent strength in their beings and so conduct
themselves as to conserve their energies. Whether by instinct,
reason or blind force they always know just what to do and how to
make the most of their heredity and environment. Their efficiency
rating is one hundred per cent.
As the whole life of all plants is a scientific progression, we can
only consider in the brief limits of this chapter some of the more
startling instances of the marvelous sense they exhibit in dealing
with Nature’s forces.
Probably one of the reasons we do not always think of plants in
the human, sympathetic way we should, is that we are inclined to
regard them as quiet, static objects, playthings of every wind that
blows upon them. Such is far from the case. Life is motion and the
plants are very much alive and very much in motion. From the tiniest
cell to the largest tree they exhibit constant, pulsating movements.
Many of the movements are described through so small a space as
ordinarily to escape our notice, but a little observation makes them
quite apparent. They all have a well-directed, scientific purpose.
What is plant growth itself but motion upward and outward? If a
telescope or an instrument such as Sir Jaghadish Bose’s
crescograph be trained on a healthy plant, it is possible to see the
growth actually take place before the eye somewhat as it is
managed in motion pictures. Travelers aver that if a Banana Plant be
cut off close to the ground and the surrounding soil well supplied with
water, the sturdy creature will make such strenuous efforts to destroy
the effects of its mutilation that its growth may easily be perceived
with the unaided eye, and a full-sized leaf produced in a single day.
Leaves and flowers are usually quite mobile. When they go to
sleep, they droop and fold their edges together very carefully,
sometimes to such an extent as to make themselves almost
invisible. Even such an astute man as Linnaeus was once
completely deceived by some sleeping specimens of Lotus. They
were very fine red flowers and he was proud of them. Taking a friend
to view them one evening by lantern-light, what was his dismay to
find that they had completely disappeared. He concluded that they
had been stolen or eaten by insects and went away, only to find
them in full array on his return the next morning. It took several
nocturnal visits to unravel the mystery and discover that the flowers
folded themselves and retired so adroitly into the surrounding foliage
each evening that they were completely hidden.
The Acacia is a plant which closes up at night; the same
phenomenon is very striking in the Oxalis. The common Bean sleeps
standing: that is, its leaves close upward instead of downward. The
little blue Veronica flower, so strikingly brilliant and attractive in the
daytime, tucks itself in so snugly at bedtime that it becomes quite
inconspicuous. A Marigold called Calendula Pluvialis even contracts
its corolla every time the sun is veiled by a passing cloud. These
sleep movements all have a scientific purpose. Their main object,
just as in animals, is to reduce bodily activities to a low ebb and so to
give the plant a chance to recuperate for another day’s efforts. The
contraction of all surfaces cuts down the radiation of heat and
moisture and presents less resistance to outside elements. The plant
is in a quiescent, somnolent state.
There are other movements of leaves and flowers the object of
which is not quite so apparent. For instance, there is the Hedysarum
Gyrans or Oscillating Sainfoin. Each of its leaves has three folioles.
The center one is very large and stands bolt upright, except at night,
when it condescends to bend its head in sleep. The two lateral
folioles are in perpetual oscillation both day and night. Nothing but a
very hot sun seems able to stop their movement. Possibly, this plant
is a fresh air fiend which requires a steady atmospheric flow upon its
respiratory surfaces! The two lateral folioles of each leaf are
delegated to act as fans and blow a constant supply of air upon their
majestic brother.
Similar oscillations have been noticed in some Orchids, where a
part of the flower’s corolla rises and falls with a regular rhythm not
unlike the beating of a human pulse.
The stamens and pistils of flowers sometimes have the power of
movement. If an insect, wandering about in the flower of the
Barberry Tree (Berberis Vulgaris), happens to touch the base of a
stamen, it bends forward with a quick, spring-like motion and
presently straightens up again. The evident intent is to shower some
pollen on the little intruder with the hope that he may carry its vital
principle to some neighbour of the same species.
In the Parnassia Palustris, fortunate observers have sometimes
seen the five stamens bend forward and beat on the head of the
pistil in rotation as if on an anvil. Perhaps outside pollen-carrying
agencies have passed this particular flower by and, in desperation, it
is resorting to self-fertilization.
The Junger Mania, a plant allied to the Mosses, shows knowledge
of the laws of mechanics when it uses a natural spring coiled in a
small tube to project its seeds out into the world. Seeds of fresh-
water Algae swim about for a few hours after leaving their mother-
plant, vibrating their cilia with great rapidity. It is the ability of certain
one-celled plants to move about freely which causes considerable
discussion as to whether they are really not animals. The Diatoms
are examples. They propel themselves through the water by
oscillating their whole bodies from side to side. To reverse their
direction they go backward like a ferryboat.
The ancients as far back as Aristotle recognized the sensitiveness
of plants to light and their eager use of its life-giving properties. In
fact, one has only to watch the Sun-Flower follow the orb of day
across the heavens to realize that there must be something vital in
sunlight for the plants. What interests us is that they have the instinct
or the knowledge to so present their surfaces to the light that they
receive a maximum benefit from its influences. From the aristocratic
indoor potted plant to the wild trees and shrubs on the edge of a
thicket, we notice a vigorous straining toward the light. Each leaf is
tilted at just the right angle to receive the largest possible share of
energy, for the leaves are starch factories for which the sun furnishes
the motive power.
Botanists tell us that this heliotropism or turning motion toward the
light is due to the tendency of most leaves to arrange themselves
perpendicularly to the sun’s rays. Tendrils may be apheliotropic or
tend to turn away from the light. Morning Glories or Wistaria, which
climb up whatever support is handy, exhibit insensibility to light no
matter from what angle it strikes. Stems, flower and leaves of all
plants each give a different and scientific reaction to light in a way
which looks much like directing thought.
Nothing is more scientific than the skill with which plants co-
operate with gravity in constructing their root systems. The roots are
often trained to grow out horizontally and resist gravity for a certain
distance. Then they gracefully yield to its pulling power, and, curving
their tips downward, grow straight toward the center of the earth. Any
secondary roots which are sent out again start horizontally to repeat
the above process on a smaller scale. All this makes for an efficient,
well-balanced root-system.
A curious motion which is not thoroughly understood is a slight
gyratory movement observable in the tips of all living plants. It is
possible that it is connected in some way with the earth’s rotation or
is it merely a kind of groping, feeling gesture? In the case of roots,
where the same gyrations occur, it undoubtedly serves that purpose.
A revolving root tip makes a very efficient drill with which the hardy
plant may bore a way through refractory soil. It is claimed that the
great whirling sweeps made by tendrils of various climbers are
merely amplifications of the circumnutation occurring in all plant
terminals.
Before leaving the subject of scientific movement in the plant
world, it will be of interest to briefly consider some of the vegetable
motions which are called forth by the stimulus of touch. Almost
everyone is familiar with the Sensitive Plant and its double rows of
tiny leaves. Touch any one of them and the whole group will instantly
begin to contract and bend toward the stalk. We say begin, for so
slow is the transmission of the impulse that one can readily see its
progress, as one after another of the leaves respond.
A motion which has forethought and design behind it occurs in the
leaves of the famous and crafty Venus Fly-Trap. Two sections of
leaves edged with teeth-like nerve-hairs form the two halves of an
enticing-looking bowl and cover. The slightest contact with one of the
delicate hairs will cause the trap to shut together and imprison any
sweet-toothed member of the insect world which has happened to
stray inside. An aquatic form of the same thing occurs in a species of
Bladderwort which spreads a leaf-net cunningly shaped to look like a
fish’s mouth. Frightened baby-fishes, accustomed to seek their
mother’s throat in time of danger, sometimes swim in and, brushing
certain nerve-hairs near the entrance, cause the lips to close and
leave them to slow dissolution. Both sinister and scientific are the
movements of carnivorous plants.
Far from being static or quiescent, the plant world is a kingdom of
energetic, vibratory motion—a motion which is cool and calculating
and which rarely fails to accomplish its purpose. Even the
protoplasm of microscopic plant cells is in constant movement. If a
thin slice of Sycamore bark be placed under a microscope, a regular
circulation of cell-liquid, suggestive of blood circulation in animals,
can be observed.
Plants show great skill in their use of water. It is their storage of
liquid in their cells which makes their soft bodies rigid and so makes
movement possible. This property sometimes called turgidity was
discovered by the scientist De Vries in 1877, the same year that
Pfeffer established the theory of osmosis. This latter is a
phenomenon which physicists find very difficult to explain and
involves the transmutation of one liquid into another through the
medium of an intervening membrane.
Some plants have acquired the faculty of storing water in their
bodies, on which, camel-like, they can subsist for long periods of
time. A certain large tree-cactus of the American desert sometimes
stores up as much as seventeen hundred pounds or five barrels of
water in the wet season. When drought comes, its roots dry up and it
lives entirely on its internal resources. It is said that an eighteen-foot
specimen can exist for a year on its stored-up liquid. A branch on
such a plant may live and bloom after the trunk is dead. Many
ordinary plants, such as Turnips, Carrots, and Beets, store water
along with starch and dextrose in their underground tubers. Such
subterranean reservoirs are preferable to those above ground.
Plants have paid particular attention to the manipulation of gases.
They maintain an internal atmosphere of their own composed of
oxygen, nitrogen and carbon dioxide in proportions varying greatly
from those of the outside air. If the stem of a Water Lily be broken
below the surface of a pond, gas bubbles will often be observed to
issue from the wound, indicating that the internal gas pressure of this
particular plant is greater than that of the external air. In other cases,
the reverse is true and we find partial vacuums within the bodies of
plants.
Man long ago found it impossible to “live on air” but the plants
have solved the difficulty of aerial existence and have become
creatures of the air rather than the earth, so far as their food is
concerned. The great bulk of the largest tree is preponderantly
composed of carbon, which has been slowly and labouriously
extracted from the air. The mineral salts and water which have been
filtered out of the ground by the roots are essential but are present in
a much lesser quantity.
It is well known that plants breathe in carbon dioxide and breathe
out oxygen. This can be graphically demonstrated by placing a plant
in a glass jar of carbon dioxide inverted in water. If its life processes
are quickened by exposure to sunlight, the plant will replace the CO₂
with oxygen in a day. A more striking example is furnished by any
aquatic plant accustomed to growing submerged in ponds and rivers.
Placed in a water-filled bottle inverted in a pan of water, it will
generate oxygen so rapidly that the bubbles can be seen forming on
the leaves when the sun is allowed to strike them fully. The bottle will
become filled with oxygen in a few hours, and its presence can be
demonstrated with the usual ember test.
Opposed to the absorption of carbon dioxide and the breathing out
of oxygen, which is really a digestive operation, the plants, queerly
enough, carry on a directly opposite process which involves the
absorption of oxygen and the breathing out of carbon dioxide. This is
a respiratory process akin to breathing in animals. It is carried on in
such a relatively small way that it does not seriously affect the
statement that “plants breathe in carbon dioxide and breathe out
oxygen” and so are purifiers of the air which man and animals
contaminate.
Besides this general use of gases common to nearly all plants, a
few of the members of the vegetable world specialize in the
production of protective and poisonous vapours of various
composition. One of the most interesting of these is the Gas Plant of
the South American jungles. This beautiful white-flowered inhabitant
of the tropics is entirely protected from leaf-destroying insects and
birds by the poisonous vapours it constantly pours forth.
The plants are expert chemists, and the reactions in which they
engage are, on the whole, much simpler than those which go on in
the bodies of animals. Vegetable tissue is largely carbon, hydrogen,
oxygen and nitrogen. It is a curious fact that instead of using the
abundant carbon compounds present in decomposed animal and
vegetable matter of the soil the plants get most of their carbon from
the carbon dioxide of the air. Inversely, they largely disregard the
seventy-eight per cent nitrogen of the air, and extract that element

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9 Mass Spectrometry with High-Performance 16 Water Hardness Testing by Complexometric

15 Vitamin C Determination by Indophenol 22 Fat Characterization 185

23 Proteins: Extraction, Quantitation, 27 CIE Color Specifications Calculated

1.1 INTRODUCTION cal ­relationship between the analyte concentration and

­alances are usually sensitive to 0.001–0.00001 g,

1.4.1 Operation 10. With plunger fully depressed, withdraw

Reproducibilitya Maximum volume Lowest recommended volume

volumetric measurements can result in significant

dilutions and concentrations whenever possible to 1. 6

1.7.3  ounding Off Single Arithmetic

“Weak with nice sense, the chaste Mimosa stands,

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9 Mass Spectrometry with High-Performance 16 Water Hardness Testing by Complexometric

15 Vitamin C Determination by Indophenol 22 Fat Characterization 185

23 Proteins: Extraction, Quantitation, 27 CIE Color Specifications Calculated

1.1 INTRODUCTION cal relationship between the analyte concentration and

alances are usually sensitive to 0.001–0.00001 g,

1.4.1 Operation 10. With plunger fully depressed, withdraw

1.7.3 ounding Off Single Arithmetic