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Issue 5 • 2011
AnalytixFood Color and Other Dye Standards
• Food Dye Analysis
• New UHPLC Solvents
• Listeria Testing
• Kjeldahl Determination
• Water in Polymers
• Reference Materials for
Herbal Medicine
2E
dit
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al
Why do we need Color Standards?
Dear Colleague,
Each of us is infl uenced by colors, at all stages
of life. There are artifi cial colors in our food, our
clothes, the cosmetics we use, the toys we buy.
While in former centuries the coloring of food
and consumer goods had been done with
natural dyes, the invention of the fi rst synthetic
organic dye, mauveine, by William Henry
Perkins in 1856, opened the door to a huge
market for synthetic dyes. Since then approx.
15,000 colorants have been produced on an
industrial scale, quickly replacing traditional
natural dyes.
With the increasing amount of this large group
of organic chemicals practically everywhere in
our daily life, the potential risk of damaging the
environment – and ourselves – increases, too.
In order to minimize such possible damage,
national and international associations have
been founded, throughout the textile manu-
facturing process, e.g. the Ecological and
Toxicological Association of the Dyestuf f
Manufacturing Industry (ETAD), founded in
1974, which coordinates the ecological and toxi-
cological efforts of organic-colorant manu-
facturers. For textile products, a variety of
ecolabeling schemes imposing environmental
requirements on a voluntary basis have been
created e.g. the Oeko-Tex® Standard label,
comprising various tests for harmful sub-
stances in textiles, based on the latest scientifi c
fi ndings, and Milieukeur and Eko of the Nether-
lands. On the legislation side, the new Euro-
pean Union (EU) regulation EC 1907/2006
(REACH regulation) deals with carcinogenic
and sensibilizing compounds in textiles.
Evaluation of food additives for safety in use has been con-
tinuing for over 50 years, both at the international level and
by national regulatory and advisory bodies. The earliest
regulation was the Pure Food and Drug Act of 1906, a
United States federal law. In most countries, food additives
cannot be marketed unless they have undergone a full
safety evaluation.
Our R&D and Product Management Team works hard to
keep pace with changing legislation in diff erent countries
around the world, so as to supply you with the latest, highly
purifi ed, well-characterized standards.
Thank you for trusting us to provide the high-quality ana-
lytical products you require. We look forward to serving you
in the future.
With kind regards,
Ingrid Hayenga
Senior Scientist, R&D
Ingrid Hayenga
Senior Scientist, R&D
Analytix is published fi ve times per year by Sigma-Aldrich Chemie GmbH,
MarCom Europe, Industriestrasse 25, CH-9471 Buchs SG, Switzerland
Publisher: Sigma-Aldrich Marketing Communications Europe
Publication director: Ingo Haag, PhD
Editor: Daniel Vogler
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33
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Featured Article
4 Color Standards
Microbiology
8 Listeria monocytogenes – A Frugal Pathogen
Elemental Analysis
12 Nitrogen Determination According to the Kjeldahl Method
Chromatography
15 Exclusively designed for UHPLC: LC-MS Ultra CHROMASOLV®
Standards
16 New Analytical Standards for the Analysis of Herbal Medicinal Products
Food Analysis
17 Enzymatic Food Analysis
Titration
21 Water Determination in Polymers and Plastics
Karl Fischer Titration with HYDRANAL® Reagents
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Color Standards
Introduction
Food colors
Food colors are food additives which are used primarily for
the following reasons:
• To make up for color loss following exposure
to light, air, moisture or variations in temperature.
• To enhance naturally occurring colors.
• To add colors to food that would otherwise be
colorless or colored differently.
Food colors are identifi ed by their name and a code number,
the numbering being based on the internationally agreed
system adopted by the Codex Alimentarius Commission
(CAC, updated 2007).
Textile dyes
The textile industry is characterized by a form of cooperation
in which each stage of production from the raw material to
the fi nished textile product is often located in a diff erent
place in the world. This extremely fragmented structure is
refl ected in the complex supply relationships between all the
companies involved throughout the textile processing chain.
On top of this, diff erent environmental regulations apply in
the individual countries involved in textile production.
At the beginning of the 1990s, the Oeko-Tex® Standard 100,
developed by the Austrian Textile-Research Institute (ÖTI)
and the German Research Institute Hohenstein, was intro-
duced as a response to the need of the general public for
textiles which did not pose a health risk. “Poison in textiles”
and other negative headlines were widespread and indis-
criminately branded all chemicals used in textile manufac-
turing as negative and dangerous to health. This is where
the basic concept behind the Oeko-Tex Standard 100
comes into play: the aim is to iron out the global diff erences
relating to the assessment of harmful substances. Currently,
the International Oeko-Tex Association consists of a group
of 15 well-known research institutes in Europe and Japan,
with representative agencies and contact offices in over
50 countries worldwide.
Regulation within EU, USA and around the world
Food colors
Within the European Union (EU), each food color authorized
for use is subject to a rigorous scientifi c safety assessment.
In late 1988, the European Community adopted a frame-
work directive which laid out the criteria by which food
additives, including food colors, would be assessed. The
framework directive provided for the adoption of more
specifi c technical directives establishing the list of additives
which could be used, the foods in which they could be
used and any maximum levels (Council Directive of
21 December 1988, 89/107/EEC). In 2008, a new framework
was agreed to with the adoption of Regulation 1333/2008,
enacted on January 20, 2010.
Under Commission Regulation 1331/2008, all food additives
must undergo a safety evaluation by the European Food and
Safety Authority (EFSA) prior to their authorization by EU risk
managers. According to Commission Regulation 1333/2008,
all food additives authorized for use in the EU before January
2009 should be subject to a new risk assessment by EFSA.
Commission Regulation 257/2010 establishes a program for
the re-evaluation of approved food additives, foremost being
food colors, because these were among the fi rst additives to
be assessed by the former Scientifi c Committee on Food.
In some European countries, even some of the approved
European food colors are either banned or subject to a vol-
untary phase out, e.g. the so-called Southampton Six in the
UK (Tartrazin, Quinoline Yellow, Sunset Yellow FCF, Car-
moisine, Ponceau 4R, Allura Red AC, and Brilliant Blue FCF).
The UK Food Standard Agency recommendation follows a
Southampton study which looked into the eff ect of these
colors on children’s behavior. The colors are listed in
Table 1. Food containing these colors must carry the warn-
ing: “May have an adverse eff ect on activity and attention in
children.” In Switzerland, 17 colors are permitted by the
legislation as food additives.
Ingrid Hayenga, Senior Scientist, R&D [email protected]
Matthias Nold, Product Manager Analytical Standards [email protected]
5
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Within the US, the FD&C Act (United States Federal Food,
Drug and Cosmetic Act) is a set of laws passed by Congress
in 1938. This legislation gave authority to the U.S. Food and
Drug Administration (FDA) to oversee the safety of food,
drugs and cosmetics. This act also makes the certifi cation of
some food color additives mandatory. The FDA lists nine
FD&C food colors; two of them are restricted to specific
uses. There are numerous D&C (Drug & Cosmetics) color-
ings allowed only in drugs for external application or in cos-
metics. Color additives derived from natural sources, such
as vegetables, minerals or animals, and man-made counter-
parts of natural derivatives are exempt from certifi cation. As
in Europe, both artificial and natural color additives are
subject to rigorous standards of safety before their approval
for use in foods.
In Japan, the Japan Food Chemical Research Foundation
was established to contribute to the safety of food. Twelve
diff erent colors are allowed as food additives.
The standards in the Australia New Zealand Food Standards
Code (FSANZ) are legislative instruments under the Legisla-
tive Instruments Act 2003. Chapter Standard 1.3.1 – Food
additives, Amendment No. 121-2011, regulates the use of
food additives, such as colors, in the production and pro-
cessing of food. There are 12 synthetic colors permitted
singularly or in combination to a total maximum level of
290 mg/kg in processed food and to a total maximum level
of 70 mg/kg in beverages.
Besides these approved dyes, there are banned dyes that are
still being used illicitly as additives in food products, e.g. the
Sudan dyes, red dyes normally used for coloring solvents,
oils, waxes, petrol, and polishes for shoes and fl ooring.
Table 1 lists the artifi cial food colors permitted in a number
of countries around the world. Table 2 lists the banned col-
ors that are still being used illicitly as food additives.
Textiles and leather dyes
In the coloring process of textile and leather, azo dyes are
often used. Some of these dyes have the capacity to release
certain aromatic amines, which pose cancer risks others are
sensibilizing. For this reason, the EU has laid down legislation
to prevent exposure to these 22 aromatic amines, listed in
Table 3. Allergenic disperse dyes and carcinogenic dyes are
also banned in textile materials from synthetic fi bers with
skin contact, listed in Table 4. The prohibition on the use of
these dyes is laid down in Annex XVII to the EU Regulation
(EC) 1907/2006 (REACH regulation).
Besides food and textile/leather dyes, Sigma-Aldrich also
off ers high-purity dyes for various applications, see Table 5.
We are constantly expanding our portfolio of dyes with
interes t ing produc ts . Please v is i t our website at
sigma-aldrich.com/fooddyes for an up-to-date product
listing.
(continued on page 6)
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E No Cat. No. Description Quality Package
Size
EU UK US Canada Australia/
New Zealand
Japan
102 03322 Tartrazin ≥ 99.0% 25 mg approved banned FD&C Yellow No 5 FD&C Yellow # 5 permitted Food Yellow No 4
104 Quinoline Yellow WS approved banned permitted
105 93883 Fast Yellow AB ≥ 97.0% 25 mg banned
107 00298 Yellow 2G ≥ 98.0% 25 mg approved
110 68775 Sunset Yellow FCF ≥ 95.0% 25 mg approved banned FD&C Yellow No 6 FD&C Yellow # 6 permitted Food Yellow No 5
111 73668 Orange GGN ≥ 97.0% 25 mg approved
120 11298 Cochineal (Natural Red 4) ≥ 96.0% 25 mg approved
121 89774 Citrus Red 2 ≥ 98.5% 25 mg banned only to color the
skin of oranges
122 52245 Carmoisine ≥ 98.0% 25 mg approved banned permitted
123 87612 Amaranth ≥ 98.0% 25 mg approved FD&C Red No 2 FD&C Red # 2 Food Red No 2
124 18137 Ponceau 4R ≥ 99.0% 25 mg approved banned permitted Food Red No 102
125 92395 Ponceau SX ≥ 98.0% 25 mg approved FD&C Red # 4
126 96365 Ponceau 6R ≥ 99.0% 25 mg approved
127 87613 Erythrosine ≥ 99.0% 25 mg approved FD&C Red No 3 FD&C Red # 3 Food Red No 3
128 40462 Red 2G ≥ 98.0% 25 mg banned
129 38213 Allura Red AC ≥ 98.0% 25 mg approved banned FD&C Red No 40 FD&C Red # 40 permitted Food Red No 40
130 Indanthrene blue RS approved
131 74748 Patent Blue V ≥ 97.0% 25 mg approved
132 73436 Indigo carmine ≥ 99.0% 25 mg approved FD&C Blue No 2 FD&C Blue # 2 permitted Food Blue No 2
133 30659 Brilliant Blue FCF ≥ 96.0% 25 mg approved banned FD&C Blue No 1 FD&C Blue # 1 permitted Food Blue No 1
142 06737 Green S ≥ 98.0% 25 mg approved permitted
143 68724 Fast Green FCF ≥ 97.0% 25 mg approved FD&C Green No 3 FD&C Green # 3 permitted Food Green No 3
151 11220 Brilliant Black BN ≥ 96.0% 25 mg approved permitted
152 56637 Food Black 2 (Black 7984) ≥ 98.0% 25 mg approved
154 Brown FK approved
155 Brown HT approved permitted
180 90689 Lithol Rubine BK ≥ 97.0% 25 mg approved
Orange B only in hot dog and
sausage casings
P2759 Phloxine (CI 45410) 25 g Food Red No 104
330000 Rose Bengale (CI 45440) 95% 1 g Food Red No 105
230162 Acid Red 52 (CI 45100) 75% 5 g Food Red No 106
Table 1 Artificial food colors and corresponding analytical standards
Cat. No. Description Purity Package Size
11298 Carminic Acid ≥ 96.0% 25 mg
89774 Citrus Red 2 ≥ 98.5% 25 mg
93883 Fast Yellow AB ≥ 97.0% 25 mg
68775 Sunset Yellow ≥ 95.0% 25 mg
59659 Toluidine Red ≥ 98.0% 25 mg
40462 Red 2G ≥ 98.0% 25 mg
51383 Sudan I ≥ 96.0% 25 mg
7937 Sudan II ≥ 96.0% 25 mg
68562 Sudan III ≥ 96.0% 25 mg
67386 Sudan IV ≥ 96.0% 25 mg
43207 Sudan Orange G ≥ 96.0% 25 mg
53373 Sudan Red 7B ≥ 96.0% 25 mg
91282 Sudan Red G ≥ 96.0% 25 mg
73225 Butter Yellow ≥ 98.0% 25 mg
44426 Metanil Yellow ≥ 98.0% 100 mg
40446 Para Red ≥ 97.0% 100 mg
49904 Ponceau 3R ≥ 98.0% 25 mg
34184 d5-Sudan I enrichment > 98 atom % D 10 mg
34161 d6-Sudan IV enrichment > 98 atom % D 10 mg
34163 d6-Sudan IV Solution 100 μg/ml in acetonitrile 2 ml
34181 d5-Sudan I Solution 101 μg/ml in acetonitrile 2 ml
Table 2 Banned food colors, non-deuterated and deuterated
Cat. No. Description Package Size
31598 4-Aminodiphenyl 250 mg
31614 Benzidin 100 mg
46282 4-Chloro-2-methylaniline 250 mg
31618 2-Naphtylamine 100 mg
31629 o-Aminoazotoluene 250 mg
45984 2-Methyl-5-nitroaniline 250 mg
35823 4-Chloroaniline 1 g
32831 2,4-Diaminoanisole 100 mg
31640 4,4’-Diaminodiphenylmethane 250 mg
48525 3,3’-Dichlorobenzidine Ampule of 100 mg
33430 3,3’-Dimethoxybenzidine 10 g, 50 g
31659 o-Tolidine 100 mg
46106 4,4’-Methylene-bis-(2-methylaniline) 100 mg
46111 2-Methoxy-5-methylaniline 250 mg
31673 4,4’-Methylene-bis-(2-chloroaniline) 100 mg
46117 4,4’-Oxydianiline 250 mg
46126 4,4’-Diaminodiphenyl sulfide 250 mg
45979 o-Toluidine 250 mg
45922 2,4-Diaminotoluene 250 mg
31039 2,3,5-Trimethylaniline solution Ampule of 2 ml, contains 100 ng/μl
46130 4-Aminoazobenzene 250 mg
31597 o-Anisidine 250 mg
Table 3 Banned aromatic amines
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Cat. No. Description C.I. number Purity Package Size
Basic Red 9 42500
22308 Acid Red 26/Ponceau 2R/Ponceau Xylidine 16150 ≥ 96.0% 25 mg
46364 Basic Violet 14 (Crystal Violet) 42510 250 mg
C1144 Direct Black 38 (Chlorazol Black) 30235 Dye content > 45% 5 g, 25 g
S476269 Direct Blue 6 22610 1 ea (1 g)
75768 Direct Red 28 22120 ≥ 97.0% 25 mg
215643 Disperse Blue 1 64500 Dye content 30% 5 g
215651 Disperse Blue 3 61505 Dye content 20% 50 g
Disperse Blue 7 62500
Disperse Blue 26 64305
17992 Disperse Blue 35 5 g
Disperse Blue 102
28241 Disperse Blue 106 5 g, 25 g
21620 Disperse Blue 124 5 g
S468061 Disperse Brown 1 1 ea (5 g)
29173 Disperse Orange 1 11080 ≥ 96.0% 25 mg
53882 Disperse Orange 3 11005 ≥ 96.0% 25 mg
42994 Disperse Orange 11 60700 ≥ 96.0% 25 mg
50323 Disperse Orange 37 ≥ 96.0% 25 mg
Disperse Orange 76
11074 Disperse Red 1 11110 ≥ 96.0% 25 mg
S944556 Disperse Red 11 62015 1 ea (50 mg)
Disperse Red 17 11210
S462934 Disperse Yellow 1 10345 1 ea (250 mg)
11344 Disperse Yellow 3 11855 ≥ 96.0% 25 mg
38464 Disperse Yellow 9 10375 ≥ 96.0% 25 mg
Disperse Yellow 39
Disperse Yellow 49
Table 4 Banned azo dyes
Cat. No. Description Purity Package Size Application
49823 Acid Red 73 ≥ 97.0% 25 mg Hair coloring
51362 Auramine ≥ 97.0% 25 mg Fluorescent stain, paints and rubber products
75768 Congo Red ≥ 97.0% 25 mg Microscopy/banned for use in paper industry
69669 Leucomalachite green ≥ 98.0% 25 mg Used as a detector for latent blood in forensic science
38800 Malachite green chloride ≥ 96.0% 25 mg Dye for paper, silk, leather; banned for the use in aquaculture
49547 Naphtol Yellow S ≥ 99.0% 25 mg Stain for collagen
69143 Orange II sodium salt ≥ 98.0% 25 mg Wool and cotton dye; microscopy
79285 Oil Orange SS ≥ 98.0% 25 mg Microscopy
79754 Rhodamine B ≥ 97.0% 25 mg Tracer dye within water
49639 Solvent Yellow 124 ≥ 98.0% 50 mg Used in the European Union as a fuel dye marker
Table 5 Dyes for various other applications
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only a mild, fl u-like illness, but the infection can lead to a
fatal outcome through miscarriage or stillbirth, or can cause
premature delivery or infection of the newborn. Extensive
research has been done in recent years concerning the
mechanism by which this pathogen invades its host. It was
found that the L. monocytogenes penetrates the mucosa
directly via enterocytes, or indirectly via invasion of Peyer’s
patches (1). L. monocytogenes replicates rapidly in the
cytosol of host cells like macrophages and lymphocytes
(see Figure 1) (2).
ETH Zürich researchers have generated a stable non-revert-
ing cell wall defi cient
life form of L. mono-cytogenes. The bacte-
ria are in a so-called
L-form and are sur-
p r i s i n g l y a b l e t o
reproduce and pro-
life rate. The cell wall
defi cient cells are sur-
rounded by a single
membrane only; they
become spher ica l
and enlarge greatly
(see Figure 2). Com-
pared to the usual
L . m o n o c y t o g e n e s
form, the gene expression is quite diff erent and the meta-
bolic functions are down-regulated. These cells can not be
detected with classical plating media (3).
The nature of Listeria monocytogenesL. monocytogenes is a Gram-positive, non spore forming, rod-
shaped flagellate but it is not able to produce flagella at
37 °C. In order to move into a host’s body, the pathogen
needs a polymerization with host cell actin at one end of
the bacterium (see Figure 1). It is a ubiquitous organism; it
exists in plants, soil and the guts of birds, fi sh, shellfi sh and
some mammals, including humans. Some studies suggest
that 1–10% of humans may be intestinal carriers of L. mono-cytogenes. Special risk materials are raw or processed meat,
raw milk products, raw or smoked fish, ready-prepared
salad and long-term vacuum packed food. Listeria species
are killed by proper heating steps. However, the bacterium
is relatively resistant to other treatments like freezing and
drying.
Listeria is relatively insensitive to high concentration of salts and acids and can also multiply at refrigerator temperatures and under vacuum.
Due to its viability, Listeria is currently becoming an increas-
ingly major problem consequent to changes in consumer
behavior regarding food consumption and the demand for
extended shelf life. An increasing variety of food products
and the trend towards “ready-to-eat” and “ready-to-cook”
products, as well as longer storage at cooling temperatures
in the range of 4 °C to 8 °C, are some of the basic reasons for
such food safety issues. Furthermore, new preparation tech-
nologies, such as “Cook & Chill” and “sous vide”, along with
new processes to extend shelf life, have led to increasing
problems with Listeria. Typical high-risk foods are raw foods
such as milk or vegetables and food that is subjected to
minimal further processing, such as soft cheeses, sausages,
pâtés and post-processed contaminated milk products.
Listeriosis is a serious infection caused by Listeria monocy-togenes. In recent years, it has been recognized that Listeria
is an important public health problem. The disease aff ects
primarily persons of advanced age, pregnant women,
newborns, and adults with weakened immune systems.
Listeriosis manifests in flu-like symptoms, fever, muscle
aches, and gastrointestinal symptoms such as nausea or
diarrhea. If infection spreads to the nervous system, symp-
toms such as headache, stiff neck, confusion, loss of bal-
ance, or convulsions can occur. A severe course of the
disease may lead to blood poisoning, encephalitis and
meningitis. It has also been found that the bacterium can
cause acute, self-limiting febrile gastroenteritis in healthy
individuals (1). Infected pregnant women may experience
Listeria monocytogenes – A Frugal Pathogen
Jvo Siegrist, Product Manager Microbiology [email protected]
Figure 1 Intracellular movement and cell-to-cell spread of L. mono-
cytogenes cells (green) driven by the polarized polymerization of
actin tails (red). (Source: M. Schuppler & M. Loessner, ETH Zürich)
Figure 2 L-form of Listeria monocytogens
(source: M. Loessner and Y. Briers, ETH
Zürich)
Gram-positive, catalase-positive, oxidase-negative,
non-spore forming, short rhods, motile at 30 °C or less
Mannitol fermentation Rhamnose fermentation
Hemolysis
Xylose fermentation Xylose fermentation Xylose fermentation
Listeriagrayi
Listeriawelshimeri
Listeriainnocua
Listeriaivanovii
londoniensis
Listeriaivanovii
Listeriaseeligeri
Listeriamono-
cytogenes
9
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Biochemical Tests and Cultural Methods
The biochemical profi le of Listeria includes: catalase positive,
oxidase negative, fermentation of carbohydrates to acid but
not to gas, hydrolysis of esculin and sodium hippurate,
methyl red positive, ammonia production from arginine,
negative reaction for hydrogen sulfi de production, indole
negative, nitrate reductase negative, no gelatin liquefaction,
no hydrolysis of starch and no urea hydrolysis. A typical dif-
ferentiation of Listeria ssp., especially for L. monocytogenes,
is by phenotype, starting with the β-hemolysis test
(L. monocytogenes is positive) and followed then by detec-
tion of carbohydrate fermentation ability. Af ter the
β-hemolysis test, it is also possible to test for a positive
rhamnose, methyl-α-D-mannopyranoside fermentation
and CAMP-test. In the CAMP-test, some Listeria species
show the ability to enhance the haemolysis of Staphylococcus aureus. More details on this method can be found in the
online data sheet for Rhamnose Broth (Fluka® 80547, see
also Table 2e). Another possibility for phenotype identifi -
cation is the testing of the fermentation ability of rhamnose,
xylose and mannitol (see the identification flow chart in
Figure 3).
An interesting topic and a smart solution for confi rmation
of L. monocytogenes are the chromogenic media. There are
diverse, commercially available chromogenic media like the
Agar Listeria acc. Ottavani and Agosti (ALOA). Most of them
use the following systems for diff erentiation:
1. Detection of β-glucosidase activity (by X-glu =
5-bromo- 4-chloro-3-indolyl-β-D-glucopyranoside) and
also Rhamnose fermentation (by indicator phenol red)
on a selective media. Listeria monocytogenes and Listeria innocua result in blue colonies with yellow background,
while Listeria ivanovii shows only blue colonies.
2. Screen the presence of β-glucosidase (by X-glu) and
phosphatidylinositol specific phospholipase C on a
selective media. Listeria monocytogenes and Listeria ivanovii result in greenish-blue colonies with an opaque
halo, while Listeria innocua shows only greenish-blue
colonies (recommended by ISO 11290-2).
Cat. No. Description Testing features
88597 Catalase Test
(H2O2, 3% solution)
Presence of catalase
77730 Gram Staining Kit Cell wall properties
40405 Hippurate Disks Hydrolysis of hippuric acid
01869 Hippurate Strips Kit Hydrolysis of hippuric acid
94438 Mannitol disks Fermentation abilities
07345 Oxidase Reagent acc.
Gaby-Hadley A
Presence of oxidase
07817 Oxidase Reagent acc.
Gaby-Hadley B
Presence of oxidase
18502 Oxidase Reagent acc.
Gordon-McLeod
Presence of oxidase
40560 Oxidase Strips Presence of oxidase
70439 Oxidase Test Presence of oxidase
93999 Rhamnose disks Fermentation abilities
07411 Xylose disks Fermentation abilities
Table 1 Biochemical tests
See more about the detection systems of the chromo-
genic and other confi rmation media in Tables 2c and 2e.
To give the media selectivity, phenyl ethanol and a high
concentration of lithium chloride and sodium chloride are
added to the media. Also, antibiotics like moxalactam, nali-
dixic acid, polymyxin B sulfate, ceftazidime, amphoteri-
cin B, acrifl avine, cycloheximide, colistin sulphate, cefotetan
and fosfomycin are taken to inhibit growth of fungi, Gram-
negative and other Gram-positive bacteria.
Figure 3 Schematic of biochemical identification for Listeria spp. (Source: Handbook of Listeria monocytogenes, 2008)
(continued on page 10)
10
Primary Enrichment Secondary Enrichment
Sample
Plating
Purification
Confirmation
Enrichment of Listeria in
sample with Fraser Broth
Incubation: 30 °C 24±2h
Select 5 presumptive colonies for confirmation. If well
separated colonies are not available , streak one colony
on Tryptone Soya Yeast Extract Agar
Purified colonies are confirmed with standard tests:
• Gram staining
• Motility test
• Carbohydrate fermentation test
• β hemolysis test and CAMP test (lysis tests)
Streaking of enriched cultures on Oxford Agar plate
Incubation: 35 °C 24h
Observe for black colonies at 24 and 48h
Streaking of enriched cultures on PALCAM Listeria
Selective Agar plate
Incubation: 30 and 35 or 37 °C 24 – 48h
Observe for green colonies with black halo at 24 and 48h
In Fraser Broth
Incubation:
35 or 37 °C 48±2h
Products for Enrichment Steps • Fraser Broth, Base (Fluka® 69198)
• Fraser Selective Supplement
(Fluka 18038)
• Fraser Supplement (Fluka 90836)
Products for Plating • Oxford Agar (Fluka 75805)
• Oxford-Listeria Selective
Supplement (Fluka 75806)
• PALCAM Listeria Selective
Agar plate (Fluka 75977)
• PALCAM Listeria Selective
Supplement (Fluka 03396)
Purification Medium • Tryptone Soya Yeast Extract Agar
(Fluka 93395)
Products for Confirmation • Gram Staining Kit (Fluka 77730)
• Listeria Motility Medium
(Fluka 55265)
• Carbohydrate Consumption Broth
(Fluka 07410)
• Blood Agar Base No. 2 (Sigma® B1676)
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EN ISO 11290-1 and EN
ISO 11290-2 (Micro-
biology of Food and
Animal Feeding Stuff s)
describe a horizontal
method for the detec-
tion and enumeration
of Listeria monocyto-genes. A fl ow chart of
the process appears in
Figure 4. The method
involves a general four-step process: enrichment, identifi ca-
tion, isolation and confi rmation. All the ISO recommended
and other common media are listed and described in
Tables 2a-e.
Cat. No. Description ISO
08105 Buffered Peptone Water (ISO) 11290-2
91366 Universal Pre-Enrichment Broth
Table 2a Non-selective Enrichment Media
References
[1] Food-Borne Pathogenic Microorganisms and Natural Toxins
Handbook: The “Bad Bug Book”. U.S. FDA/CFSAN. Center for
Food Safety and Applied Nutrition, Food and Drug
Administration, College park, MD (2003).
[2] Cossart, P.; Bierne, H.; The use of host cell machinery in the
pathogenesis of Listeria monocytogenes. Curr. Opin. Immunol.
(England), 13(1), 96-103 (2001).
[3] Verbrauchertipps: Schutz vor lebensmittelbedingten Infektio-
nen mit Listerien, Bundesinstitut für Risikobewertung (2008).
[4] C.L. Birmingham et al., Listeriolysin O allows Listeria
monocytogenes replication in macrophage vacuoles, Nature
451: 350-354 (2008).
[5] L. Dongyou, Handbook of Listeria monocytogenes, CRC Press
(2008).
[6] Simone Dell’Era, et al., Listeria monocytogenes L-forms
respond to cell wall deficiency by modifying gene expression
and the mode of division, Molecular Microbiology, Vol. 73,
Issue 2, pages 306–322, July 2009.
[7] M. Schuppler, M.J. Loessner, The Opportunistic Pathogen
Listeria monocytogenes: Pathogenicity and Interaction with
the Mucosal Immune System, International Journal of
Inflammation Volume 2010 (2010).
Figure 5 HiCrome™ Listeria Agar
(left L. ivanovii, right L. monocytogenes)
Figure 4 ISO Protocol (EN-ISO 11290-1:1996) for detection and enumeration of Listeria monocytogenes
11
Mic
rob
iolo
gy
Cat. No. Description ISO
50595 Buffered Listeria Enrichment Broth Base
62351 Listeria Selective Enrichment Supplement according to IDF/FIL
69198 Fraser Broth, Base 11290-1
18038 Fraser Selective Supplement 11290-1
90836 Fraser Supplement 11290-1
F6672 Fraser secondary enrichment broth base 11290-1
F2674 Fraser enrichment supplement 11290-1
62353 Listeria Enrichment Broth according to FDA/IDF-FIL
62351 Listeria Selective Supplement according to IDF-FIL
62348 Listeria Selective Supplement according to FDA
59859 PALCAM Listeria Selective Enrichment Broth, Vegitone (see Figure 4)
91986 PALCAM Listeria Selective Supplement according to Van Netten et al.
94485 UVM Listeria Selective Enrichment Broth, modified*
90554 UVM Listeria Selective Enrichment Broth, modified I & II
50195 Listeria UVM Supplement I
50279 Listeria UVM Supplement II
Table 2b Selective Enrichment Media
Cat. No. Description Features ISO
77408 Listeria mono Differential Agar, Base (ALOA, chromogenic
media acc. ISO)
Presence of phosphatidylinositol specific phospholipase
C of Listeria monocytogenes, selective media
11290-2
03708 Listeria mono Enrichment Supplement I (Use with 77408) 11290-2
92301 Listeria mono Selective Supplement I „ 11290-2
91603 Listeria mono Selective Supplement II „ 11290-2
62355 Listeria Selective Agar* selective media
62653 LPM Agar* „
43963 Moxalactam Supplement (Use with 43963)
75805 Oxford Agar esculin hydrolysis, selective media 11290-1
75806 Oxford-Listeria Selective Supplement (Use with 75805)
75977 PALCAM Listeria Selective Agar esculin hydrolysis, selective media 11290-1
15776 PALCAM Listeria Selective Agar, Vegitone (Use with 75977)
91986 PALCAM Listeria Selective Supplement according to
Van Netten et al.
„ 11290-1
Table 2c Identification Media
Cat. No. Description ISO
93395 Tryptone Soya Yeast Extract Agar 11290-1/2
Table 2d Purification Media
Cat. No. Description Features ISO
B1676 Blood Agar Base No. 2 Lysis test
07410 Carbohydrate Consumption Broth Fermentation ability
53707 HiCrome™ Listeria Agar Base, modified (chromogenic
media, see Figure 5)
β-glucosidase activity, rhamnose fermentation, selective
media
59688 HiCrome Listeria Selective Supplement (Use with 53707)
92302 Listeria mono Confirmatory Agar, Base (chromogenic
media)
Presence of phosphatidylinositol specific phospholipase C
of Listeria monocytogenes and fermentation of
α-methyl-D-mannoside, selective media
15895 Listeria mono Enrichment Supplement II (Use with 92302)
92301 Listeria mono Selective Supplement I „
91603 Listeria mono Selective Supplement II „
55265 Listeria Motility Medium Motility test 11290-1/2
80547 Carbohydrate Utilization Broth* Fermentation ability 11290-1/2
80301 Rhamnose Broth Supplement (Uses with 80547) 11290-1/2
02046 Methyl α-D-mannopyranoside Supplement „
Table 2e Confirmation Media (Differentiation)
* not sold in USA
sigma-aldrich.com/microbiology
12
sigma-aldrich.com/kjeldahl
Ele
me
nta
l A
na
lysi
s
the amount of nitrogen present in the original sample, is
determined by back titration. The method is described by
various standard methods such as ASTM D3179, ISO 332 and
ISO 333.
(CHNO) + H2SO4 CO2 + SO2 + H2O + (NH4)2SO4
[Sample] [catalyst]
(organic nitrogen) (inorganic nitrogen)
Figure 1 Kjeldahl reaction (condensed)
The Kjeldahl method is often used to report the protein con-
tent of a sample based on the total nitrogen after applying
the so-called Jones factors [1–3]. If the sample contains
nitrite or nitrate, it must be reduced with Arnd’s alloy (11066)
in weak acid to produce a neutral solution prior to analysis [4].
There are several suitable Kjeldahl catalysts. The mercury-
and selenium-free catalyst (31835) is popular for environmental
and toxicological reasons. The selenium-containing catalyst
according to Wieninger (31108) is used for very resistant
samples, such as heteroaromatic compounds, mineral oils
and fats. The Missouri catalyst (31831) is an environmentally
friendly alternative because it has a low copper content,
although the reaction is signifi cantly slower.
Kjeldahl digestions convert nitrogen-containing organic
compounds, such as amino acids, into ammonia com-
pounds by fi rst heating them in concentrated sulfuric acid
(84727). A catalyst speeds up the decomposition. Free
ammonia is released by adding concentrated sodium
hydroxide solution (30531), which is evaporated by steam
distillation. The amount of ammonia present, and therefore
Nitrogen Determination According to the Kjeldahl Method
Cat. No. Brand Description Package Size
31835 Fluka® Kjeldahl Catalyst
Mercury- and selenium-free
Contains Na2S2O8/CuSO4
250 tablets
(2.5 g)
31831 Fluka Kjeldahl Catalyst according to Missouri
Contains 99.5% K2SO4 and 0.5% CuSO4
250 tablets
(2.5 g)
31108 Fluka Kjeldahl Catalyst according to Wieninger
Contains 96.5% Na2SO4, 1.5% CuSO4 and 2.0% Se
250 tablets
(2.5 g)
31821 Fluka Disintegrating mixture for Kjeldahl
Contains H2SO4 and Se
1 L
11066 Fluka Arnd’s alloy
Contains 60% Cu and 40% Mg
50 g
Product Table A selection of digestion reagents. Please find the complete product list of Kjeldahl reagents at sigma-aldrich.com/kjeldahl
Michael Jeitziner, Market Segment Manager, Analytical Reagents & Standards [email protected]
References:
[1] Merrill, A. L. and Watt, B. K. Energy value of foods: Basis and
derivation, revised. U.S. Department of Agriculture,
Agriculture Handbook 74, 1973.
[2] Protein (Crude) Determination in Animal Feed: Copper
Catalyst Kjeldahl Method. (984.13) Official Methods of
Analysis. 1990. Association of Official Analytical Chemists.
15th Edition.
[3] Protein (Crude) Determination in Animal Feed: CuSO4/TiO2
Mixed Catalyst Kjeldahl Method. (988.05) Official Methods of
Analysis. 1990. Association of Official Analytical Chemists.
15th Edition.
[4] Arnd T. Zur Bestimmung des Stickstoffs salpeter- und
salpetrigsaurer Salze mit Kupfer-Magnesium. Angew. Chem.
1920, 33, 296 –298.
13
sigma-aldrich.com/selectophore
Ch
rom
ato
gra
ph
y
Get our New Headspace Grade Solvents and Save 30%When developing a headspace method, parameters such
as sample solvent, extraction temperature, extraction time,
sample volume and headspace volume are optimized.
Because the composition and purity of the sample solvent
have signifi cant eff ects on the recovery and quality of the
chromatogram, we have developed solvents specifi cally for
GC-HS applications. Their purity and handling specifi cations
meet the requirements of European Pharmacopoeia (Ph.Eur.)
and United States Pharmacopeia (USP), as well as the Inter-
national Conference on Harmonization of Technical
Requirements for Registration of Pharmaceuticals for
Human Use (ICH) guidelines. All solvents are microfi ltered at
0.2 μm and packed under inert gas for longer shelf life.
Product Brand Name Abbreviation Package Size
44901 Fluka® N,N-Dimethylacetamide, GC-Headspace tested DMA 1 L
51779 Fluka Dimethyl sulfoxide, GC-Headspace tested DMSO 1 L, 2.5 L
51781 Fluka N,N-Dimethylformamide, GC-Headspace tested DMF 1 L
53463 Fluka Water, GC-Headspace tested 1 L
67484 Fluka 1,3-Dimethyl-2-imidazolidinone, GC-Headspace tested DMI 100 mL, 1 L
NEW 68809 Fluka Cyclohexanone, GC-Headspace tested CYHAONE 1 L
NEW 69337 Fluka 1-Methyl-2-pyrrolidinone, GC-Headspace tested NMP 1 L
NEW 80708 Fluka Benzyl alcohol, GC-Headspace tested BA 1 L
To take advantage of this off er, please quote Promotion Code SAQ on your order. Off er valid until January 31, 2012.
New Selectophore® Brochure
2011 availableBasic Principles, Technical Instructions, Applications & Product Listings
Ionophores and auxiliary reagents for the preparation of ion-selective
membranes:
• Ionophores
• Plasticizers
• Additives
• Solvents
• Calibration standards
• Membranes & Cocktails
• Equipment
To order this brochure, please visit sigma-aldrich.com/selectophore or
check the box on the reply card to receive your free copy.
sigma-aldrich.com/icsigma-aldrich.com/derivatization
Don’t forget to order your 2012 –2014 Aldrich Handbook.
For reliable, high-quality chemicals you can trust, add your free
copy of the Aldrich Handbook to your laboratory by visiting:
Aldrich.com/handbookrequest
77007
The new Aldrich® Handbook contains the widest selection of Chemistry
and Materials Science products and is your resource for chemical structures,
literature references, and extensive chemical and physical data. Our compli-
mentary catalog includes new and innovative reagents and building blocks,
plus a focused line of Labware products to support your chemistry needs.
The Aldrich Handbook’s portfolio supports the research community with:
• More than 40,000 research chemicals
• Over 4,000 new products
• 10,000 chemical structures
• 8,500 updated literature citations
• Extensive chemical and physical data
New Brochure Derivatization ReagentsBroad offering for accurate analysis on GC, HPLC or TLC
Over 400 Derivatizing Reagents
• Silylation, Acylation and Alkylation reagents for GC
• UV/VIS, Fluorescent and Electrochemical derivatives for HPLC
• Optically pure derivatizing reagent for Chiral
• Derivatizing reagents for TLC applications
• Accessories for derivatizing reaction
To order your free copy of the New Derivatization Guide, and for product and ordering information,
visit: sigma-aldrich.com/derivatization
14D
eri
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15
sigma-aldrich.com/lc-ms
Ch
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0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 Time [min]
-2.5
0.0
2.5
5.0
7.5
10.0
Intens.[mAU]
-2
0
2
4
6
8
NEW
• Positive and negative Ion mode testing for all
MS applications
• Most narrow and consistent specifications
We ensure UHPLC suitability, which is achieved by considering
parameters of the preparation process far beyond fi ltration.
The new LC-MS Ultra CHROMASOLV grade solvents are
exclusively tested for high performance with UHPLC gradi-
ent separations plus UV, positive and negative mode MS
detection. Table 1 shows the new products available to
enhance your analytical performance.
Recent innovations in HPLC and mass spectrometry (MS),
including Fused-Core® particles and ultra-high perfor-
mance/pressure liquid chromatography (UHPLC) systems,
have pushed the limits of speed (throughput), effi ciency,
and sensitivity.
Sigma-Aldrich introduces the new LC-MS Ultra CHROMASOLV
product line, providing an outstanding quality and an ultra-
pure mobile phase that off ers advantages such as:
• Tested specific to UHPLC applications
• Extremely small drift in UHPLC gradient analysis
Exclusively designed for UHPLC: LC-MS Ultra CHROMASOLV®
Figure 1 UV drift of solvent baseline resulting from a gradient application of LC-MS Ultra CHROMASOLV Acetonitrile/water (red) compared
with classical HPLC Acetonitrile/water (blue). A gradient drift in UHPLC shows different values, due to an increased sensitivity compared to
classical methods. A regular gradient solvent would result in different chromatograms by running on HPLC versus UHPLC set-up.
Cat. No. Brand Name Description Package Size
14261 Fluka® Acetonitrile LC-MS Ultra CHROMASOLV, ≥99.9%, gradient-tested for UHPLC, UV & MS 1 L, 2 L
14262 Fluka Methanol LC-MS Ultra CHROMASOLV, ≥99.9%, gradient-tested for UHPLC, UV & MS 1 L, 2 L
14263 Fluka Water LC-MS Ultra CHROMASOLV, gradient-tested for UHPLC 1 L, 2 L
14264 Fluka Trifluoroacetic acid LC-MS Ultra eluent additive, ≥ 99.0%, suitable for UHPLC-MS 1 mL, 2 mL
14265 Fluka Formic acid LC-MS Ultra eluent additive, ≥ 98%, suitable for UHPLC-MS 1 mL, 2 mL
14266 Fluka Ammonium formate LC-MS Ultra eluent additive, suitable for UHPLC-MS 25 g
14267 Fluka Ammonium acetate LC-MS Ultra eluent additive, suitable for UHPLC-MS 25 g
Table 1 New LC-MS Ultra CHROMASOLV solvents and LC-MS Ultra eluent additives.
For further product information, please visit sigma-aldrich.com/lc-ms
16
sigma-aldrich.com/medicinalplants
New Analytical Standards for the Analysis of Herbal Medicinal Products
In recent months, Sigma-Aldrich extensively expanded its
off ering of analytical standards and primary reference stan-
dards for active ingredients and marker compounds found in
plant derived medicinal products. The table below lists the
latest additions to this rapidly growing product line.
On our webpage at sigma-aldrich.com/medicinalplants,
you can fi nd an up-to-date list of these products, catalogued
not only by alphabetical order, but also by substance class
and by genus of a large number of medicinal plants.
Description Cat. No. Package Size
-Acetylboswellic acid 56208 10 mg
β-Acetylboswellic acid 49873 10 mg
3-O-Acetylboswellic acid ( +β) 96729 10 mg
3-O-Acetyl-11-keto-β boswellic acid 74607 10 mg
Allantoin 93791 50 mg
(+) L-Alliin 72805 10 mg
Aloe Emodin 93938 10 mg
Amentoflavone 18571 10 mg
Andrograpanin 19443 10 mg
Andrographolide 90281 10 mg
Arbutin 66468 50 mg
Bacopasaponin C 76092 10 mg
Bacopaside-II 44698 10 mg
Bacoside-A (Mixture) 76091 10 mg
Bacoside-A4 53889 10 mg
Bacosine 69528 10 mg
Benzyl benzoate 68183 1 mL
Bilobalide (-) 79593 10 mg
β-Boswellic acid 80342 10 mg
Boswellic acid ( +β) 63850 10 mg
Caftaric acid 88656 10 mg
Carvacrol 42632 50 mg
Carveol 61370 50 mg
Casticin 16382 10 mg
Chicoric acid 06957 10 mg
Chrysophanol 01542 25 mg
Cinnamyl acetate 42759 1 mL
trans-Cinnamyl alcohol 93066 50 mg
R(+) Citronellal 72638 1 mL
Corilagin 75251 10 mg
Cryptotanshinone 80709 10 mg
Curcumin 08511 10 mg
Curcumol 36236 10 mg
Daidzein 16587 10 mg
Daidzin 42926 10 mg
14-Deoxy-11,12 -didehydro
Andrographolide
55549 10 mg
Dihydrocarvone 09164 50 mg
Diosmin 61386 50 mg
Echinacoside 07538 10 mg
Elemolic acid (α+β) 73527 10 mg
Description Cat. No. Package Size
β-Elemonic acid 00708 10 mg
Eleutheroside B 90974 10 mg
Eleutheroside E 08198 10 mg
Emodin 30269 10 mg
(-) Epicatechin 68097 10 mg
(-) Epicatechin 3-gallate 78059 10 mg
(-) Epigallocatechin 3-gallate 93894 10 mg
(-) Gallocatechin 01388 10 mg
Genistein 92136 10 mg
Genistin 73822 10 mg
Ginkgolic acid C13:1 49962 10 mg
Ginkgolid B 94970 10 mg
Hesperidin 50162 10 mg
trans-Isoferulic acid 05407 10 mg
Jujubogenin isomer of
bacopasaponin C
42488 10 mg
11-Keto-β-boswellic acid 78535 10 mg
Lupeol 18692 10 mg
Luteolin 72511 10 mg
trans-oMethoxy-zimtaldehyd (trans-o) 61384 50 mg
2-Methyl-3-buten-2-ol 05392 1 mL
Methyleugenol 04607 50 mg
7-O-Methylwogonin 41442 10 mg
Myricitrin 67268 10 mg
Neoandrographolide 49879 10 mg
Oleanolic acid 42515 10 mg
Oleuropein 92167 10 mg
Oxybenzone 59647 50 mg
Physcion 93893 10 mg
Pyrogallol 06931 50 mg
Salvianolic acid A 97599 10 mg
Salvianolic acid B 49724 10 mg
Schaftosid 42925 5 mg
Scutellarin 73577 10 mg
Sennosid C 73235 10 mg
Sennosid D 16383 10 mg
Serratol 78689 10 mg
Sesamin 59867 10 mg
Tanshinone I 80714 10 mg
Tanshinone II A 51704 10 mg
Sta
nd
ard
s
17
sigma-aldrich.com/enzym-food-kits
Fo
od
An
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Enzymatic Food Analysis
Jvo Siegrist, Product Manager Microbiology [email protected]
Enzymatic methods for food analysis are highly specifi c and
off er considerable time and cost savings over other meth-
ods, especially from the sample preparation standpoint.
Sigma-Aldrich off ers a wide variety of convenient kits and
reagents for rapid and reliable enzymatic food analysis.
Because of the importance of enzymatic methods in food
production and assurance of quality and safety, there are
many textbooks, national and international industry associ-
ations and government agencies devoted to the subject.
Like many other analytical methods, enzymatic analysis was
used fi rst in clinical diagnosis, then adapted for the determi-
nation of food ingredients. The high specifi city of enzymes
enables the analysis in complex sample matrixes without
complicated sample preparation techniques. This makes
enzymatic food analysis a highly valuable tool because it
saves time, reduces costs and gives reliable results indepen-
dent of the sample matrix. Additionally, enzymatic methods
use non-hazardous reagents, are environmentally friendly
and can be automated for in-line process monitoring.
Applications: Carbohydrate and dietary fiber levels
Qualitative and quantitative analysis in the food and bever-
age industry is extremely important from quality, storage,
nutrition and safety standpoints. The levels of certain carbo-
hydrates, like glucose, lactose, fructose, sucrose and starch,
aff ect intolerance conditions, diabetes and obesity. The pres-
ence of unwanted carbohydrates or their hydrolysis pro-
ducts can alter the manufacturing process or reduce
product shelf life. They can also indicate microbial contami-
nation (e.g. yeasts) or improper processing (e.g. overheat-
ing). For fruit juice and wine, raw materials that have variable
sugar content infl uence the quality of the fi nished product
and should therefore be monitored.
Did you know …
that most fructose is produced using enzymes?
In the food industry, fructose is commonly produced by
digesting cornstarch with -amylase, glucoamylase and
glucose isomerase. The result is a mixture of 42% fruc-
tose, 50 –52% glucose, and small amounts of various
other sugars.
Figure 1 Enzyme assay
Brand Cat. No. Kit Package Size Detection method & wavelength
Sigma SCA20 Sucrose Assay Kit Sufficient for ~20 assays NADH; 340 nm
Sigma GAGO20 Glucose (GO) Assay Kit Sufficient for ~20 assays H2O2; 540 nm
Sigma GAHK20 Glucose (HK) Assay Kit Sufficient for ~20 assays NADH; 340 nm
Sigma FA20 Fructose Assay Kit Sufficient for ~20 assays NADH; 340 nm
Sigma STA20 Starch (GO/P) Assay Kit Sufficient for ~20 assays H2O2; 540 nm
Sigma SA20 Starch (HK) Assay Kit Sufficient for ~20 assays NADH; 340 nm
Sigma TDF100A Total Dietary fiber Assay Kit Sufficient for ~100 assays gravimetric
Fluka® TDFC10 Total Dietary fiber Assay Control Kit Sufficient for ≥10 assays gravimetric
Table 1 Enzymatic assay kits
Brand Cat. No. Product Required for Kit Number
Sigma-Aldrich 258105 Sulfuric acid, ACS reagent GAGO20, STA20
Sigma-Aldrich 154938 Dimethyl sulfoxide, ACS reagent STA20
Sigma-Aldrich 459844 Ethyl alcohol, ACS reagent STA20, TDF100A
Sigma-Aldrich 184519 Petroleum ether, ACS reagent TDF100A
Sigma-Aldrich 320110 Acetone, ACS reagent TDF100A
Sigma-Aldrich S0876 Sodium phosphate dibasic, anhydrous TDF100A
Sigma-Aldrich S0751 Sodium phosphate monobasic, anhydrous TDF100A
Sigma-Aldrich S2567 Sodium hydroxide, 1.0 M TDF100A
Sigma H3162 Hydrochloric acid, 1.0 M TDF100A
Table 2 Additional required reagents (continued on page 18)
18
sigma-aldrich.com/enzym-food-kits
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od
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Determination of dietary fiber is another important food analysis.
Consuming high-fi ber foods, like fruits, vegetables, nuts and grains, is
recommended to treat or prevent such maladies as constipation,
haemorrhoids and diverticulitis. Water-soluble fi ber also helps decrease
blood cholesterol levels. From a chemical perspective, dietary fi ber is a
mixture of complex organic substances, including hydrophilic com-
pounds, like soluble and insoluble polysaccharides and non-digestable
oligosaccharides, and a range of non-swellable, relatively hydrophobic
compounds, like cutins, suberins and lignins. Verifying a high content of
dietary fiber in food permits a higher quality grading and access to
higher-end product markets.
Detection principle of enzymatic reactions
Enzymatic methods to determine analyte concentration typically
employ photometry to measure the concentration changes of specifi c
products or substrates during the enzyme-catalysed reaction. Concen-
tration of compound of interest is measured using the reaction
stoichiometry.
Some commonly employed enzyme systems include:
• Nicotinamide adenine dinucleotide (NADH/NAD+)
coenzyme system
• Activity of dehydrogenases using the NAD+/NADH system is
measured by monitoring the changes in absorbance at 340 nm.
NADH has an absorption maximum at 340 nm, while the oxidized
form NAD+ does not absorb at this wavelength.
• H2O2 production (Oxidase/peroxidase system)
• Oxidases cleave H2O2 from molecules (e.g. glucose) and
o-dianisidine is oxidized with the enzyme peroxidase and H2O2.
Oxidized o-dianisidine reacts with sulfuric acid to form a more
stable pink-colored product that can be measured at 540 nm.
Carbohydrate assay kits
The kits for the enzymatic assay of sucrose, fructose and starch are
based on the enzymatic determination of glucose. The fi rst step is the
conversion or degradation of the carbohydrate to glucose or glucose
derivate. The glucose concentration is then determined according to
the two diff erent detection systems: glucose oxidase (GO) or hexokinase
(HK) in combination with the glucose-6-phosphate dehydrogenase
reaction.
Figure 2 Natural fruit juice
contains dietary fiber and
many different sugars
Fiber
AOAC Method 920.86: Fiber (Crude) in Flour – Ceramic Fiber Filter Method, Enzymatic-Gravimetric Method
AOAC Method 985.29: Total Dietary Fiber in Foods – Enzymatic-Gravimetric Method
AOAC Method 991.42: Insoluble Dietary Fiber in Food and Food Products – Enzymatic-Gravimetric Method, Phosphate Buffer
AOAC Method 991.43: Total, Soluble, and Insoluble Dietary Fiber in Food – Enzymatic-Gravimetric Method, MES-TRIS Buffer
AOAC Method 992.16: Total Dietary Fiber – Enzymatic-Gravimetric Method
AOAC Method 993.19: Soluble Dietary Fiber in Food and Food Products – Enzymatic-Gravimetric Method (Phosphate Buffer)
AOAC Method 2009.01: Total Dietary Fiber in Foods – Enzymatic–Gravimetric-Liquid Chromatographic Method
AACC Method 32-05: Total Dietary Fiber
AACC Method 32-07: Soluble, Insoluble, and Total Dietary Fiber in Foods and Food Products
AACC Method 32-21: Insoluble and Soluble Dietary Fiber in Oat Products – Enzymatic-Gravimetric Method
SLMB 468: Determination of Dietary Fiber in Special Foods – Enzymatic-Gravimetric Method
Sugar (Glucose, Fructose, Sucrose … )
AOAC Method 969.39 Glucose in Corn Syrups and Dextrose Products – Glucose Oxidase Method
AOAC Method 985.09 Glucose and Fructose in Wine – Enzymatic Method
ICUMSA Method GS 2-4 (2007): The Determination of Glucose + Fructose in White Sugar by the Hexokinase Method
ICUMSA Draft Method No. 8 (2007): The Determination of the Apparent Total Sucrose coming from Sucrose, Glucose and Fructose in Molasses by
an Enzymatic Method
ICUMSA Method GS 8/4/6-4 (2007): The Determination of Glucose and Fructose in Beet Juices and Processing Products by an Enzymatic Method
SLMB 305: Determination of D-Glucose, D-Fructose, Saccharose, Lactose and Sorbitol in Ice Cream – Enzymatic Method
SLMB 840: Determination of Diverse Sugars in Wine – Enzymatic Method
ISO 13965/1998: Meat and Meat Products – Determination of Starch and Glucose Contents – Enzymatic Method
Starch
AOAC Method 2002.02: Resistant Starch in Starch and Plant Materials – Enzymatic Digestion
AOAC-AACC Method 996.11: Starch (Total) in Cereal Products – Amyloglucosidase-alpha-Amylase Method
AACC-AOAC 979.10: Starch in Cereals – Glucoamylase Method
AACC Method 76-13: Total Starch Assay Procedure (Amyloglucosidase/alpha-Amylase Method)
SLMB 467: Determination of Starch and Starch Decomposition Products in Special Foods – Enzymatic Method
ICC Standard Method 128/1: Procedure for the Determination of Starch after Enzymatic Decomposition
ICC Standard Method 164: Measurement of Damaged Starch by Using Enzymatic Kit
ISO 15914/2004: Animal Feed Stuffs – Enzymatic Determination of Total Starch Content
ISO 13965/1998: Meat and Meat Products – Determination of Starch and Glucose Content – Enzymatic Method
Table 3 Standard Official Methods
19
sigma-aldrich.com/enzym-food-kits
Fo
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Total dietary fiber assay kit
The total dietary fi ber content is determined by a combination of enzy-
matic and gravimetric methods. Samples of dried, fat-free foods are
gelatinized with heat-stable -amylase and then enzymatically digested
with protease and amyloglucosidase to remove protein and starch in
the sample. Ethanol is added to precipitate the soluble dietary fi ber. The
residue is then fi ltered and washed with ethanol and acetone. After dry-
ing, the residue is weighed. Half of the sample is analysed for protein
and the other half is ashed. Total dietary fi ber equals weight of residue
minus weight of protein and ash.
HN
CH3O H
2O
Pink Color (540 nm)H
2SO
4
OCH3
NH
+H
2O
2
+
O-Dianisidine
Glucose Gluconic Acid
Glucose
Oxidase
Peroxidase
Glucose Glucose-6-phosphate 6-PhosphogluconateHexokinase
ATP ADP
Glucose-6-phosphate
dehydrogenase
NAD NADH
Glucose
Glucose Fructose
Glucose-6-phosphate 6-PhosphogluconateHexokinase
Invertase
ATP ADP
Glucose-6-phosphate
dehydrogenase
+
NAD NADH
Sucrose
Glucose-6-phosphate 6-Phosphogluconate
Glucose-6-phosphateFructose-6-phosphate
Glucose-6-phosphate
dehydrogenase
NAD NADH
FructoseHexokinase
ATP ADP
Phosphoglucose
isomerase
Figure 3 Glucose (GO) assay kit
Detection of glucose via glucose oxidase (GO) and peroxidase, monitored at 540 nm.
Figure 4 Glucose (HK) assay kit
Detection of glucose via hexokinase (HK) and glucose-6-phosphate
dehydrogenase by formation of NADH, monitored at 340 nm.
Figure 5 Sucrose assay kit
Sucrose is hydrolyzed to glucose and fructose by invertase. Glucose and fructose
are phosphorylated with ATP in the hexokinase reaction. Glucose-6-phosphate
and NAD are then converted to 6-phosphogluconate and NADH via glucose-6-
phosphate dehydrogenase, monitored at 340 nm.
Figure 6 Fructose assay kit
Fructose is phosphorylated by ATP in a reaction catalyzed by hexokinase. The
resulting fructose-6-phosphate is then converted to glucose-6-phosphate by
phosphoglucose isomerase. Glucose-6-phosphate and NAD is converted to
6-phosphogluconate and NADH, monitored at 340 nm.
HN
CH3O H
2O
Pink Color (540 nm)
H2O
2
H2SO
4
OCH3
NH
+
+
H2O
2
+
O-Dianisidine
Glucose Gluconic Acid
Amylose
Polymer of a-(1-4)-D-glycopyranosyl units
Starch
Amylopectin
Polymer of α-(1-4)-D-glycopyranosyl units with
approximately 4 % α-(1-6) branching.
Glucose
Oxidase
Peroxidase
α-Amylase
Amyloglucosidase
α-Amylase
Glucose
OH
OOH
CH2OH
OH
O
OH
O
CH2OH
OH
O
OH
O
CH2OH
OH
O
OH
O
n
CH2OH
OH
O
OH
OHO
CH2
OH
O
HOOH
CH2OH
OH
O
O
OH
O
CH2OH
OH
O
OH
O
CH2OH
OH
O
OH
O
CH2OH
OH
O
OH
O
CH2OH
OH
O
n
Amyloglucosidase
Amyloglucosidase
(terminal (1-6) residues)
Glucose Glucose-6-phosphate 6-PhosphogluconateHexokinase
ATP ADP
Glucose-6-phosphate
dehydrogenase
NAD NADH
OH
OHO
CH2
OH
O
HOOH
CH2OH
OH
O
O
OH
O
CH2OH
OH
O
n
Amyloglucosidase
(terminal (1-6) residues)
Amylopectin
Polymer of α-(1-4)-D-glycopyranosyl units with
approximately 4 % α-(1-6) branching. Amylose
Polymer of a-(1-4)-D-glycopyranosyl units
Starch
Amylo-
glucosidaseGlucose
OHOH
O
CH2OH
OH
O
OH
O
CH2OH
OH
O
n
Amyloglucosidase
Figure 7 Starch (GO/P) assay kit
The hydrolysis of starch to glucose is catalyzed by -amylase and amyloglucosi-
dase. Glucose is then converted to gluconic acid and H2O2 by glucose oxidase;
detection of H2O2 via peroxidase reaction at 540 nm (proportional to the original
starch concentration).
Figure 8 Starch (HK) assay kit
The hydrolysis of starch to glucose is catalyzed by amyloglucosidase. Glucose is
phosphorylated by ATP in a reaction catalyzed by hexokinase. Glucose-6-phosphate
and NAD is converted to 6-phosphogluconate and NADH; detection of NADH at
340 nm (proportional to the original starch concentration).
Additional information and instruction bulletins to all our enzymatic assay
kits are available on our website sigma-aldrich.com/enzym-food-kits
References:
[1] Trowell, H. Definitions of Fiber. The Lancet 1974, 303 (7856), 503.
[2] Trowell, H., Southgate, D. A.; Wolever, T. M. S.; Leeds, A. R., Gassull, M. A.;
Jenkins, D. A. Dietary Fiber Redefined. The Lancet, 1976, 307 (7966), 967.
[3] Van Soest, P.J. and McQueen, R.W. The chemistry and estimation of fiber.
Proc. Nutr. Soc.1973, 32, 123 – 130.
[4] Official Methods of Analysis of AOAC International, 16th Edition, Volume II,
Section 45.4.07, Method 985.29 (1997).
[5] Matissek, R.; Schnepel, F.; Steiner, G. Lebensmittelanalytik. 2nd Edition (1992),
p.397.
TOTAL DIETARY FIBER ASSAY
Heat Stable, α-Amylase and Amyloglucosidase, incubation at pH 6.0, 6 h, 37 °C
Adjust pH to 8.2, incubation 20 min, 90 °C
Protease incubation 30 min, 60 °C
Adjust pH to 4.5 and add internal Standard
Add 4 volumes ethanol for 1 h, then filter
Low molecuar weight soluble
dietary fiber determination
High molecular weight
dietary fiber determination
Calculation of Total Dietary Fiber
CHO Determination
Figure 9 Flow chart of Total Dietary Fiber assay kit (acc. AOAC Method 2009.01)
20
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Request the new catalog now!
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To order your free copy of the RTC Catalog please tick the box on the BRC, Code NLB.
Do you want reliable quantitative results?Try our NEW organic TraceCERT® CRMs!
• Products for HPLC, GC and qNMR
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21
sigma-aldrich.com/hydranal
Tit
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Water Determination in Polymers and PlasticsKarl Fischer Titration with HYDRANAL® Reagents
Polymers and plastic compounds may absorb atmospheric
moisture during production, transport, and storage. This
may seriously aff ect their handling, structure, and proper-
ties. For certain production processes water content limits
are defined and must be adhered to; otherwise, quality
defects such as surface cords, lack of bonding, blistering, or
other eff ects can occur in the fi nished products. Controlling
the water content is a crucial factor in the production of
high-quality end products.
Karl Fischer (KF) titration is a suitable method for water con-
tent determination in polymers and plastics. However, the
standard KF procedure may have to be varied in many
cases; sample solubility can be enhanced by the addition of
various solvents as solubilizers, or by carrying out the titra-
tion at increased temperatures. For insoluble samples, the
KF oven can be used. If the sample is absolutely insoluble
and even the oven treatment is not applicable, external
extraction of water from the sample with a suitable solvent
can be the method of choice.
Karl Fischer oven method for insoluble samples
Some samples are insoluble in the working media of KF
titration, and they release their water only at high tempera-
tures, making them unsuitable for direct KF titration. Other
compounds may cause side reactions with components of
the KF reagents like iodine or methanol and thereby falsify
the titration results. Examples of such substances are plas-
tics, oils with additives or a number of inorganic salts. For
correct water content determination in these substances,
the indirect KF method is recommended: the sample water
is driven out at temperatures between 50 and 300 °C in an
oven and transferred to the titration cell by means of a
dried, inert carrier gas. An important condition for this
method is a stable sample matrix at the chosen temperature.
Decomposition of the sample should be avoided or at least
any decomposition products should not interfere with the
KF reaction (e.g. no formation of water).
Since the investigated substances often contain only trace
amounts of water, the drying oven is most commonly com-
bined with a coulometer, although it can be used with a
volumeter as well. The sample size will depend on the water
content of the sample and on the type of equipment used
for KF titration. We recommend coulometric titrations for
amounts of water in the range of 500–3000 μg, and volu-
metric titrations for water content of 1–10 mg per sample.
Smaller amounts of water will increase the standard devia-
tion of the determinations; higher amounts may lead to
condensation in the tubing.
Determination of water content in polymer
and plastic compounds
Many polymers have poor solubility and are mainly analyzed
through the use of a KF oven, although the temperature
must be optimized for each product. Some plastic powders
release water easily and can be titrated in suspension, prefer-
ably at an increased temperature. In some cases, samples
may need grinding with a laboratory mill before titration, or
a homogenization device can be used inside of the titration
vessel to grind the sample. Particular plastics are soluble in
chloroform, formamide, N-methylpyrollidone or in ketones.
As long as these solvents are suitable for use in combination
with the usual alcohols in the KF titration, volumetric titration
is possible. Table 1 shows recommended variations of KF
titration for selected polymer and plastic samples.
Andrea Felgner, Market Segment Manager HYDRANAL [email protected]
Thomas Wendt, HYDRANAL Technical Service [email protected]
(continued on page 22)
22
sigma-aldrich.com/hydranal
Tit
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on
Sigma-Aldrich offers over 650 application reports for KF
t itrat ion. A ful l l is t can be found on our website
sigma-aldrich.com/hydrana . Application repor ts
a re av a i l a b l e o n re q u e s t ; p l ea s e co nt a c t u s a t
[email protected] or use the contact form on our
website.
More detailed information can be obtained from the
respective Application Reports (designated Lxxx in the
tables) or on our HYDRANAL® Multimedia Guide CD (use the
attached reply card to receive your free copy).
New HYDRANAL team at Sigma-Aldrich
After a long and successful career at Sigma-Aldrich,
Helga Hoff mann retired as of April this year. We would
like to take this opportunity to introduce your new
Hydranal Team:
Andrea Felgner – Market Segment Manager HYDRANAL
Thomas Wendt – Head of HYDRANAL Technical Service
Laboratory
If you have any questions or wish to discuss any
HYDRANAL matter, please don’t hesitate to contact us
at our general e-mail: [email protected], or on the
HYDRANAL helpline +49-5137-8238 353. We will be
happy to help you with technical inquiries related to
HYDRANAL reagents and KF titration.
Sample Recommendation
Determination with KF oven
ABS-Copolymer (Acrylonitrile-Butadiene-Styrene-Copolymer) (L313)
Plastic film (polyamide and ethylene vinyl alcohol) (L526)
Polyamide 66 (L167)
Polyethylene granulates (L193)
Polyethylene terephthalate
Polypropylene (L194)
Polyurethane granulate (L303)
PVC, plasticized (L204)
Determination with standard KF procedure
Methylmethacrylate
Poloxamer (L521)
Polyetherpolyol (L377)
Determination by addition of solubilizer
Acrylamide Copolymer Vol. KFT: mixture of Methanol dry and Formamide dry, and titration at increased temperature (40 °C) (L389)
Crospovidone Vol. KFT: Working Medium K (L472) or KF oven
Epoxy resin (mixture of bisphenol A
and epichlorohydrin, liquid)
Vol. KFT: LipoSolver CM or Solvent CM (L466)
Methylcyano acrylate Vol. KFT: Working Medium K with addition of Salicylic acid (L420)
Phenol-Urea-Formaldehyde-
Condensate
Vol. KFT: mixture of Methanol dry or Solvent and Formamide dry, and titration at increased temperature
(50 °C) (L130)
Polyacrylamide Copolymers Vol. KFT: mixture of Methanol dry and Formamide dry, and titration at increased temperature (50 °C) (L246)
Polycarbonate Vol. KFT: mixture of Working Medium K and trichloroethylene (L129) or KF oven (L127)
Polycarbonatediol Vol. KFT: LipoSolver CM, or Solvent CM, or Coulomat Oil (L533)
Polyisobutylene 900 Vol. KFT: Working Medium K (L002) or Coul. KFT: dissolve sample in Xylene before titration
Poly-L-lactate (PLLA/Polylactic acid) Vol. KFT: dissolve sample in chloroform before titration in LipoSolver CM or KF oven (L577)
Polymethyl methacrylate Vol. KFT: mixture of Methanol dry and Chloroform, and titration at increased temperature (50 °C) (L215)
Urea formaldehyde resin Vol. KFT: mixture of Solvent and Formamide dry, and titration at increased temperature (50 °C) or KF oven
Table 1 Recommended variations of volumetric (vol.) and coulometric (coul.) KF titration (KFT) for selected polymer and plastic samples
(for KF reagents refer to Table 3)
23
sigma-aldrich.com/hydranal
Tit
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on
Compound name Report No.
Caprolactam 238
Caprolactone 410
Chewing gum 308
Cold seal cover 423
Cyanoacrylate adhesives 118
Epoxy Styrol – Varnish 323
Organic plastic 401
Olefin copolymer in mineral oil 185
Plastics with the Karl Fischer oven 328
PLGA, mixture of Poly-lactate, gluconic acid and starch 475
Polyacrylate (liquid) with sodium silicate 253
Polyacrylic acid 266
Polyacrylic acid 314
Polyamide 126
Polyamide dissolved in NMP, Xylene and Cyclohexanol 226
Polybutene 188
Polycarbonate 127
Polyelectrolyte, high molecular I (PLEX 5372 E) 257
Polyelectrolyte, high molecular II (PLEX 5477 E) 256
Polyester film 125
Polyester Resin LP 24/23 for the Lime Industry 239
Polyethylene cross-linked 128
Polyethylene cross-linked 640
Polyethylene glycol-based Grease with additives 477
Polyethylene imine 522
Polymer comprising maleic and acrylic acid 199
Polymers, various (epoxy powder, polyamide 6,
polyamide 66, polybutadiene terephthalate)
174
Polypropylene filled 181
Polyurethane – chlorinated 124
Polyurethane component A for adhesives 119
Polyvinyl alcohol 490
Polyvinylacetate-based dispersion 278
Rubber, vulcanized 176
Thiokol 245
Table 2 More application reports for polymers and plastic
compounds
HYDRANAL® Seminars 2012
As a service to the scientifi c community, we routinely
offer seminars to provide training on the chemistry
behind the Karl Fischer technique and information
specific to the HYDRANAL product line. For 2012,
seminars have been scheduled in many cities around
the world. Please visit sigma-aldrich.com/events for
detailed information, schedule and registration.
Our annual two-day KF titration seminar in Seelze,
Germany, will be held February 7–8, 2012, featuring
expert speakers from a variety of fields, representa-
tives from leading instrument manufacturers, and
practical demonstrations. This seminar is a must for
any practicing KF analyst!
Cat. No. Description
Reagents for volumetric KF titration
34805 HYDRANAL-Composite 5 (one-component titrating agent)
34741 HYDRANAL-Methanol dry (one-component working
medium)
37817 HYDRANAL-Methanol Rapid (one-component working
medium)
37855 HYDRANAL-LipoSolver CM (one-component working
medium for titration in non-polar substances, fats and oils)
34817 HYDRANAL-Working Medium K (one-component working
medium for titration in aldehydes and ketones)
34801 HYDRANAL-Titrant 5 (two-component titrating agent)
34800 HYDRANAL-Solvent (two-component working medium)
34812 HYDRANAL-Solvent CM (two-component working
medium for titration in oils)
Reagents for coulometric KF titration
34739 HYDRANAL-Coulomat AG-Oven (anolyte solution, for cells
with and without diaphragm)
34868 HYDRANAL-Coulomat Oil (anolyte solution for titration in
oils, for cells with diaphragm)
34840 HYDRANAL-Coulomat CG (catholyte solution)
Standards for KF titration
34849 HYDRANAL-Water Standard 10.0 (liquid standard for
volumetric KF titration, 1 g contains 10.0 mg = 1.0% water,
exact value on CoA)
34828 HYDRANAL-Water Standard 1.0 (liquid standard for
coulometric KF titration, 1 g contains 1.0 mg = 0.1% water,
exact value on CoA)
34847 HYDRANAL-Water Standard 0.1 (liquid standard for
coulometric KF titration, 1 g contains 0.1 mg = 0.01%
water, exact value on CoA)
34748 HYDRANAL-Water Standard KF-Oven 220-230 °C (solid
standard for control of KF oven, 5.55 ± 0.05% water, exact
value on CoA)
34693 HYDRANAL-Water Standard KF-Oven 140-160 °C (solid
standard for control of KF oven, ~5% water, exact value
on CoA)
Auxiliaries
37863 HYDRANAL-Chloroform (solvent for KF titration)
34724 HYDRANAL-Formamide dry (solvent for KF titration)
37866 HYDRANAL-Xylene (solvent for KF titration)
37865 HYDRANAL-Salicylic acid (buffer substance for KF titration)
32035 HYDRANAL-Benzoic acid (buffer substance for KF titration)
37859 HYDRANAL-Buffer Base (buffer substance for KF titration)
34804 HYDRANAL-Buffer Acid (buffer substance for KF titration)
34241 HYDRANAL-Molecular sieve 0.3 nm (drying agent for air
and gases for KF titration)
Table 3 Selected HYDRANAL reagents (Fluka® brand)
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