Current protocols in food analytical chemistry ronald e wrolstad, terry e acree, haejung an, eric a decker, michael h penner, david s reid, steven j schwartz, charles f shoemaker

APPENDIX 1ABBREVIATIONS AND USEFUL DATA APPENDIX 1A Abbreviations Used in This Manual AACC American Association of Cereal Chemists ACS American Chemical Society AED atomic emission detec

APPENDIX 1

ABBREVIATIONS AND USEFUL DATA

APPENDIX 1A

Abbreviations Used in This Manual

AACC American Association of Cereal

Chemists

ACS American Chemical Society

AED atomic emission detection

AEDA aroma extract dilution analysis

AMC 7-amido-4-methylcoumarin

ANS anilinonapththalene sulfonate

AOAC Association of Official Analytical

Chemists

AOCS American Oil Chemists’ Society

AOM active oxygen method

AOS allene oxide synthase

BCA bicinchoninic acid

BET Brunauer-Emmet-Teller (equation)

BGG bovine gamma globulin

BHA butylated hydroxyanisole

BHC branched hydrocarbons

BHT butylated hydroxytoluene

Brij 35 polyoxyethylene 23-lauren ether

°Brix measure of sugar content as

determined by refractometer with a Brix scale

BSA bovine serum albumin

BV biological value

CAPT compensated attached proton test

CD conjugated diene; circular dichroism

CDTA

trans-1,2-diaminocyclohexane-N,N,N′,N′-tetraacetic acid

CETAB cetyltrimethylammonium bromide

Chl a and b chlorophyll a and b

CI chemical ionization

CID collision-induced dissociation

CIE Commission Internationale de

l’Éclairage (International Commission for

Illumination)

CI/MS chemical ionization/mass

spectrometry

CLA conjugated linoleic acids

CLSM confocal laser-scanning microscopy

CMC critical micelle concentration

COSY correlation spectroscopy

DAD diode array detector DCI direct exposure chemical ionization DCM dichloromethane

DEI direct exposure electron impact Deoxy Mb deoxymyoglobin

DH degree of hydrolysis DHP dihydroxy pigment DMF dimethylformamide DMSO dimethylsulfoxide DNPH 2,4-dinitrophenylhydrazine DOPA 3,4-dihydroxyphenylalanine dpd delphinidin

DPO diphenol oxidase DQF-COSY double quantum filtered

correlation spectroscopy

DSA drop shape analysis DSC differential scanning calorimetry DTNB 5,5′-dithiobis(2-nitrobenzoic acid)

DTT dithiothreitol DVT drop volume tensiometer

EC Enzyme Commission EDTA ethylenediaminetetraacetic acid

EI electron impact EI/MS electron impact/mass spectrometry ELSD evaporative light-scattering detector

EM expressible moisture EPA (U.S.) Environmental Protection

Agency

ERH equilibrium relative humidity ESI electrospray ionization FAB/MS fast atom bombardment mass

spectrometry

FAME fatty acid methyl ester

FC Folin-Ciocalteau FDA (U.S.) Food and Drug Administration FFA free fatty acids

FID free induction decay; flame ionization

detection

FOX ferrous oxidation/xylenol orange

method

FP fecal protein FPD flame photometric detection FPLC fast protein liquid chromatography FTIR Fourier-transform infrared

(spectrometry)

Current Protocols in Food Acid Chemistry (2003) A.1A.1-A.1A.3

Abbreviations and Useful Data

g gravity (in expressions of relative

centrifugal force)

GAB Guggenheim-Anderson-DeBoer

(equation)

GC gas chromatography GC/FID gas-liquid chromatography with

flame ionization detection

GC/MS gas-liquid chromatography with

mass selective detection

GC/O gas chromatography/olfactometry GLC gas-liquid chromatography GOPOD glucose oxidase/peroxidase

(reagent)

HBS hydroxybenzenesulfonamide HDPE high-density polyethylene HEC hydroxyethylcellulose

IS internal standard ISO International Standard Organization IUB International Union of Biochemistry IUBMB International Union of

Biochemistry and Molecular Biology

IUPAC International Union of Pure and

LC-APCI-MS liquid chromatography/

atmospheric pressure chemical ionizationmass spectrometry

LED light-emitting diode LOX lipoxygenase LSIMS liquid secondary ion mass

spectrometry

LVE linear viscoelastic region

MA malonaldehyde; malondialdehyde MALDI matrix-assisted laser

desorption/ionization

MALDI-TOF MS matrix-assisted laser

desorption/ionization time-of-flight massspectrometer

MCAC metal-chelate affinity

chromatography

MCC microcrystalline cellulose MDGC multidimensional gas chromato-

MOPS 3 -(N-morpholino)propane sulfonic

OU odor units Oxy Mb oxymyoglobin PBS phosphate-buffered saline PDA photodiode array PDCAAS protein digestibility–corrected

amino acid score

PDMS polydimethylsiloxane

PE pectinesterase PEG polyethylene glycol PER protein efficiency ratio PGase polygalacturonase pgd pelargonidin

pI isoelectric point

PIPES piperazine-N,Nsulfonic acid)

′-bis(2-ethane-PL pectic lyase PMSF phenylmethylsulfonyl fluoride

Abbreviations

Used in This

Manual

RAS retronasal aroma stimulator

RDA recommended dietary allowance

RF radio frequency

RFI relative fluorescence intensity

RI retention index

RNU relative nitrogen utilization

ROESY rotational nuclear Overhäuser

RVP relative vapor pressure

S sieman (unit of conductance)

SD standard deviation

SDE simultaneous distillation extraction

SDS sodium dodecyl sulfate

SFC solid fat content

SFI solid fat index

SHAM salicylhydroxamic acid

SIM selected ion monitoring

SNIF-NMR site-specific natural isotope

fractionation measured by nuclear magnetic

resonance spectroscopy

SP-HPLC straight-phase

high-perform-ance liquid chromatography

SPME solid-phase microextraction

SV saponification value

TA titratable acidity TBA thiobarbituric acid TBARS thiobarbituric acid-reactive

substances

TBS Tris-buffered saline TCA trichloracetic acid

TD true digestibility TEA triethylamine TFA trifluoroacetic acid THF tetrahydrofuran TLC thin-layer chromatography

TLCK N α-p-tosyl-L-lysine chloromethylketone

TMCS trimethylchlorosilane imidazole TMG tetramethylguanidine

TMP 1,1,3,3-tetramethoxypropane TMS trimethylsilyl

TNBS trinitrobenzenesulfonic acid TOCSY total correlation spectroscopy TPA texture profile analysis

TPCK N-tosyl-L-phenylalaninechloromethyl ketone

TRF theoretical relative response factor Tris tris(hydroxymethyl)aminomethane Tris ⋅Cl Tris hydrochloride

TTS time-temperature superposition

U unit (of enzyme activity) UHP ultra high purity USDA United States Department of

Agriculture

UV ultraviolet WHC water holding capacity

WUA water uptake ability

A.1A.3

Abbreviations and Useful Data

APPENDIX 2

LABORATORY STOCK SOLUTIONS,

EQUIPMENT, AND GUIDELINES

APPENDIX 2A

Common Buffers and Stock Solutions

This section describes the preparation of buffers and reagents used in the manipulation

of nucleic acids.

For preparation of acid and base stock solutions, see Tables A.2A.1 and A.2A.2 as well

as individual recipes.

GENERAL GUIDELINES

When preparing solutions, use deionized, distilled water and (for most applications)

reagents of the highest grade available Sterilization is recommended for most

applica-tions and is generally accomplished by autoclaving Materials with components that are

volatile, altered or damaged by heat, or whose pH or concentration are critical should be

sterilized by filtration through a 0.22- µm filter In many cases such components are added

from concentrated stocks after the solution has been autoclaved Where specialized

sterilization methods are required, this is indicated in the individual recipes.

CAUTION: It is important to follow laboratory safety guidelines and heed manufacturers’

precautions when working with hazardous chemicals; consult institutional safety officers

and appropriate references for further details.

STORAGE

Most simple stock solutions can be stored indefinitely at room temperature if reasonable

care is exercised to keep them sterile; where more rigorous conditions are required, this

is indicated in the individual recipes.

Table A.2A.1 Molarities and Specific Gravities of Concentrated Acids and Basesa

weight

% by weight

Molarity (approx.)

1 M solution (ml/liter)

Specific gravity

108.882.1

1.541.70

Table A.2A.2 pKa Values and Molecular Weights for Some Common Biological Buffers

range

Mol wt.(g/mol)

Citric acidb C6H7O7− (H2Cit−) 4.74 (pKa2) —

Citric acidb C6H6O7− (HCit2−) 5.40 (pKa3) —

bAvailable as a variety of salts, e.g., ammonium, lithium, sodium.

Current Protocols in Food Analytical Chemistry

A.2A.2

SELECTION OF BUFFERS

Table A.2A.2 reports pKa values for some common buffers Note that polybasic buffers,

such as phosphoric acid and citric acid, have more than one useful pKa value When

choosing a buffer, select a buffer material with a pKa close to the desired working pH (at

the desired concentration and temperature for use) In general, effective buffers have a

range of approximately 2 pH units centered about the pKa value Ideally the dissociation

constant—and therefore the pH—should not shift with a change in concentration or

temperature If the shift is small, as for MES and HEPES, then a concentrated stock

solution can be prepared and diluted without adjustment to the pH Buffers containing

phosphate or citrate, however, show a significant shift in pH with concentration change,

and Tris buffers show a large change in pH with temperature For convenience,

concen-trated stock solutions of these buffers can still be used, provided that a pH adjustment is

made after any temperature and concentration adjustments All adjustments to pH should

be made using the appropriate base—usually NaOH or KOH, depending on the

corre-sponding free counterion Tetramethylammonium hydroxide can be used to prepare

buffers without a mineral cation Many common buffers are supplied both as a free acid

or base and as the corresponding salt By mixing precalculated amounts of each, a series

of buffers with varying pH values can conveniently be prepared.

Citrate-phosphate buffer (McIlvaine’s buffer)

Solution A: 19.21 g/liter citric acid (0.1 M final)

Solution B: 53.65 g/liter Na2HPO4⋅7H2O or 71.7 g/liter Na2HPO4⋅12H2O

Referring to Table A.2A.3 for desired pH, mix the indicated volumes of solutions

A and B, then dilute with water to 100 ml Filter sterilize, if necessary, using a 0.2

µm filter and store up to 1 month 4°C.

DTT (dithiothreitol), 1 M

Dissolve 1.55 g DTT in 10 ml water and filter sterilize Store in aliquots at −20°C.

Do not autoclave to sterilize.

EDTA (ethylenediaminetetraacetic acid), 0.5 M (pH 8.0)

Dissolve 186.1 g disodium EDTA dihydrate in 700 ml water Adjust pH to 8.0 with

10 M NaOH ( ∼50 ml; add slowly) Add water to 1 liter and filter sterilize.

Begin titrating before the sample is completely dissolved EDTA, even in the disodium salt

form, is difficult to dissolve at this concentration unless the pH is increased to between 7

and 8 Heating the solution may also help to dissolve EDTA.

Potassium acetate buffer, 0.1 M

Solution A: 11.55 ml glacial acetic acid per liter (0.2 M) in water.

Solution B: 19.6 g potassium acetate (KC2H3O2) per liter (0.2 M) in water.

Referring to Table A.2A.4 for desired pH, mix the indicated volumes of solutions A and B, then dilute with water to 100 ml Filter sterilize if necessary Store up to 3 months at room temperature.

This may be made as a 5- or 10-fold concentrate by scaling up the amount of sodium acetate

in the same volume Acetate buffers show concentration-dependent pH changes, so check the

pH by diluting an aliquot of concentrate to the final concentration.

To prepare buffers with pH intermediate between the points listed in Table A.2A.4, prepare closest higher pH, then titrate with solution A.

Table A.2A.3 Preparation of Citrate-Phosphate Buffers

Desired pH Solution A (ml) Solution B (ml)

aAdapted with permission from Fasman (1989).

Current Protocols in Food Analytical Chemistry

A.2A.4

Common Buffers

and Stock

Solutions

Potassium phosphate buffer, 0.1 M

Solution A: 27.2 g KH2PO4 per liter (0.2 M final) in water.

Solution B: 34.8 g K2HPO4 per liter (0.2 M final) in water.

Referring to Table A.2A.5 for desired pH, mix the indicated volumes of solutions

A and B, then dilute with water to 200 ml Filter sterilize if necessary Store up to

3 months at room temperature.

This buffer may be made as a 5- or 10-fold concentrate simply by scaling up the amount of

potassium phosphate in the same final volume Phosphate buffers show

concentration-de-pendent changes in pH, so check the pH of the concentrate by diluting an aliquot to the final

concentration.

To prepare buffers with pH intermediate between the points listed in Table A.2A.5, prepare

closest higher pH, then titrate with solution A.

Table A.2A.4 Preparation of 0.1 M Sodium

and Potassium Acetate Buffersa

DesiredpH

Solution A(ml)

Solution B(ml)

aAdapted by permission from CRC (1975).

Table A.2A.5 Preparation of 0.1 M Sodium and Potassium Phosphate Buffersa

Desired

pH

Solution A(ml)

Solution B(ml)

DesiredpH

Solution A(ml)

Solution B(ml)

SDS, 20% (w/v)

Dissolve 20 g SDS (sodium dodecyl sulfate or sodium lauryl sulfate) in water to 100

ml total volume with stirring Filter sterilize using a 0.45- µm filter.

It may be necessary to heat the solution slightly to fully dissolve the powder.

Sodium acetate, 3 M

Dissolve 408 g sodium acetate trihydrate (NaC2H3O2⋅3H2O) in 800 ml H2O Adjust pH to 4.8, 5.0, or 5.2 (as desired) with 3 M acetic acid (see Table A.2A.1) Add H2O to 1 liter

Filter sterilize

Sodium acetate buffer, 0.1 M

Solution A: 11.55 ml glacial acetic acid per liter (0.2 M) in water.

Solution B: 27.2 g sodium acetate (NaC2H3O2⋅3H2O) per liter (0.2 M) in water Referring to Table A.2A.4 for desired pH, mix the indicated volumes of solutions A and B, then dilute with water to 100 ml Filter sterilize if necessary Store up to 3 months at room temperature.

This may be made as a 5- or 10-fold concentrate by scaling up the amount of sodium acetate

in the same volume Acetate buffers show concentration-dependent pH changes, so check the

pH by diluting an aliquot of concentrate to the final concentration.

To prepare buffers with pH intermediate between the points listed in Table A.2A.4, prepare closest higher pH, then titrate with solution A.

Sodium phosphate buffer, 0.1 M

Solution A: 27.6 g NaH2PO4⋅H2O per liter (0.2 M final) in water.

Solution B: 53.65 g Na2HPO4⋅7H2O per liter (0.2 M) in water.

Referring to Table A.2A.5 for desired pH, mix the indicated volumes of solutions A and B, then dilute with water to 200 ml Filter sterilize if necessary Store up to 3 months at room temperature.

This buffer may be made as a 5- or 10-fold concentrate by scaling up the amount of sodium phosphate in the same final volume Phosphate buffers show concentration-dependent changes in pH, so check the pH by diluting an aliquot of the concentrate to the final concentration.

To prepare buffers with pH intermediate between the points listed in Table A.2A.5, prepare closest higher pH, then titrate with solution A.

Tris ⋅Cl, 1 M

Dissolve 121 g Tris base in 800 ml H2O Adjust to desired pH with concentrated HCl Adjust volume to 1 liter with H2O

Filter sterilize if necessary Store up to 6 months at 4 °C or room temperature

Approximately 70 ml HCl is needed to achieve a pH 7.4 solution, and ∼42 ml for a solution that is pH 8.0.

IMPORTANT NOTE: The pH of Tris buffers changes significantly with temperature, decreasing approximately 0.028 pH units per 1°C Tris-buffered solutions should be adjusted

to the desired pH at the temperature at which they will be used Because the pK a of Tris is 8.08, Tris should not be used as a buffer below pH ∼7.2 or above pH ∼9.0.

Always use high-quality Tris (lower-quality Tris can be recognized by its yellow appearance when dissolved).

Current Protocols in Food Analytical Chemistry

A.2A.6

Common Buffers

and Stock

Solutions

LITERATURE CITED

Chemical Rubber Company, 1975 CRC Handbook of Biochemistry and Molecular Biology, Physical and

Chemical Data, 3d ed., Vol 1 CRC Press, Boca Raton, Fla

Fasman, G.D (ed.) 1989 Practical Handbook of Biochemistry and Molecular Biology CRC Press, Boca

Raton, Fla

Mohan, C (ed.), 1997 Buffers: A Guide for the Preparation and Use of Buffers in Biological Systems,

Calbiochem, San Diego, Calif

Laboratory Stock Solutions, Equipment, and Guidelines

APPENDIX 2B

Laboratory Safety

Persons carrying out the protocols in the

laboratory may encounter various hazardous or

potentially hazardous materials including:

ra-dioactive substances; toxic chemicals and

car-cinogenic, mutagenic, or teratogenic reagents;

and pathogenic and infectious biological

agents Most governments regulate the use of

these materials; it is essential that they be used

in strict accordance with local and national

regulations Cautionary notes are included in

many instances throughout the manual, and

some specific guidelines for working safely

with chemicals are provided below (and

refer-ences therein) However, we emphasize that

users must proceed with the prudence and

pre-cautions associated with good laboratory

prac-tice, under the supervision of personnel

respon-sible for implementing laboratory safety

pro-grams at their institutions and in compliance

with designated guidelines of federal, state, and

local officials

HAZARDOUS CHEMICALS

It is not possible in the space available to list

all the precautions to be taken when handling

hazardous chemicals Many texts have been

written about laboratory safety; see Literature

Cited for a selected list of examples Obviously,

all national and local laws should be obeyed as

well as all institutional regulations Controlled

substances are regulated by the Drug

Enforce-ment Administration By law, Material Safety

Data Sheets must be readily available All

labo-ratories should have a Chemical Hygiene Plan

[29CFR Part 1910.1450] and institutional safety

officers should be consulted as to its

implemen-tation Help is (or should be) available from your

institutional Safety Office Use it

Chemicals should be stored properly For

example, flammable chemicals (e.g., ethanol,

methanol, acetone, methyl ethyl ketone,

petro-leum distillates, toluene, benzene, and other

materials labeled flammable) should be stored

in approved flammable storage cabinets, and

flammable chemicals requiring refrigeration

should be stored in explosion-proof

refrigera-tors Oxidizers should be segregated from other

chemicals, and corrosive acids (e.g., sulfuric,

hydrochloric, nitric, perchloric, and

hydroflu-oric acids) should also be stored in a separate

cabinet, well-removed from the flammable

or-ganics

Facilities should be appropriate for the

han-dling of hazardous chemicals In particular,

hazardous chemicals should only be handled inchemical fume hoods, not in laminar flow cabi-nets The functioning of these fume hoodsshould be periodically checked Laboratoriesshould also be equipped with safety showersand eye-washing facilities Again, this equip-ment should be tested periodically to make surethat it functions correctly Other safety equip-ment may be required depending on the nature

of the materials being handled In addition,researchers should be trained in the properprocedures for handling hazardous chemicals

as well as other areas of laboratory operations,e.g., handling of compressed gases, use of cryo-genic liquids, operation of high voltage powersupplies, etc

Before starting work, have a plan for dealingwith spills or accidents; coming up with a goodplan on the spur of the moment is difficult Forexample, have the appropriate decontaminat-ing or neutralizing agents prepared and close athand Small spills can probably be cleaned up

by the researcher In the case of larger spills,the area should be evacuated and help soughtfrom those experienced and equipped for deal-ing with spills, e.g., your institutional safetydepartment

Protective equipment should include, at aminimum, eye protection, a lab coat, andgloves Sandals, open-toed shoes, and shortsshould not be worn In certain circumstancesother items of protective equipment may benecessary, e.g., a face shield Different types ofgloves exhibit different chemical resistanceproperties; listings of these properties are avail-able (Forsberg and Keith, 1989) Glovesshould, however, be regarded as the last line ofdefense and should be changed if they becomecontaminated, because many types of chemi-cals pass relatively freely through rubber Ifpossible, handling procedures should be de-signed so that gloves do not become contami-nated All common-sense precautions should

be observed, e.g., do not pipet by mouth, keepunauthorized persons away from hazardouschemicals, prohibit eating and drinking in thelab, etc

Order hazardous chemicals only in ties that are likely to be used in a reasonabletime Buying large quantities at a lower unitcost is no bargain if someone (perhaps you) has

quanti-to pay quanti-to dispose of surplus quantities tute alcohol-filled thermometers for mercury-

Substi-Contributed by George Lunn

Current Protocols in Food Analytical Chemistry (2001) A.2B.1-A.2B.2

Copyright © 2001 by John Wiley & Sons, Inc.

A.2B.1

Laboratory Stock Solutions, Equipment, and Guidelines

filled thermometers The latter are a hazardouschemical spill waiting to happen.

Although any number of chemicals monly used in laboratories are toxic if usedimproperly, the toxic properties of a number ofreagents require special attention Many chemi-cals are considered carcinogenic, corrosive,flammable, lachrymatory, mutagenic, oxidiz-ing, teratogenic, or toxic Chemicals labeledcarcinogenic range from those accepted by ex-pert review groups as causing cancer in humans

com-to those for which only minimal evidence ofcarcinogenicity exists Oxidizers may reactviolently with oxidizable material, e.g., hydro-carbons, wood, and cellulose Before using anychemical, thoroughly investigate all of its char-acteristics Material Safety Data Sheets arereadily available; they list some hazards butvary widely in quality A number of texts de-scribing hazardous properties are listed in Fur-ther Reading In particular, Sax’s DangerousProperties of Industrial Materials, 8th ed (Le-wis, 1992) and Bretherick’s Handbook of Re-active Chemical Hazards, 4th ed (Bretherick,1990) give comprehensive listings of knownhazardous properties However, these texts listonly the known properties Many chemicalshave been tested only partially or not at all

Prudence dictates, therefore, that unless there

is good reason for believing otherwise, allchemicals should be regarded as volatile,highly toxic, flammable human carcinogensand should be handled with care

Waste should always be disposed of in cordance with all applicable regulations Wasteshould be segregated according to institutionalrequirements, for example, into solid, aqueous,nonchlorinated organic, and chlorinated or-ganic material A collection (Lunn and San-sone, 1994) of techniques for the disposal ofchemicals in laboratories has been publishedrecently Incorporation of these procedures intolaboratory protocols can help to minimizewaste disposal problems

ac-LITERATURE CITED

Bretherick, L 1990 Bretherick’s Handbook of active Chemical Hazards, 4th ed Butterworths,London

Re-Forsberg, K and Keith, L.H 1989 Chemical tective Clothing Performance Index Book JohnWiley & Sons, New York

Pro-Lewis, R.J., Sr 1992 Sax’s Dangerous Properties ofIndustrial Materials, 8th ed Van Nostrand-Rein-hold, New York

Lunn, G and Sansone, E.B 1994 Destruction ofHazardous Chemicals in the Laboratory, 2nd ed

John Wiley & Sons, New York

KEY REFERENCES

General safety

Freeman, N.T and Whitehead, J 1982 Introduction

to Safety in the Chemical Laboratory AcademicPress, New York

Furr, A.K (ed.) 1990 CRC Handbook of tory Safety, 3rd ed CRC Press, Boca Raton, Fla.Fuscaldo, A.A., Erlick, B.J., and Hindman, B (eds.)

Labora-1980 Laboratory Safety, Theory and Practice.Academic Press, New York

Miller, B.M (ed.) 1986 Laboratory Safety, ples and Practices American Society for Micro-biology, Washington, D.C

Princi-Occupational Health and Safety 1993 NationalSafety Council, Chicago

Pal, S.B (ed.) 1985 Handbook of LaboratoryHealth and Safety Measures Kluwer AcademicPublishers, Hingham, Mass

Young, J.A (ed.) 1987 Improving Safety in theChemical Laboratory: A Practical Guide JohnWiley & Sons, New York

Laboratory safety for hazardous chemicals

American Chemical Society, Committee on cal Safety 1990 Safety in Academic ChemistryLaboratories, 5th ed American Chemical Soci-ety, Washington, D.C

Chemi-Forsberg and Keith, 1989 See above

National Research Council, Committee on ous Substances in the Laboratory 1981 PrudentPractices for Handling Hazardous Chemicals inLaboratories National Academy Press, Wash-ington, D.C

Hazard-Properties and disposal procedures for hazardous chemicals

Aldrich Chemical Co 2001 Aldrich Catalog book of Fine Chemicals Aldrich Chemical Co.,Milwaukee, Wis

Hand-Bretherick, L (ed.) 1986 Hazards in the ChemicalLaboratory, 4th ed Royal Society of Chemistry,London

Bretherick, 1990 See above

Budavari, S (ed.) 1996 The Merck Index, 12th ed.Merck & Co., Rahway, N.J

Lewis, 1992 See above

Lunn, and Sansone, 1994 See above

Contributed by George LunnBaltimore, Maryland

Laboratory Safety

APPENDIX 2C

Standard Laboratory Equipment

Special equipment is itemized in the materials list of each protocol Listed below are

standard pieces of equipment in the modern food science laboratory—i.e., items used

extensively in this manual and thus not usually included in the individual materials lists.

See SUPPLIERS APPENDIX for contact information for commercial vendors of laboratory

Biohazard disposal containers and bags

Blender (e.g., Waring Blendor)

Bottles, glass and plastic

Bunsen burners

Centrifuges, low-speed (6,000 rpm) and

speed (20,000 rpm) refrigerated centrifuges,

ultracentrifuge (20,000 to 80,000 rpm), and

microcentrifuge that holds standard 0.5- and

1.5-ml microcentrifuge tubes

NOTE: Centrifuge speeds are provided as g or

as rpm (with example rotor models)

throughout the manual.

Cold room (4°C) or cold box

Computer (PC or Macintosh) and printer

Conical centrifuge tubes, 15- and 25-ml plastic

Cuvettes, plastic disposable, glass, and quartz

Darkroom and developing tank, or X-Omat

automatic X-ray film developer (Kodak)

Desiccators (including vacuum desiccators)

and desiccant

Dry ice

Filtration apparatus, for collecting acid

precipitates on nitrocellulose filters or

Gel electrophoresis equipment, horizontal

full-size and minigel apparatus, vertical

full-size and minigel apparatus for

polyacrylamide protein gels, and specialized

equipment for two-dimensional protein gels

Grinder (e.g., coffee grinder)

Heat-sealable plastic bags and apparatus

Heating blocks, thermostat-controlled metal

heating block that holds test tubes and/or

microcentrifuge tubes

Hoods, chemical and microbiological

Hot plates, with or without magnetic stirrer

Gloves, plastic and latex, disposable and

asbestos

Graduated cylinders

Ice buckets Ice maker Immersion oil for microscopy Kimwipes, or equivalent lint-free tissues

Lab coats Laboratory glass ware Light box, for viewing gels and autoradiograms

Liquid nitrogen and Dewar flask Magnetic stirrers (with heater is useful)

Markers, including indelible markers and china-marking pencils

Microcentrifuge, Eppendorf-type, maximum speed 12,000 to 14,000 rpm

Microcentrifuge tubes, 1.5-ml and 0.5-ml

Microscope, standard optical model (optionally with epifluorescence or phase-contrast illumination)

Microscope slides and coverslips Microwave oven, to melt agar and agarose

Mortar and pestle Muffle furnace Ovens, drying, vacuum, and microwave

Paper cutter, large size, for 46 × 57-cm Whatman paper sheets

Paper towels Parafilm Pasteur pipets and bulbs

pH meter and pH standard solutions

pH paper Pipet bulbs, or battery-operated pipetting devices—e.g., Pipet-Aid (Drummond Scientific)

Pipets, Pasteur and graduated, glass and plastic, serological (1- to 25-ml)

Pipettors, adjustable delivery, volume ranges 0.5 to 10 µl, 10 to 200 µl, and 200 to

1000 µl

Plastic wrap, UV transparent (e.g., Saran Wrap)

Polaroid camera Power supplies, 300-V for polyacrylamide gels; 2000- to 3000-V for some applications

Racks, for test tubes and microcentrifuge tubes

Radiation shield, Lucite or Plexiglas

Radioactive waste containers, for liquid and solid waste

Razor blades

Current Protocols in Food Analytical Chemistry (2001) A.2C.1-A.2C.2

Copyright © 2001 by John Wiley & Sons, Inc.

Supplement 2

A.2C.1

Laboratory Stock Solutions, Equipment, and Guidelines

Refrigerator, 4°C

Ring stands and rings Rotator, end-over-end Rubber bands Rubber policemen Rubber stoppers Safety glasses Scalpels and blades Scintillation counter Scissors

Shakers, orbital and platform Spectrophotometer, UV and visible Speedvac evaporator (Savant) Stir-bars, assorted sizes

Tape, masking and electrician’s Thermometers

Timer

UV transilluminator Vacuum aspirator Vacuum line Volumetric flasks Vortex mixers Wash bottles, plastic and glass Water baths, variable temperature up to 80°C

Water purification equipment, e.g., Milli-Q

system (Millipore) or equivalent

X-ray film cassettes and intensifying screens

Standard

Laboratory

Equipment

Almost a century ago, the first mass

spec-trometers were used to prove the existence of

isotopes of the elements During the first half

of the 20th century, physicists and physical

chemists used mass spectrometers to help

char-acterize new elements and the fission products

of radioactive elements as they were created or

discovered Other applications included the

analysis of isotopic enrichment of elements and

their inorganic derivatives As this era of mass

spectrometry reached maturity, by the 1940s,

the analysis of organic molecules emerged as a

new application of mass spectrometry

Begin-ning in 1945, organic mass spectrometers using

electron impact (EI) ionization became

com-mercially available and were used primarily by

the petroleum industry Toward the late 1950s,

organic mass spectrometers began to be used

for the analysis of a wider variety of organic

molecules, and gradually became a

fundamen-tal analytical tool for the characterization of

synthetic organic compounds

During the 1960s, high-resolution,

double-focusing magnetic sector instruments became

available from multiple manufacturers and

were widely used in organic chemistry for exact

mass measurements and elemental

composi-tion analysis EI was used for generating

struc-turally significant fragment ions for compoundidentification, and rules for structure elucida-tion using mass spectrometry were developed(for a thorough review of EI and ion fragmen-tation pathways, see McLafferty and Turecek,1993) Biomedical and food chemistry appli-cations of mass spectrometry were developedduring this time Chemical ionization (CI),which was developed by researchers in thepetroleum industry (Field, 1990), was quicklyadopted as a softer ionization alternative to EI,useful in reducing fragmentation so that mo-lecular weights could be confirmed more easily

CI became another standard ionization nique for mass spectrometry (see FigureA.3A.1 for a guide to the selection of ionizationtechniques in mass spectrometry)

tech-GAS CHROMATOGRAPHY/MASS SPECTROMETRY (GC/MS)

With the introduction of computerized datasystems for data acquisition, reduction, andstorage during the 1960s, the efficiency of massspectrometric analysis grew rapidly and con-tinues to grow to this day The use of computersfor data reduction and analysis helped gas chro-matography/mass spectrometry (GC/MS) be-come a practical and powerful tool for qualita-

Supplement 2

Contributed by Richard B van Breemen

Current Protocols in Food Analytical Chemistry (2001) A.3A.1-A.3A.7

Copyright © 2001 by John Wiley & Sons, Inc.

Figure A.3A.1 Flow chart illustrating the selection of a suitable ionization technique for the mass

spectrometric analysis of a sample Abbreviations: APCI, atmospheric pressure chemical ionization;

CI, chemical ionization; EI, electron impact; FAB, fast atom bombardment; MALDI, matrix-assisted

laser desorption/ionization

A.3A.1

Commonly Used Techniques

tive and quantitative analysis of compounds inmixtures Both EI and CI were immediatelyuseful for GC/MS, since both of these ioniza-tion methods require that the analytes be in thegas phase When capillary GC was incorpo-rated into GC/MS, this technique reached ma-turity The advantages of GC/MS includespeed, selectivity, and sensitivity Typically,GC/MS may be used to select, identify, andquantify organic compounds in complex mix-tures at the femtomole level Compounds areselected using a combination of chroma-tographic separation and mass selection, andwhen using tandem mass spectrometry(MS/MS; see discussion below), the fragmen-tation pathway may be used for additional se-lectivity The speed of GC/MS is determined

by the chromatography step, which typicallyrequires from several minutes to one hour peranalysis Although GC/MS remains importantfor the analysis of many organic compounds,this technique is limited to volatile and ther-mally stable compounds (see chromatogra-phy/MS selection flow chart in Fig A.3A.2)

Therefore, thermally unstable compounds—

including food pigments such as carotenoidsand chlorophylls and biomolecules such as pro-teins, carbohydrates, and nucleic acids—can-not be analyzed in their native forms usingGC/MS (for more details regarding GC/MSand its applications, see Watson, 1997)

DESORPTION IONIZATION MASS SPECTROMETRY

During the 1970s and early 1980s, tion ionization techniques such as field desorp-tion (FD), desorption EI, desorption CI (DCI),and laser desorption were developed to extendthe utility of mass spectrometry towards theanalysis of more polar and less volatile com-pounds (see Watson, 1997, for more informa-tion regarding desorption ionization techniquesincluding DCI and FD) Although these tech-niques helped extend the mass range of mass

desorp-spectrometry beyond a traditional limit of m/z

1000 and toward ions of m/z 5000 (Fig.

A.3A.1), the first breakthrough in the analysis

of polar, nonvolatile compounds occurred in

1982 with the invention of fast atom ment (FAB; Barber et al., 1982) FAB and itscounterpart, liquid secondary ion mass spec-trometry (LSIMS), facilitate the formation ofabundant molecular ions, protonated mole-cules, and deprotonated molecules of nonvola-tile and thermally labile compounds such aspeptides, chlorophylls, and complex lipids up

bombard-to approximately m/z 12,000 FAB and LSIMS

use energetic particle bombardment (fast atoms

or ions from 3,000 to 20,000 V of energy) toionize compounds dissolved in nonvolatile ma-trices such as glycerol or 3-nitrobenzyl alcoholand desorb them from this condensed phaseinto the gas phase for mass spectrometric analy-sis Molecular ions and/or protonated mole-cules are usually abundant and fragmentation

is minimal

sample

APCI, electrospray, particle beam, CF-FAB

GC/MS

Figure A.3A.2 Selection of chromatography-mass spectrometry system for the analysis of a

sample Abbreviations: APCI, atmospheric pressure chemical ionization; CF, continuous flow; CI,chemical ionization; EI, electron impact; FAB, fast atom bombardment; GC/MS, gas chromatogra-phy/mass spectrometry; LC/MS, liquid chromatography/mass spectrometry

Introduction to

Mass

Spectrometry for

Food Chemistry

Introduced in the late 1980s, matrix-assisted

laser desorption/ionization (MALDI) has

helped solve the mass-limit barriers of laser

desorption mass spectrometry so that singly

charged ions may be obtained up to m/z 500,000

and sometimes higher (Hillenkamp et al.,

1991) For most commercially available

MALDI mass spectrometers, ions up to m/z

200,000 are readily obtained Like FAB and

LSIMS, MALDI samples are mixed with a

matrix to form a solution that is loaded onto the

sample stage for analysis Unlike the other

matrix-mediated techniques, the solvent is

evaporated prior to MALDI analysis, leaving

sample molecules trapped in crystals of solid

phase matrix The MALDI matrix is selected

to absorb the pulse of laser light directed at the

sample Most MALDI mass spectrometers are

equipped with a pulsed UV laser, although IR

lasers are available as an option on some

com-mercial instruments Therefore, matrices are

often substituted benzenes or benzoic acids

with strong UV absorption properties During

MALDI, the energy of the short but intense UV

laser pulse obliterates the matrix and in the

process desorbs and ionizes the sample Like

FAB and LSIMS, MALDI typically produces

abundant protonated or deprotonated

mole-cules with little fragmentation

LIQUID

CHROMATOGRAPHY/MASS

SPECTROMETRY (LC/MS)

By the time that GC/MS had become a

standard technique in the late 1960s, LC/MS

was still in the developmental stages

Produc-ing gas-phase sample ions for analysis in a

vacuum system while removing the HPLC

mo-bile phase proved to be a challenging task Early

LC/MS techniques included a moving belt

in-terface to desolvate and transport the HPLC

eluate into a CI or EI ion source, or a direct inlet

system in which the eluate was pumped at a low

flow rate of 1 to 3 µl/min into a CI source

However, neither of these systems was robust

enough or suitable for a broad enough range of

samples to gain widespread acceptance

Since FAB (or LSIMS) requires that the

analyte be dissolved in a liquid matrix, this

ionization technique was easily adapted for

infusion of solution-phase samples into the

FAB ionization source, in an approach known

as continuous-flow FAB Continuous-flow

FAB was connected to microbore HPLC

col-umns for LC/MS applications (Ito et al., 1985)

Since this method is limited to microbore

HPLC applications at flow rates of <10 µl/min

and requires considerable operator tion, it is not ideal for the analysis of largesample sets Instead, more robust techniqueshave been developed to fulfill this requirement

interven-However, continuous-flow FAB is still in use

in some laboratories

Like continuous-flow FAB, the popularity

of particle beam interfaces is diminishing, butsystems are still available from commercialsources During particle beam LC/MS, theHPLC eluate is sprayed into a heated chamberconnected to a vacuum pump As the dropletsevaporate, aggregates of analyte (particles)form and pass through a momentum separatorthat removes the lower-molecular-weight sol-vent molecules Finally, the particle beam en-ters the mass spectrometer ion source where theaggregates strike a heated plate from which theanalyte molecules evaporate and are ionizedusing conventional EI or CI ionization Particlebeam LC/MS is limited to the analysis of vola-tile and thermally stable compounds that areamenable to flash evaporation and EI or CImass spectrometry Therefore, this approach isnot used for polar compounds in food chemistrysuch as carbohydrates, sugars, peptides, pro-teins, or nucleic acids (Fig A.3A.2)

Since thermospray became the first widelyutilized LC/MS technique (during the late1970s and early 1980s), this technique should

be mentioned here Thermospray facilitates theinterfacing of standard analytical HPLC sys-tems at flow rates up to 1 ml/min with massspectrometers Although the interface betweenthe HPLC and mass spectrometer is inefficientand exhibits low sensitivity for most analytes,thermospray has been useful for the LC/MSanalysis of many types of small molecules

During thermospray, the HPLC eluate issprayed through a heated capillary into a heateddesolvation chamber at reduced pressure Gasphase ions remaining after desolvation of thedroplets are extracted through a skimmer intothe mass spectrometer for analysis The sensi-tivity of thermospray is poor since there is nomechanism or driving force to enhance thenumber of sample ions entering the gas phasefrom the spray during desolvation Also, ther-mally labile compounds tend to decompose inthe heated source These problems were solvedwhen thermospray was replaced by elec-trospray during the late 1980s

During the 1990s, electrospray ionization(ESI) and atmospheric pressure chemical ioni-zation (APCI) became the standard interfacesfor LC/MS Unlike thermospray, particle beam,

or continuous-flow FAB, ESI and APCI

A.3A.3

Commonly Used Techniques

faces operate at atmospheric pressure and donot depend upon vacuum pumps to removesolvent vapor As a result, they are compatiblewith a wide range of HPLC flow rates Also, nomatrix is required Both APCI and ESI arecompatible with a wide range of HPLC col-umns and solvent systems Like all LC/MS

systems, the solvent system should containonly volatile solvents, buffers, or ion-pairagents, to reduce fouling of the mass spec-trometer ion source In general, APCI and ESIform abundant molecular ion species (FiguresA.3A.1 and A.3A.2) When fragment ions are

8150

5000

100

computer-reconstructedmass chromatogram of m/z 269

Introduction to

Mass

Spectrometry for

Food Chemistry

formed, they are usually more abundant in

APCI than ESI mass spectra

The APCI interface uses a heated nebulizer

to form a fine spray of the HPLC eluate, which

is much finer than the particle beam system but

similar to that formed during thermospray A

cross-flow of heated nitrogen gas is used to

facilitate the evaporation of solvent from the

droplets The resulting gas-phase sample

mole-cules are ionized by collisions with solvent

ions, which are formed by a corona discharge

in the atmospheric pressure chamber

Molecu-lar ions, M+ or M−., and/or protonated or

de-protonated molecules can be formed The

rela-tive abundance of each type of ion depends

upon the sample itself, the HPLC solvent, and

the ion source parameters Next, ions are drawn

into the mass spectrometer analyzer for

meas-urement through a narrow opening or skimmer,

which helps the vacuum pumps to maintain

very low pressure inside the analyzer while the

APCI source remains at atmospheric pressure

During ESI, the HPLC eluate is sprayed

through a capillary electrode at high potential

(usually 2000 to 7000 V) to form a fine mist of

charged droplets at atmospheric pressure As

the charged droplets migrate towards the

open-ing of the mass spectrometer due to electrostatic

attraction, they encounter a cross-flow of

heated nitrogen that increases solvent

evapora-tion and prevents most of the solvent molecules

from entering the mass spectrometer

Molecu-lar ions, protonated or deprotonated molecules,

and cationized species such as [M+Na]+ and

[M+K] can be formed (for additional tion on ESI, see Cole, 1997) In addition tosingly charged ions, ESI is unique as an ioni-zation technique in that multiply charged spe-cies are common and often constitute the ma-jority of the sample ion abundance The relativeabundance of each of these species dependsupon the chemistry of the analyte, the pH, thepresence of proton-donating or -accepting spe-cies, and the levels of trace amounts of sodium

informa-or potassium salts in the mobile phase In trast, APCI, MALDI, EI, CI, and FAB/LSIMSusually produce singly charged species A con-sequence of forming multiply charged ions is

con-that they are detected at lower m/z values (i.e.,

|z| >1) than the corresponding singly charged

species This has the benefit of allowing mass

spectrometers with modest m/z ranges to detect

and measure ions of molecules with very highmasses For example, ESI has been used tomeasure ions with molecular weights of hun-dreds of thousands or even millions of daltons

on mass spectrometers with m/z ranges of only

a few thousand (for a review of LC/MS niques, see Niessen, 1999)

tech-An example of the LC/MS analysis of a plantextract is shown in Figure A.3A.3 In this case,negative ion ESI-MS was used in combinationwith C18 reversed-phase HPLC separation Ex-

tracts of the botanical Trifolium pratense (red

clover) are used as dietary supplements bymenopausal and post-menopausal women (Liu

et al., 2001) The two-dimensional map trates the amount of information that may be

illus-ion source

sample compounds a and

b and impurity c

a

MS analyzer 1

m/z

M+⋅

CID

MS analyzer 2

b c

b

Figure A.3A.4 Scheme illustrating the selectivity of MS/MS and the process by which

collision-induced dissociation (CID) facilitates fragmentation of preselected ions

A.3A.5

Commonly Used Techniques

acquired using hyphenated techniques such asLC/MS In the time dimension, chromatogramsare obtained and a sample computer-recon-structed mass chromatogram is shown for the

signal at m/z 269 One intense chromatographic

peak was detected in this chromatogram eluting

at 12.4 min In the m/z dimension, the negative

ion electrospray mass spectrum recorded at

12.4 min shows a base peak at m/z 269 Based

on comparison to authentic standards (data not

shown), the ion of m/z 269 was shown to

cor-respond to the deprotonated molecule ofgenistein, which is an estrogenic isoflavone(Liu et al., 2001) Since almost no fragmenta-tion of the genistein ion was observed, addi-tional characterization would require collision-induced dissociation (CID) and tandem massspectrometry as discussed in the next section

TANDEM MASS SPECTROMETRY (MS/MS) AND HIGH RESOLUTION

Desorption ionization techniques like FABand MALDI and LC/MS ionization techniqueslike ESI and APCI facilitate the molecularweight determination of a wide range of polarand nonpolar, low- and high-molecular-weightcompounds However, the “soft” ionizationcharacter of these techniques means that most

of the ion current is concentrated in molecularions and few structurally significant fragmentions are formed In order to enhance the amount

of structural information in these mass spectra,collision-induced dissociation (CID) may beused to produce abundant fragment ions frommolecular ion precursors formed and isolatedduring the first stage of mass spectrometry

Then, a second mass spectrometry analysis may

be used to characterize the resulting productions This process is called tandem mass spec-trometry or MS/MS and is illustrated in FigureA.3A.4

Another advantage of the use of tandemmass spectrometry is the ability to isolate aparticular ion such as the molecular ion of the

analyte of interest during the first mass trometry stage This precursor ion is essentiallypurified in the gas phase and is free of impuri-ties such as solvent ions, matrix ions, or otheranalytes Finally, the selected ion is fragmentedusing CID and analyzed using a second massspectrometry stage In this manner, the result-ing tandem mass spectrum contains exclusivelyanalyte ions without impurities that might in-terfere with the interpretation of the fragmen-tation patterns In summary, CID may be usedwith LC/MS/MS or desorption ionization andMS/MS to obtain structural information such

spec-as amino acid sequences of peptides and sites

of alkylation of nucleic acids, or to distinguishstructural isomers such as β-carotene and ly-copene

The most common types of MS/MS ments available to researchers in food chemis-try include triple quadrupole mass spectrome-ters and ion traps Less common but commer-cially produced tandem mass spectrometersinclude magnetic sector instruments, Fouriertransform ion cyclotron resonance (FTICR)mass spectrometers, and quadrupole time-of-flight (QTOF) hybrid instruments (TableA.3A.1) Beginning in 2001, TOF-TOF tandemmass spectrometers became available from in-strument manufacturers These instrumentshave the potential to deliver high-resolutiontandem mass spectra with high speed andshould be compatible with the chip-based chro-matography systems now under development

instru-In addition to MS/MS with CID to obtainstructural information, it is also useful to usehigh-resolution exact mass measurements toconfirm the elemental compositions of ions.Essentially, exact mass measurements permitthe unambiguous composition analysis of low-molecular-weight compounds (mol wt <500)

through precise and accurate m/z

measure-ments The types of mass spectrometers ble of exact mass measurements include mag-netic sector mass spectrometers, QTOF hybrid

capa-Table A.3A.1 Types of Mass Spectrometers and Tandem Mass Spectrometersa

aFTICR, Fourier transform ion cyclotron resonance; QTOF, quadropole time-of-flight; TOF, time-of-flight.

Introduction to

Mass

Spectrometry for

Food Chemistry

mass spectrometers, reflectron TOF

instru-ments, and FTICR mass spectrometers (Table

A.3A.1) Some of these instruments permit the

simultaneous use of tandem mass spectrometry

and exact mass measurement of fragment ions

These include FTICR instruments, QTOF, and

the TOF-TOF

CONCLUSION

Mass spectrometry has become an essential

analytical tool for a wide variety of biomedical

applications such as food chemistry and food

analysis Mass spectrometry is highly sensitive,

fast, and selective By combining mass

spec-trometry with HPLC, GC, or an additional stage

of mass spectrometry (MS/MS), the selectivity

increases considerably As a result, mass

spec-trometry may be used for quantitative as well

as qualitative analyses In this manual, mass

spectrometry is mentioned frequently, and

ex-tensive discussions of mass spectrometry

ap-pear, for example, in units describing the

analy-ses of carotenoids (UNIT F2.4) and chlorophylls

(UNIT F4.5) In particular, these units include

examples of LC/MS and MS/MS and the use

of various ionization methods

LITERATURE CITED

Barber, M., Bordoli, R.S., Elliott, G.J., Sedgwick

R.D., and Tyler, A.N 1982 Fast atom

bombard-ment mass spectrometry Anal Chem

54:645A-657A

Cole, R.B (ed.) 1997 Electrospray Ionization

Mass Spectrometry John Wiley & Sons, New

York

Field, F 1990 Early days of chemical ionization J.

Am Soc Mass Spectrom 1:277-283.

Hillenkamp, F., Karas, M., Beavis, R.C., and Chait,

B.T 1991 Matrix-assisted laser

desorption/ioni-zation mass spectrometry of biopolymers Anal.

Chem 63:1193A-1203A.

Ito, Y., Takeuchi, T., Ishii, D., and Goto, M 1985

Direct coupling of micro high-performance uid chromatography with fast atom bombard-

liq-ment mass spectrometry J Chromatogr.

346:161-166

Liu, J., Burdette, J.E., Xu, H., Gu, C., van Breemen,R.B., Bhat, K.P.L., Booth, N., Constantinou,A.I., Pezzuto, J.M., Fong, H.H.S., Farnsworth,N.R., and Bolton, J.L 2001 Evaluation of estro-genic activity of plant extracts for the potential

treatment of menopausal symptoms J Agric.

Food Chem 49:2472-2479.

McLafferty, F.W and Turecek, F 1993 tion of Mass Spectra, 4th ed University ScienceBooks, Mill Valley, Calif

Interpreta-Niessen, W.M 1999 State-of-the-art in liquid

chro-matography-mass spectrometry J Chromatogr.

A 856:179-189.

Watson, J.T 1997 Introduction to Mass try, 3rd ed Lippincott-Raven, Philadelphia, Pa

Spectrome-KEY REFERENCES

McLafferty and Turecek, 1993 See above

This classic text describes fragmentation pathways and mechanisms for ions formed using electron impact (EI) ionization In addition, this edition con- tains additional information regarding desorption ionization and the corresponding related fragmen- tation mechanisms.

Watson, 1997 See above

This textbook provides an overview of biomedical mass spectrometry with particular emphasis on GC/MS and quantitative methods In addition, de- scriptions are provided of the various types of mass spectrometers and ionization techniques that are used for biomedical applications.

Contributed by Richard B van BreemenUniversity of Illinois at ChicagoChicago, Illinois

A.3A.7

Commonly Used Techniques

SELECTED SUPPLIERS OF REAGENTS AND EQUIPMENT

Listed below are addresses and phone numbers of commercial suppliers who have been recommended for particular items used inour manuals because: (1) the particular brand has actually been found to be of superior quality, or (2) the item is difficult to find inthe marketplace Consequently, this compilation may not include some important vendors of biological supplies For comprehensive

listings, see Linscott’s Directory of Immunological and Biological Reagents (Santa Rosa, CA), The Biotechnology Directory (Stockton Press, New York), the annual Buyers’ Guide supplement to the journal Bio/Technology, as well as various sites on the

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14

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CPFA Supplement 8 Current Protocols Selected Suppliers of Reagents and Equipment

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16

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CPFA Supplement 8 Current Protocols Selected Suppliers of Reagents and Equipment

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18

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CPFA Supplement 8 Current Protocols Selected Suppliers of Reagents and Equipment

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