ABBREVIATIONS AND USEFUL DATA

APPENDIX 1

Abbreviations Used in This Manual

APPENDIX 1A

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
AP alkaline phosphatase
APCI atmospheric pressure chemical
ionization
ASTM American Society for Testing and
Materials
ATR attenuated total reflection
AU absorbance units
AV acid value
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-diaminocyclohexaneN,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
CP-HPLC chiral-phase high-performance
liquid chromatography

CPA cis-parinaric acid
CT conjugated triene
CV coefficient of variation

cyd cyanidin
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
Copyright © 2003 by John Wiley & Sons, Inc.

Abbreviations
and Useful Data

A.1A.1


Abbreviations
Used in This
Manual


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
HEPES
N-[2-hydroxyethyl]piperazineN′-[2-ethanesulfonic acid]
HIC hydrophobic-interaction
chromatography
HMBC heteronuclear multiple bond correlation
HMDS hexamethyldisilazane
HPLC high-performance liquid chromatography
HRGC high -resolution gas chromatography
HRP horseradish peroxidase
HS headspace
HS-SPME headspace solid-phase
microextraction
HSQC heteronuclear single quantum
coherence

i.d. inner diameter
IDA isotope dilution assay
IEF isoelectric focusing
IgG immunoglobulin G
IPA indole-3-propionic acid
IRMS isotope ratio mass spectrometry
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
Applied Chemistry
IV iodine value
KM Michaelis constant
Kat Katal (catalytic unit for enzyme
activity)
LC-APCI-MS liquid chromatography/
atmospheric pressure chemical ionization
mass 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 mass
spectrometer
MCAC metal-chelate affinity
chromatography
MCC microcrystalline cellulose
MDGC multidimensional gas chromatography
2-ME 2-mercaptoethanol
Me HODES methyl hydroxyoctadecadienoates
MES 2-(N-morpholino)ethanesulfonic acid
MetMb metmyoglobin
MFP metabolic fecal protein
MHP monohydroxy pigment
MOPS 3-(N-morpholino)propane sulfonic
acid
MS mass spectrometry; mass selective
(detection)
MS/MS tandem mass spectrometry
mvd malvidin
MWCO molecular weight cutoff
m/z mass-to-charge ratio
NBS National Bureau of Standards
NIST National Institute of Standards and
Technology
NMR nuclear magnetic resonance
NO-heme nitrosylheme
NOESY nuclear Overhäuser enhancement
spectroscopy
NPLC normal-phase HPLC
NPR net protein ratio
NPU net protein utilization

OAV odor activity value
o.d. outer diameter
OSI oil stability index
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,N′-bis(2-ethanesulfonic acid)
PL pectic lyase
PMSF phenylmethylsulfonyl fluoride

A.1A.2
Supplement 10

Current Protocols in Food Acid Chemistry


p-NA paranitroanilide
pnd peonidin
psi pounds per square inch
ptd petunidin

PTFE polytetrafluoroethylene
PUFA polyunsaturated fatty acid
PV peroxide value
PVDF polyvinylidene difluoride
PVP polyvinylpyrrolidone
PVPP polyvinylpolypyrolidone
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
enhancement spectroscopy
RP-HPLC reversed-phase HPLC
RPER relative protein efficiency ratio
RS resistant starch
RT retention time
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-performance 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 Nα-p-tosyl-L-lysine chloromethyl
ketone
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-phenylalanine
chloromethyl 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

Abbreviations
and Useful Data

A.1A.3
Current Protocols in Food Acid Chemistry

Supplement 10


LABORATORY STOCK SOLUTIONS,
EQUIPMENT, AND GUIDELINES

APPENDIX 2

Common Buffers and Stock Solutions

APPENDIX 2A

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 applications 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

Acid/base
Acids
Acetic acid
(glacial)
Formic acid
Hydrochloric acid
Nitric acid
Perchloric acid
Phosphoric acid
Sulfuric acid
Bases
Ammonium

hydroxide
Potassium hydroxide
Potassium hydroxide
Sodium hydroxide

Molecular
weight

% by
weight

Molarity
(approx.)

1 M solution
(ml/liter)

Specific
gravity

60.05

99.6

17.4

57.5

1.05


46.03

98.00
98.07

90
98
36
70
60
72
85
98

23.6
25.9
11.6
15.7
9.2
12.2
14.7
18.3

42.4
38.5
85.9
63.7
108.8
82.1
67.8

54.5

1.205
1.22
1.18
1.42
1.54
1.70
1.70
1.835

35.0

28

14.8

67.6

0.90

56.11
56.11
40.0

45
50
50

11.6

13.4
19.1

82.2
74.6
52.4

1.447
1.51
1.53

36.46
63.01
100.46

aCAUTION: Handle strong acids and bases carefully.

Current Protocols in Food Analytical Chemistry (2001) A.2A.1-A.2A.7
Copyright © 2001 by John Wiley & Sons, Inc.

Laboratory Stock
Solutions,
Equipment, and
Guidelines

A.2A.1


Table A.2A.2


pKa Values and Molecular Weights for Some Common Biological Buffersa

Name

Chemical formula or IUPAC name

pKa

Useful pH Mol. wt.
range
(g/mol)

Phosphoric acid
Citric acidb
Formic acid
Succinic acid
Citric acidb
Acetic acid
Citric acidb
Succinic acid
MES
Bis-Tris

H3PO4
C6H8O7 (H3Cit)
HCOOH
C4H6O4
C6H7O7− (H2Cit−)
CH3COOH
C6H6O7− (HCit2−)

C4H5O4−
2-(N-Morpholino]ethanesulfonic acid
bis(2-Hydroxyethyl)iminotris
(hydroxymethyl)methane
N-(2-Acetamido)-2-iminodiacetic
acid
Piperazine-N,N′-bis(2-ethanesulfonic
acid)
N-(Carbamoylmethyl)-2-aminoethanesulfonic acid
1,3-Diaza-2,4-cyclopentadiene
C7H12O4
3-(N-Morpholino)propanesulfonic
acid
NaH2PO4

2.12 (pKa1)
3.06 (pKa1)
3.75
4.19 (pKa1)
4.74 (pKa2)
4.75
5.40 (pKa3)
5.57 (pKa2)
6.15
6.50

—
—
—
—

—
—
—
—
5.5-6.7
5.8-7.2

195.2
209.2

6.60

6.0-7.2

190.2

6.80

6.1-7.5

302.4

6.80

6.1-7.5

182.2

7.00
7.20

7.20

—
—
6.5-7.9

68.08
160.2
209.3

7.21 (pKa2)

—

120.0

KH2PO4

7.21 (pKa2)

N-tris(Hydroxymethyl)methyl-2aminoethanesulfonic acid
N-(2-Hydroxyethyl)piperazine-N′(2-ethanesulfonic acid)
N-(2-Hydroxyethyl)piperazine-N′(2-hydroxypropanesulfonic acid)
C2H6N2O⋅HCl
N-tris(Hydroxymethyl)methylglycine
C4H8N2O3
Tris(hydroxymethyl)aminomethane
N,N-bis(2-Hydroxyethyl)glycine
H3BO3
2-(N-Cyclohexylamino)ethanesulfonic acid

3-(Cyclohexylamino)-1-propanesulfonic acid
Na2HPO4

7.40

6.8-8.2

229.3

7.55

6.8-8.2

238.3

7.80

7.1-8.5

268.3

8.10
8.15
8.20
8.30
8.35
9.24
9.50

7.4-8.8

7.4-8.8
7.5-8.9
7.0-9.0
7.6-9.0
—
8.6-10.0

110.6
179.2
132.1
121.1
163.2
61.83
207.3

10.40

9.7-11.1

221.3

12.32 (pKa3)

—

142.0

12.32 (pKa3)

—


174.2

ADA
PIPES
ACES
Imidazole
Diethylmalonic acid
MOPS
Sodium phosphate,
monobasic
Potassium phosphate,
monobasic
TES
HEPES
HEPPSO
Glycinamide HCl
Tricine
Glycylglycine
Tris
Bicine
Boric acid
CHES
CAPS
Sodium phosphate,
dibasic
Potassium phosphate,
dibasic

K2HPO4


98.00
192.1
46.03
118.1
60.05

136.1

aSome data reproduced from Buffers: A Guide for the Preparation and Use of Buffers in Biological Systems (Mohan, 1997) with
permission of Calbiochem.
bAvailable as a variety of salts, e.g., ammonium, lithium, sodium.

A.2A.2
Current Protocols in Food Analytical Chemistry


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, concentrated 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 corresponding 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.
RECIPES
Ammonium acetate, 10 M
Dissolve 385.4 g ammonium acetate in 150 ml H2O
Add H2O to 500 ml
Sterilize by filtration
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.

HCl, 1 M
Mix in the following order:
913.8 ml H2O
86.2 ml concentrated HCl (Table A.2A.1)
KCl, 1 M

74.6 g KCl
H2O to 1 liter

Laboratory Stock
Solutions,
Equipment, and
Guidelines

A.2A.3
Current Protocols in Food Analytical Chemistry


MgCl2 , 1 M
20.3 g MgCl2⋅6H2O
H2O to 100 ml
MgCl2 is extremely hygroscopic. Do not store opened bottles for long periods of time.

MgSO4 , 1 M
24.6 g MgSO4⋅7H2O
H2O to 100 ml
NaCl, 5 M
292 g NaCl
H2O to 1 liter
NaOH, 10 M
Dissolve 400 g NaOH in 450 ml H2O
Add H2O to 1 liter
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

Common Buffers
and Stock
Solutions

Preparation of Citrate-Phosphate Buffers

Desired pH

Solution A (ml)

Solution B (ml)

2.6
2.8
3.0
3.2
3.4
3.6
3.8
4.0
4.2

4.4
4.6
4.8
5.0
5.2
5.4
5.6
5.8
6.0
6.2
6.4
6.6
6.8
7.0

44.6
42.2
39.8
37.7
35.9
33.9
32.3
30.7
29.4
27.8
26.7
25.2
24.3
23.3
22.2

21.0
19.7
17.9
16.9
15.4
13.6
9.1
6.5

5.4
7.8
10.2
12.3
14.1
16.1
17.7
19.3
20.6
22.2
23.3
24.8
25.7
26.7
27.8
29.0
30.3
32.1
33.1
34.6
36.4

40.9
43.6

aAdapted with permission from Fasman (1989).

A.2A.4
Current Protocols in Food Analytical Chemistry


Table A.2A.4 Preparation of 0.1 M Sodium
and Potassium Acetate Buffersa

Desired
pH

Solution A
(ml)

Solution B
(ml)

3.6
3.8
4.0
4.2
4.4
4.6
4.8
5.0
5.2

5.4
5.6

46.3
44.0
41.0
36.8
30.5
25.5
20.0
14.8
10.5
8.8
4.8

3.7
6.0
9.0
13.2
19.5
24.5
30.0
35.2
39.5
41.2
45.2

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)

Desired
pH

Solution A
(ml)

Solution B
(ml)

5.7
5.8
5.9
6.0
6.1
6.2
6.3
6.4
6.5
6.6
6.7

6.8

93.5
92.0
90.0
87.7
85.0
81.5
77.5
73.5
68.5
62.5
56.5
51.0

6.5
8.0
10.0
12.3
15.0
18.5
22.5
26.5
31.5
37.5
43.5
49.0

6.9
7.0

7.1
7.2
7.3
7.4
7.5
7.6
7.7
7.8
7.9
8.0

45.0
39.0
33.0
28.0
23.0
19.0
16.0
13.0
10.5
8.5
7.0
5.3

55.0
61.0
67.0
72.0
77.0
81.0

84.0
87.0
90.5
91.5
93.0
94.7

aAdapted by permission from CRC (1975).

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-dependent 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.
Laboratory Stock
Solutions,
Equipment, and
Guidelines

A.2A.5
Current Protocols in Food Analytical Chemistry


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 pKa 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).
Common Buffers
and Stock
Solutions

A.2A.6
Current Protocols in Food Analytical Chemistry


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

A.2A.7
Current Protocols in Food Analytical Chemistry


Laboratory Safety
Persons carrying out the protocols in the
laboratory may encounter various hazardous or
potentially hazardous materials including: radioactive substances; toxic chemicals and carcinogenic, 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 references therein). However, we emphasize that
users must proceed with the prudence and precautions associated with good laboratory practice, under the supervision of personnel responsible for implementing laboratory safety programs 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 Enforcement Administration. By law, Material Safety
Data Sheets must be readily available. All laboratories should have a Chemical Hygiene Plan
[29CFR Part 1910.1450] and institutional safety
officers should be consulted as to its implementation. 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, petroleum 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 refrigerators. Oxidizers should be segregated from other
chemicals, and corrosive acids (e.g., sulfuric,
hydrochloric, nitric, perchloric, and hydrofluoric acids) should also be stored in a separate
cabinet, well-removed from the flammable organics.
Facilities should be appropriate for the handling of hazardous chemicals. In particular,

APPENDIX 2B
hazardous chemicals should only be handled in
chemical fume hoods, not in laminar flow cabinets. The functioning of these fume hoods
should be periodically checked. Laboratories
should also be equipped with safety showers
and eye-washing facilities. Again, this equipment should be tested periodically to make sure

that it functions correctly. Other safety equipment may be required depending on the nature
of the materials being handled. In addition,
researchers should be trained in the proper
procedures for handling hazardous chemicals
as well as other areas of laboratory operations,
e.g., handling of compressed gases, use of cryogenic liquids, operation of high voltage power
supplies, etc.
Before starting work, have a plan for dealing
with spills or accidents; coming up with a good
plan on the spur of the moment is difficult. For
example, have the appropriate decontaminating or neutralizing agents prepared and close at
hand. Small spills can probably be cleaned up
by the researcher. In the case of larger spills,
the area should be evacuated and help sought
from those experienced and equipped for dealing with spills, e.g., your institutional safety
department.
Protective equipment should include, at a
minimum, eye protection, a lab coat, and
gloves. Sandals, open-toed shoes, and shorts
should not be worn. In certain circumstances
other items of protective equipment may be
necessary, e.g., a face shield. Different types of
gloves exhibit different chemical resistance
properties; listings of these properties are available (Forsberg and Keith, 1989). Gloves
should, however, be regarded as the last line of
defense and should be changed if they become
contaminated, because many types of chemicals pass relatively freely through rubber. If
possible, handling procedures should be designed so that gloves do not become contaminated. All common-sense precautions should
be observed, e.g., do not pipet by mouth, keep
unauthorized persons away from hazardous

chemicals, prohibit eating and drinking in the
lab, etc.
Order hazardous chemicals only in quantities that are likely to be used in a reasonable
time. Buying large quantities at a lower unit
cost is no bargain if someone (perhaps you) has
to pay to dispose of surplus quantities. Substitute alcohol-filled thermometers for mercury-

Contributed by George Lunn
Current Protocols in Food Analytical Chemistry (2001) A.2B.1-A.2B.2
Copyright © 2001 by John Wiley & Sons, Inc.

Laboratory Stock
Solutions,
Equipment, and
Guidelines

A.2B.1


filled thermometers. The latter are a hazardous
chemical spill waiting to happen.
Although any number of chemicals commonly used in laboratories are toxic if used
improperly, the toxic properties of a number of
reagents require special attention. Many chemicals are considered carcinogenic, corrosive,
flammable, lachrymatory, mutagenic, oxidizing, teratogenic, or toxic. Chemicals labeled
carcinogenic range from those accepted by expert review groups as causing cancer in humans
to those for which only minimal evidence of
carcinogenicity exists. Oxidizers may react
violently with oxidizable material, e.g., hydrocarbons, wood, and cellulose. Before using any
chemical, thoroughly investigate all of its characteristics. Material Safety Data Sheets are

readily available; they list some hazards but
vary widely in quality. A number of texts describing hazardous properties are listed in Further Reading. In particular, Sax’s Dangerous
Properties of Industrial Materials, 8th ed. (Lewis, 1992) and Bretherick’s Handbook of Reactive Chemical Hazards, 4th ed. (Bretherick,
1990) give comprehensive listings of known
hazardous properties. However, these texts list
only the known properties. Many chemicals
have been tested only partially or not at all.
Prudence dictates, therefore, that unless there
is good reason for believing otherwise, all
chemicals should be regarded as volatile,
highly toxic, flammable human carcinogens
and should be handled with care.
Waste should always be disposed of in accordance with all applicable regulations. Waste
should be segregated according to institutional
requirements, for example, into solid, aqueous,
nonchlorinated organic, and chlorinated organic material. A collection (Lunn and Sansone, 1994) of techniques for the disposal of
chemicals in laboratories has been published
recently. Incorporation of these procedures into
laboratory protocols can help to minimize
waste disposal problems.

KEY REFERENCES

LITERATURE CITED

Lunn, and Sansone, 1994. See above.

Bretherick, L. 1990. Bretherick’s Handbook of Reactive Chemical Hazards, 4th ed. Butterworths,
London.
Forsberg, K. and Keith, L.H. 1989. Chemical Protective Clothing Performance Index Book. John

Wiley & Sons, New York.

General safety
Freeman, N.T. and Whitehead, J. 1982. Introduction
to Safety in the Chemical Laboratory. Academic
Press, New York.
Furr, A.K. (ed.) 1990. CRC Handbook of Laboratory Safety, 3rd ed. CRC Press, Boca Raton, Fla.
Fuscaldo, A.A., Erlick, B.J., and Hindman, B. (eds.)
1980. Laboratory Safety, Theory and Practice.
Academic Press, New York.
Miller, B.M. (ed.) 1986. Laboratory Safety, Principles and Practices. American Society for Microbiology, Washington, D.C.
Occupational Health and Safety. 1993. National
Safety Council, Chicago.
Pal, S.B. (ed.) 1985. Handbook of Laboratory
Health and Safety Measures. Kluwer Academic
Publishers, Hingham, Mass.
Young, J.A. (ed.) 1987. Improving Safety in the
Chemical Laboratory: A Practical Guide. John
Wiley & Sons, New York.

Laboratory safety for hazardous chemicals
American Chemical Society, Committee on Chemical Safety. 1990. Safety in Academic Chemistry
Laboratories, 5th ed. American Chemical Society, Washington, D.C.
Forsberg and Keith, 1989. See above.
National Research Council, Committee on Hazardous Substances in the Laboratory. 1981. Prudent
Practices for Handling Hazardous Chemicals in
Laboratories. National Academy Press, Washington, D.C.

Properties and disposal procedures for
hazardous chemicals

Aldrich Chemical Co. 2001. Aldrich Catalog Handbook of Fine Chemicals. Aldrich Chemical Co.,
Milwaukee, Wis.
Bretherick, L. (ed.) 1986. Hazards in the Chemical
Laboratory, 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.

Contributed by George Lunn
Baltimore, Maryland

Lewis, R.J., Sr. 1992. Sax’s Dangerous Properties of
Industrial Materials, 8th ed. Van Nostrand-Reinhold, New York.

Laboratory Safety

Lunn, G. and Sansone, E.B. 1994. Destruction of
Hazardous Chemicals in the Laboratory, 2nd ed.
John Wiley & Sons, New York.

A.2B.2
Current Protocols in Food Analytical Chemistry


Standard Laboratory Equipment

APPENDIX 2C


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
equipment.
Applicators, cotton-tipped and wooden
Autoclave
Balances, analytical and preparative
Beakers
Biohazard disposal containers and bags
Blender (e.g., Waring Blendor)
Bottles, glass and plastic
Bunsen burners
Centrifuges, low-speed (6,000 rpm) and highspeed (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
membranes
Flasks, glass (e.g., Erlenmeyer, beveled
shaker)
Forceps
Freezers, −20° and −80°C
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.

Laboratory Stock
Solutions,
Equipment, and
Guidelines

A.2C.1
Supplement 2


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

A.2C.2
Supplement 2

Current Protocols in Food Analytical Chemistry



COMMONLY USED TECHNIQUES

APPENDIX 3

Introduction to Mass Spectrometry for Food
Chemistry

APPENDIX 3A

Almost a century ago, the first mass spectrometers 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 characterize 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. Beginning in 1945, organic mass spectrometers using
electron impact (EI) ionization became commercially 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 fundamental analytical tool for the characterization of
synthetic organic compounds.
During the 1960s, high-resolution, doublefocusing magnetic sector instruments became
available from multiple manufacturers and
were widely used in organic chemistry for exact

mass measurements and elemental composition analysis. EI was used for generating struc-

turally significant fragment ions for compound
identification, and rules for structure elucidation using mass spectrometry were developed
(for a thorough review of EI and ion fragmentation pathways, see McLafferty and Turecek,
1993). Biomedical and food chemistry applications of mass spectrometry were developed
during this time. Chemical ionization (CI),
which was developed by researchers in the
petroleum industry (Field, 1990), was quickly
adopted as a softer ionization alternative to EI,
useful in reducing fragmentation so that molecular weights could be confirmed more easily.
CI became another standard ionization technique for mass spectrometry (see Figure
A.3A.1 for a guide to the selection of ionization
techniques in mass spectrometry).

GAS CHROMATOGRAPHY/MASS
SPECTROMETRY (GC/MS)
With the introduction of computerized data
systems for data acquisition, reduction, and
storage during the 1960s, the efficiency of mass
spectrometric analysis grew rapidly and continues to grow to this day. The use of computers
for data reduction and analysis helped gas chromatography/mass spectrometry (GC/MS) become a practical and powerful tool for qualita-

sample

mol. wt. < 1000

volatile

El, Cl,

APCI

mol. wt. > 1000

nonvolatile

APCI

mol. wt. < 5000

FAB, MALDI,
electrospray

mol. wt. > 5000

MALDI,
electrospray

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.
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.

Commonly Used
Techniques

A.3A.1

Supplement 2


tive and quantitative analysis of compounds in
mixtures. Both EI and CI were immediately
useful for GC/MS, since both of these ionization methods require that the analytes be in the
gas phase. When capillary GC was incorporated into GC/MS, this technique reached maturity. The advantages of GC/MS include
speed, selectivity, and sensitivity. Typically,
GC/MS may be used to select, identify, and
quantify organic compounds in complex mixtures at the femtomole level. Compounds are
selected using a combination of chromatographic separation and mass selection, and
when using tandem mass spectrometry
(MS/MS; see discussion below), the fragmentation pathway may be used for additional selectivity. The speed of GC/MS is determined
by the chromatography step, which typically
requires from several minutes to one hour per
analysis. Although GC/MS remains important
for the analysis of many organic compounds,
this technique is limited to volatile and thermally stable compounds (see chromatography/MS selection flow chart in Fig. A.3A.2).
Therefore, thermally unstable compounds—
including food pigments such as carotenoids
and chlorophylls and biomolecules such as proteins, carbohydrates, and nucleic acids—cannot be analyzed in their native forms using
GC/MS (for more details regarding GC/MS
and its applications, see Watson, 1997).

DESORPTION IONIZATION MASS
SPECTROMETRY
During the 1970s and early 1980s, desorption ionization techniques such as field desorption (FD), desorption EI, desorption CI (DCI),
and laser desorption were developed to extend
the utility of mass spectrometry towards the
analysis of more polar and less volatile compounds (see Watson, 1997, for more information regarding desorption ionization techniques

including DCI and FD). Although these techniques helped extend the mass range of mass
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 bombardment (FAB; Barber et al., 1982). FAB and its
counterpart, liquid secondary ion mass spectrometry (LSIMS), facilitate the formation of
abundant molecular ions, protonated molecules, and deprotonated molecules of nonvolatile and thermally labile compounds such as
peptides, chlorophylls, and complex lipids up
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) to
ionize compounds dissolved in nonvolatile matrices such as glycerol or 3-nitrobenzyl alcohol
and desorb them from this condensed phase
into the gas phase for mass spectrometric analysis. Molecular ions and/or protonated molecules are usually abundant and fragmentation
is minimal.

sample

mol. wt. < 1000

Introduction to
Mass
Spectrometry for
Food Chemistry

mol. wt. > 1000

GC/MS


LC/MS

El, Cl

APCI,
electrospray,
particle beam,
CF-FAB

MW > 5000

electrospray

LC/MS

CF-FAB,
electrospray

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 chromatography/mass spectrometry; LC/MS, liquid chromatography/mass spectrometry.

A.3A.2
Supplement 2

Current Protocols in Food Analytical 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 commercial 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 molecules 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. Producing gas-phase sample ions for analysis in a
vacuum system while removing the HPLC mobile phase proved to be a challenging task. Early
LC/MS techniques included a moving belt interface 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 columns 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 intervention, it is not ideal for the analysis of large
sample sets. Instead, more robust techniques
have been developed to fulfill this requirement.
However, continuous-flow FAB is still in use
in some laboratories.
Like continuous-flow FAB, the popularity
of particle beam interfaces is diminishing, but
systems are still available from commercial
sources. During particle beam LC/MS, the
HPLC eluate is sprayed into a heated chamber

connected to a vacuum pump. As the droplets
evaporate, aggregates of analyte (particles)
form and pass through a momentum separator
that removes the lower-molecular-weight solvent molecules. Finally, the particle beam enters the mass spectrometer ion source where the
aggregates strike a heated plate from which the
analyte molecules evaporate and are ionized
using conventional EI or CI ionization. Particle
beam LC/MS is limited to the analysis of volatile and thermally stable compounds that are
amenable to flash evaporation and EI or CI
mass spectrometry. Therefore, this approach is
not used for polar compounds in food chemistry
such as carbohydrates, sugars, peptides, proteins, or nucleic acids (Fig. A.3A.2).
Since thermospray became the first widely
utilized LC/MS technique (during the late
1970s and early 1980s), this technique should
be mentioned here. Thermospray facilitates the
interfacing of standard analytical HPLC systems at flow rates up to 1 ml/min with mass
spectrometers. Although the interface between
the HPLC and mass spectrometer is inefficient
and exhibits low sensitivity for most analytes,
thermospray has been useful for the LC/MS
analysis of many types of small molecules.
During thermospray, the HPLC eluate is
sprayed through a heated capillary into a heated
desolvation chamber at reduced pressure. Gas
phase ions remaining after desolvation of the
droplets are extracted through a skimmer into
the mass spectrometer for analysis. The sensitivity of thermospray is poor since there is no
mechanism or driving force to enhance the
number of sample ions entering the gas phase

from the spray during desolvation. Also, thermally labile compounds tend to decompose in
the heated source. These problems were solved
when thermospray was replaced by electrospray during the late 1980s.
During the 1990s, electrospray ionization
(ESI) and atmospheric pressure chemical ionization (APCI) became the standard interfaces
for LC/MS. Unlike thermospray, particle beam,
or continuous-flow FAB, ESI and APCI inter-

Commonly Used
Techniques

A.3A.3
Current Protocols in Food Analytical Chemistry

Supplement 2


faces operate at atmospheric pressure and do
not depend upon vacuum pumps to remove
solvent vapor. As a result, they are compatible
with a wide range of HPLC flow rates. Also, no
matrix is required. Both APCI and ESI are
compatible with a wide range of HPLC columns and solvent systems. Like all LC/MS

systems, the solvent system should contain
only volatile solvents, buffers, or ion-pair
agents, to reduce fouling of the mass spectrometer ion source. In general, APCI and ESI
form abundant molecular ion species (Figures
A.3A.1 and A.3A.2). When fragment ions are


100

computer-reconstructed
mass chromatogram of m /z 269
%

0
500

m/z

150

Relative abundance

8

12

100
mass spectrum
at 12.4 min

24

28

269 [M−H]−

%

189
0
100

Introduction to
Mass
Spectrometry for
Food Chemistry

20
16
Retention time (min)

140

180

220
m /z

260

300

340

Figure A.3A.3 LC/MS analysis of a dietary supplement consisting of extract of Trifolium pratense
(red clover). Reversed-phase C18 HPLC and negative ion electrospray ionization mass spectrometry were used with a quadrupole mass spectrometer analyzer (Agilent; also see Table A.3A.1). The
map illustrates the abundance of information provided by this hyphenated technique with HPLC
mass chromatograms in one dimension and mass spectra in another dimension.


A.3A.4
Supplement 2

Current Protocols in Food Analytical 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 molecules are ionized by collisions with solvent
ions, which are formed by a corona discharge
in the atmospheric pressure chamber. Molecular ions, M+. or M−., and/or protonated or deprotonated molecules can be formed. The relative 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 measurement 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 opening of the mass spectrometer due to electrostatic
attraction, they encounter a cross-flow of

heated nitrogen that increases solvent evaporation and prevents most of the solvent molecules
from entering the mass spectrometer. Molecular ions, protonated or deprotonated molecules,
and cationized species such as [M+Na]+ and

[M+K]+ can be formed (for additional information on ESI, see Cole, 1997). In addition to
singly charged ions, ESI is unique as an ionization technique in that multiply charged species are common and often constitute the majority of the sample ion abundance. The relative
abundance of each of these species depends
upon the chemistry of the analyte, the pH, the
presence of proton-donating or -accepting species, and the levels of trace amounts of sodium
or potassium salts in the mobile phase. In contrast, APCI, MALDI, EI, CI, and FAB/LSIMS
usually produce singly charged species. A consequence of forming multiply charged ions is
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 high
masses. For example, ESI has been used to
measure ions with molecular weights of hundreds 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 techniques, see Niessen, 1999).
An example of the LC/MS analysis of a plant
extract is shown in Figure A.3A.3. In this case,
negative ion ESI-MS was used in combination
with C18 reversed-phase HPLC separation. Extracts of the botanical Trifolium pratense (red
clover) are used as dietary supplements by
menopausal and post-menopausal women (Liu
et al., 2001). The two-dimensional map illustrates the amount of information that may be

a
ion

source

b
c

sample
compounds a and
b and impurity c

MS
analyzer 1

b

CID

MS
analyzer 2

M+⋅

m/z

Figure A.3A.4 Scheme illustrating the selectivity of MS/MS and the process by which collisioninduced dissociation (CID) facilitates fragmentation of preselected ions.

Commonly Used
Techniques

A.3A.5
Current Protocols in Food Analytical Chemistry


Supplement 2


acquired using hyphenated techniques such as
LC/MS. In the time dimension, chromatograms
are obtained and a sample computer-reconstructed 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 correspond to the deprotonated molecule of
genistein, which is an estrogenic isoflavone
(Liu et al., 2001). Since almost no fragmentation of the genistein ion was observed, additional characterization would require collisioninduced dissociation (CID) and tandem mass
spectrometry as discussed in the next section.

TANDEM MASS SPECTROMETRY
(MS/MS) AND HIGH RESOLUTION
Desorption ionization techniques like FAB
and MALDI and LC/MS ionization techniques
like ESI and APCI facilitate the molecular
weight determination of a wide range of polar
and nonpolar, low- and high-molecular-weight
compounds. However, the “soft” ionization
character of these techniques means that most
of the ion current is concentrated in molecular
ions and few structurally significant fragment
ions are formed. In order to enhance the amount

of structural information in these mass spectra,
collision-induced dissociation (CID) may be
used to produce abundant fragment ions from
molecular ion precursors formed and isolated
during the first stage of mass spectrometry.
Then, a second mass spectrometry analysis may
be used to characterize the resulting product
ions. This process is called tandem mass spectrometry or MS/MS and is illustrated in Figure
A.3A.4.
Another advantage of the use of tandem
mass spectrometry is the ability to isolate a
particular ion such as the molecular ion of the
Table A.3A.1

Introduction to
Mass
Spectrometry for
Food Chemistry

analyte of interest during the first mass spectrometry stage. This precursor ion is essentially
purified in the gas phase and is free of impurities such as solvent ions, matrix ions, or other
analytes. Finally, the selected ion is fragmented
using CID and analyzed using a second mass
spectrometry stage. In this manner, the resulting tandem mass spectrum contains exclusively
analyte ions without impurities that might interfere with the interpretation of the fragmentation patterns. In summary, CID may be used
with LC/MS/MS or desorption ionization and
MS/MS to obtain structural information such
as amino acid sequences of peptides and sites
of alkylation of nucleic acids, or to distinguish
structural isomers such as β-carotene and lycopene.

The most common types of MS/MS instruments available to researchers in food chemistry include triple quadrupole mass spectrometers and ion traps. Less common but commercially produced tandem mass spectrometers
include magnetic sector instruments, Fourier
transform ion cyclotron resonance (FTICR)
mass spectrometers, and quadrupole time-offlight (QTOF) hybrid instruments (Table
A.3A.1). Beginning in 2001, TOF-TOF tandem
mass spectrometers became available from instrument manufacturers. These instruments
have the potential to deliver high-resolution
tandem mass spectra with high speed and
should be compatible with the chip-based chromatography systems now under development.
In addition to MS/MS with CID to obtain
structural information, it is also useful to use
high-resolution exact mass measurements to
confirm the elemental compositions of ions.
Essentially, exact mass measurements permit
the unambiguous composition analysis of lowmolecular-weight compounds (mol. wt. <500)
through precise and accurate m/z measurements. The types of mass spectrometers capable of exact mass measurements include magnetic sector mass spectrometers, QTOF hybrid

Types of Mass Spectrometers and Tandem Mass Spectrometersa

Instrument

Resolution

m/z Range

Tandem MS

Magnetic sector
Quadrupole
Triple quadrupole

TOF
FTICR
QTOF
TOF-TOF

100,000
<4,000
<4,000
15,000
>200,000
12,000
15,000

12,000
4,000
4,000
>200,000
<10,000
4,000
>10,000

Low resolution
None
Low resolution
None
High resolution
High resolution
High resolution

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


A.3A.6
Supplement 2

Current Protocols in Food Analytical Chemistry


mass spectrometers, reflectron TOF instruments, 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 spectrometry with HPLC, GC, or an additional stage
of mass spectrometry (MS/MS), the selectivity
increases considerably. As a result, mass spectrometry may be used for quantitative as well
as qualitative analyses. In this manual, mass
spectrometry is mentioned frequently, and extensive discussions of mass spectrometry appear, for example, in units describing the analyses 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 bombardment mass spectrometry. Anal. Chem. 54:645A657A.
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/ionization 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 liquid chromatography with fast atom bombardment 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 estrogenic 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. Interpretation of Mass Spectra, 4th ed. University Science
Books, Mill Valley, Calif.
Niessen, W.M. 1999. State-of-the-art in liquid chromatography-mass spectrometry. J. Chromatogr.
A 856:179-189.
Watson, J.T. 1997. Introduction to Mass Spectrometry, 3rd ed. Lippincott-Raven, Philadelphia, Pa.

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 contains additional information regarding desorption

ionization and the corresponding related fragmentation 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, descriptions are provided of the various types of mass
spectrometers and ionization techniques that are
used for biomedical applications.

Contributed by Richard B. van Breemen
University of Illinois at Chicago
Chicago, Illinois

Commonly Used
Techniques

A.3A.7
Current Protocols in Food Analytical Chemistry

Supplement 2


SELECTED SUPPLIERS OF REAGENTS AND EQUIPMENT
Listed below are addresses and phone numbers of commercial suppliers who have been recommended for particular items used in
our manuals because: (1) the particular brand has actually been found to be of superior quality, or (2) the item is difficult to find in
the 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
Internet.
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1
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Suppliers

2
CPFA Supplement 8

Current Protocols Selected Suppliers of Reagents and Equipment


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Suppliers

3
Current Protocols Selected Suppliers of Reagents and Equipment

CPFA Supplement 8


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Suppliers

4
CPFA Supplement 8

Current Protocols Selected Suppliers of Reagents and Equipment