analytix 5/2011

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

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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

[email protected]

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

sigma-aldrich.com/analytix

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sigma-aldrich.com/analytix

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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sigma-aldrich.com/fooddyes

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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]

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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



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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sigma-aldrich.com/microbiology

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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

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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


Gaby-Hadley B

Presence of oxidase


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)


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


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


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


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

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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

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15

sigma-aldrich.com/lc-ms

Ch

rom

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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

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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


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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


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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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RTC is now a member of the Sigma-Aldrich family

Request the new catalog now!

Proficiency Testing & Quality Control Standards

Natural Matrix CRMs

Microbiology Standards

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A2LA accredited Proficiency Test Provider (cert# 2122.01)

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ACLASS accredited ISO/IEC 17043:2010 Proficiency Testing Provider (cert# AP-1469)

ISO 9001:2000 Registered Company

EPA I.D. Number WYD988870218

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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For more information and an up-to-date product list,

please visit our website at sigma-aldrich.com/organiccrm

21

sigma-aldrich.com/hydranal

Tit

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on

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]


22


Tit

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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


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)


coulometric KF titration, 1 g contains 1.0 mg = 0.1% water,

exact value on CoA)


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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