Chapter 6

Additives: Smoke Flavorings

Dietrich Meier
Contents
6.1 Introduction ....................................................................................................................110
6.1.1 Smoking with Smoke Generators .......................................................................... 111
6.1.2 Smoking with Liquid Smoke................................................................................. 111
6.1.2.1 Development of Liquid Smoke Flavor ..................................................... 111
6.1.2.2 Manufacturing of Liquid Smoke Flavor ..................................................112
6.1.2.3 Legal Marketing Aspects of Liquid Smoke Flavor ...................................113
6.2 Direct Sampling of Components from Smoked Meat ......................................................114
6.2.1 Sampling of Smoke Flavorings ..............................................................................114
6.2.1.1 Headspace-Gas Chromatography ............................................................114
6.2.1.2 Solid-Phase Micro Extraction.................................................................. 115
6.2.2 Sampling of Polycyclic Aromatic Hydrocarbons ................................................... 115
6.3 Separation and Detection of Smoke Components............................................................117
6.3.1 Polycyclic Aromatic Hydrocarbons .......................................................................117
6.3.1.1 Gas Chromatography ..............................................................................117
6.3.1.2 High-Performance Liquid Chromatography............................................118
6.3.2 Aroma Constituents from Traditional Smoking .................................................... 119
6.3.2.1 Traditional Smoking ............................................................................... 119
6.3.3 Aroma Constituents from Liquid Smoke Flavorings .............................................119
6.3.3.1 Determination of Major Chemical Parameters in Liquid
Smoke Flavorings ....................................................................................119
6.3.3.2 Determination of Single Smoke Constituents .........................................121
References ............................................................................................................................... 126

109

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6.1 Introduction
Additives used in meat and meat products should be harmless to health. They serve solely to
inuence odor and avor, color and consistency, or shelf life. Without additives it would be
impossible to distribute many foods, as they either could not be made at all or would spoil too
quickly. Additives are grouped into those that are intended to be eaten along with the food and
those that are components of coverings on the surface of the food but are usually not eaten [1].
Apart from special additives in meat such as bactericide [2], curing agent [3], and tenderizer
[4], the application of smoke is the most frequently used additive for meat. Therefore, the analysis
of smoke components will be the focus of this chapter. Smoking, together with drying and salting,
is perhaps the oldest process to preserve foodstus. It has also been called mans rst spice [5].
The smoke components eects are germicidal and desiccating. They coagulate the proteins and
thus work to preserve. Moreover, they add aroma and color to the food, making it more attractive
for the consumer. In the course of the past 50 years, preservation has become less important.
Currently smoking of sh and meat is mostly done to enhance avor [6].
In principle, smoke used for meat is the result of the pyrolytic degradation of wood, which is
basically composed of two types of polymers: (1) polysaccharides (cellulose and hemicelluloses)
and (2) lignin. Each of these polymers gives a characteristic spectrum of pyrolytic products. The
degradation products of polysaccharides are mainly furans, acids, alcohols, anhydrosugars, esters,
and aldehydes, and are predominantly responsible for the staining and bactericidal eects of
smoke, whereas the phenolic lignin degradation products such as guaiacol, syringol, and derivatives are generally responsible for the typical smoky avor.
The most typical woods used in smoke generation are beach (Fagus sylvatica, a common wood
in Europe), hickory (Carya ovata, a common wood in United States and Canada), oak (Quercus
spp.), and maple (Acer spp.). Some other species such as cherry (Prunus spp.), apple (Malus spp.),
mesquite (Prosopis glandulosa), and pine (Pinus spp.) are used in minor amounts. The list indicates
thatwith the exception of pinewoodsmoke generation is based on hardwood pyrolysis. They
contain much less extractives than softwood, whose pyrolysis products might add undesirable
smell to the smoke avor. However, more important is the dierent lignin structure. Hardwood
lignins are polymers derived from a mixture of coniferyl alcohol (Figure 6.1) and sinapyl alcohol
(Figure 6.2), whereas softwood contains only moieties of the coniferyl-type. Furthermore, the
O
OH

HO

Figure 6.1

Lignin precursor coniferylalcohol.

O
OH

HO
O

Figure 6.2

Lignin precursor sinapylalcohol.

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superior sensory properties can be attributed to the phenolic sinapyl derivatives, which provide the
typical smoke aroma.
Regardless of the process of smoke generation, the basic smoke components are chemically very
similar. They dier mainly in their amount and their ratio due to the dierent technologies of smoke
generation. Processed meat can either be smoked directly with the help of dierent types of smoke
generators or by applying liquid smoke avorings, which are becoming more prevalent [711].

6.1.1 Smoking with Smoke Generators

Technical smoke generators for direct smoking mainly work according to three operation
principles:
Glowing
Friction
Superheated steam
Thus, the aroma can be controlled via temperature and type of wood. Furthermore, not only the
pyrolysis temperature but also the temperature of the vapors in contact with the meat is important, as the nal chemical composition and the amount of toxic polycyclicaromatic hydrocarbons
(PAHs) are determined by temperature.
The higher the temperature, the shorter is the exposition time of the food in the smoke. The
preserving eect is improved by leaving the foodstus for a longer time at low smoke temperatures,
as cold smoke penetrates more easily and more deeply into meat and sh.
With respect to temperature control, three smoking technologies are known [6]:
Cold smoking (smoke temperature in the range of 1525C), used for crude sausage, crude
ham, and salami
Warm smoking (smoke temperature in the range of 2550C), used for frankfurters
Hot smoking (smoke temperature in the range of 5085C), applied for cooked ham, eel,
mackerel, and halibut

6.1.2 Smoking with Liquid Smoke

6.1.2.1

Development of Liquid Smoke Flavor

The history of liquid smoke avors (LSFs), as they are used today, starts in the early 1970s,
although early treatments of meat with LSFs goes back to 1811 [12]. LSFs were rst applied in
the United States and Eastern Europe. Their application to meat products is through dipping,
spraying, or treatment with aerosols, similar to the treatment in traditional smokehouses. The
aerosol technology was rst applied by Hickory Specialities in 1969, and was the breakthrough
for producing LSFs [12]. The liquid smoke technology has made such progress that it has been
applied in many countries throughout the world. This is due to several reasons: ease of application,
speed, uniformity of the product, reproducibility of physical and chemical properties, and cleanliness of application [13]. In addition to these advantages, and the more ecient use of resources
involved, another important reason for using smoke avorings instead of smoke directly is the
fact that the amount of (known) toxic compounds found in smoke can be controlled before being

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added to the food [14]. A decrease of the content of certain PAHs has been observed [15,16];
according to Pszczola, PAHs are virtually absent [5].
It is important to note that LSFs not only can replace traditional smoking, but can be added
to various other foodstus such as soups, sauces, savories, cheese, and spices.

6.1.2.2

Manufacturing of Liquid Smoke Flavor

Liquid smoke avorings are solutions obtained from pyrolysis of wood. Wood is thermally degraded
in the absence of oxygen, and the vapors are condensed either in water or vegetable oils. On a global
basis, there are more than half a dozen of producers. Production plants operate pyrolysis reactors
in either continuous or batch mode. The exact process conditions are corporate secrets. The volatile
smoke constituents are continuously removed from the hot reaction zone and condensed in special
equipment. The raw products are divided into dierent classes according to their solubility in
water (Figure 6.3); water-soluble condensates are called primary smoke condensates. The waterinsoluble tarry phase is cleaned mostly by extraction and called primary tar fraction. Both fractions are rened through further process steps such as extraction, distillation, and concentration
by evaporation, absorption, or membrane ltration. During condensation other water-insoluble
oily products are formed that are not utilized.
Wood

Smoke
generation

Condensation

Water soluble

Water insoluble
tar phase

Water insoluble
oil phase
(discarded)

Purification and
fractionation

Primary smoke

Primary
tar fraction

Primary smoke products

Subject of EU
regulation

Refination by:
Distillation
Concentration
Adsorption
Membrane separation
Addition

Figure 6.3 Simplied diagram of the manufacture process for LSFs. The primary smoke
condensates are the subject of E.U. regulation 2065/2003. (From Meier, D., Fleischwirtschat
International, 4, 3740, 205. With permission.)

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The application of liquid smoke is more economic than the operation of smoke generators.
Liquid smoke is not only cheaper but also can be standardized and adopted to the needs of customers.
Thus, products can be manufactured with constant quality, taste, and uniform appearance.
Regulations for the use of LSFs in dierent countries vary considerably. For example, in Germany
freshly generated smoke is classied as a food additive. For toxicological reasons, only solid foodstus are allowed to be smoked. Also in Scandinavia a special regulation exists for smoke aromas.
All other countries in and outside of the European Union (E.U.) allow the unrestricted use of liquid
smoke, as long as the maximum content (10 g/kg) of 3,4-benzo[a]pyrene is not exceeded [6].
As more liquid smoke aroma penetrated the German market, the legal situation became
complicated, as many exceptions had to be put into force. This unsatisfactory situation is now
resolved by new E.U. regulation 2065/2003, which directs the authorization and characterization
of all smoke aromas. For the rst time, the Council of Europe and the European Scientic Committee have considered sanitary and toxicological aspects.

6.1.2.3

Legal Marketing Aspects of Liquid Smoke Flavor

There is a document available to give guidance to petitioners and other interested parties wishing
to introduce smoke-avoring primary products according to the European Parliament and
Council Regulation 2065/2003 of November 10, 2003 on smoke avorings used or intended for
use in or on foods (Ocial Journal of the European Union L 309, November 26, 2003, p. 1). It
gives guidance on the administrative and technical data required, on the range of toxicological
tests generally required for smoke avoring primary products, and on the format for formal
submissions (hereafter referred to as dossiers) to the competent authority of a member state for
further transmission to the European Food Safety Authority.
As stated in Regulation 2065/2003, the use of a primary product in or on foods shall only be
authorized if it is suciently demonstrated that it does not present risks to human health. A list
of primary products authorized to the exclusion of all others in the community for use in or on
food and for the production of derived smoke avorings shall therefore be established after the
authority has issued an opinion on each primary product. Following the establishment of this list,
all new applications need a favorable opinion by the authority for inclusion in the list.
According to the E.U. regulation, the primary product should be chemically characterized as
far as it is necessary to describe and dene its identity. For this purpose the following data should
be provided:
Information on the identity (name and Chemical Abstract Service [CAS] number) and the
concentration of the major chemical constituents of the primary product
Information on the concentration in the primary product of the PAHs listed in Annex 2
Information considered adequate to characterize and to recognize the primary product
(e.g., gas chromatograms, liquid chromatograms, mass spectra, and infrared spectra)
The solvent-free fraction (% m/m = weight %) in the primary product and how it is
determined
The volatile fraction (% m/m = weight %) in the primary product and how it is determined
Information on the fraction of unidentied constituents. (e.g., solid contents and proportions
of acids, phenols, and carbonyls)
Any other information on chemical composition considered to be relevant for evaluation of
the primary product

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The state of the art and development of legislation in Europe for PAH analysis is summarized by
Wenzl et al. [17].

6.2 Direct Sampling of Components from Smoked Meat

6.2.1 Sampling of Smoke Flavorings
6.2.1.1 Headspace-Gas Chromatography
The headspace (HS) is the gas space in a chromatography vial above the sample. HS analysis
is therefore the analysis of the components present in that gas. Headspace-gas chromatography
(HS-GC) is used for the analysis of volatile and semivolatile organics in solid, liquid, and gas
samples. Common applications include alcohol in blood, monomers in polymers and plastic,
avor compounds in beverages and food products, and fragrances in perfumes and cosmetics.
It is best suited for the analysis of the very light volatiles in samples that can be eciently
partitioned into the HS gas volume from the liquid or solid matrix sample. Higher boiling volatiles
and semivolatiles are not detectable with this technique, due to their low partition in the gas HS volume. HS analysis also lends itself to automation for quality control or sample screening. This is made
possible by modern instrumentation that can reproducibly prepare samples in an ecient manner.
Complex sample matrices, which would otherwise require sample extraction or preparation,
or be dicult to analyze directly, are ideal candidates for HS-GC, because they can be placed
directly in a vial with little or no preparation.
An HS sample is normally prepared in a vial containing the sample, the dilution solvent, a matrix
modier, and the HS. Volatile components from complex sample mixtures can be extracted from
nonvolatile sample components and isolated in the HS or gas portion of a sample vial. A sample of
the gas in the HS is injected into a GC system for separation of all the volatile components. Once the
sample phase is introduced into the vial and the vial is sealed, volatile components diuse into the
gas phase until the HS has reached a state of equilibrium. The sample is then taken from the HS.
Direct HS-GC of volatiles from smoked meat is not very common. Wittkowski et al. [18]
described a method using this technique combined with GC and ame ionization detector (FID).
Their procedure is as follows: The solid sample was placed in a 250-mL Erlenmeyer ask, and after
conditioning the sample for several minutes at room temperature, 5 mL of the HS volume was
injected with a special syringe into the GC. To prevent peak broadening, the rst section of the
GC column was immersed into a Dewar ask lled with liquid nitrogen.
Hierro et al. [19] used HS in combination with GC and mass spectrometry (GC-MS) for
the analysis of volatile components from salted and smoked dried meats. In general, 110 volatile
compounds were identied and quantied. The HS-GC method, rst described by Elmore et al.
[20], is as follows.

6.2.1.1.1

Sampling Technique

Twenty-ve grams of meat sample were introduced into a glass ask and equilibrated for 30 min
at 30C. Volatiles were extracted at 30C by a nitrogen ow of 40 mL/min for 1 h and adsorbed
on a steel trap (105 mm length, 3 mm inner diameter) containing 85 mg of Tenax TA. A standard
of 131 ng of 1,2-dichlorobenzene in 1 mL of methanol was added to the trap at the end of the collection, and excess solvent and any water retained on the trap were removed by purging the trap
with nitrogen at 40 mL/min for 5 min.

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6.2.1.1.2 Separation and Detection of Volatiles

Analyses were performed on a Hewlett-Packard 5972 mass spectrometer tted with a HP5890
Series II gas chromatograph and a G1034 Chemstation (Hewlett-Packard, Palo Alto, CA). A CHIS
injection port (Scientic Glass Engineering Ltd., Staordshire, U.K.) was used to thermally desorb
the volatiles from the Tenax trap onto the front of a CP-Sil 8 CB low bleed/mass spectrometry (MS)
fused silica capillary column (60 m, 0.25 mm inner diameter, 0.25 m lm thickness, Chrompack,
Middelburg, the Netherlands). During a desorption period of 5 min, volatile compounds were
cryofocused by immersing 15 cm of column adjacent to the heater in a solid CO2 bath while the
oven was held at 40C. The bath was then removed and chromatography achieved by holding at
40C for 2 min followed by a programmed rise to 280C at 4C/min and holding for 5 min. A series
of n-alkanes (C6C22) (Sigma-Aldirch Inc., St. Louis, Missouri) was analyzed under the same
conditions to obtain linear retention index values for the aroma components. The mass spectrometer was operated in electron impact (EI) mode with an electron energy of 70 eV and an emission
current of 50 mA. Approximate quantities of the volatiles were estimated by comparing their peak
areas with those of the 1,2-dichlorobenzene internal standard (IS), obtained from the total ion
chromatograms, using a response factor of 1.

6.2.1.2 Solid-Phase Micro Extraction

Solid-phase micro extraction (SPME) is an innovative, solvent-free technology that is fast, economical,
and versatile. SPME is a ber coated with a liquid (polymer), a solid (sorbent), or a combination of
both. The ber coating removes the compounds from the sample by absorption in the case of liquid
coatings or adsorption in the case of solid coatings. The SPME ber is then inserted directly into
the hot injector of a gas chromatograph for desorption and analysis. SPME has gained widespread
acceptance as the technique of preference for many applications, including avors and fragrances,
forensics and toxicology, environmental and biological matrices, and product testing.

6.2.1.2.1 Sampling Technique

In principle the meat sample is places in a vial and sealed with a septum. The piercing needle of the
SPME device (Supelco Inc., Bellefonte, PA) is drilled through the septum, and the needle is inserted
into the vial. The needle tip is adjusted in such a way that the ber does not have contact with the
meat when the plunger is extended to expose the SPME ber into the HS. Now sampling time starts,
and after a certain time the ber is retracted into the needle, the manifold is removed from the vial,
and the needle is introduced into the hot injector of a gas chromatograph to desorb the volatiles.
Several papers have been published using SPME for volatile constituents of cooked meat
[2123] or contaminants, and even for the analysis of PAHs [2428]. Volatiles from wood pyrolysis
liquids (bio-oils) have been analyzed by Meier using SPME-GC-MS [29]. Although the sampling
technique is rather simple, there is no information in the literature on the use of SPME for the
analysis of smoke aroma components. More general information, with a practical SPME applications guide, is available from Supelco (www.sigma-aldrich.com/supelco).

6.2.2 Sampling of Polycyclic Aromatic Hydrocarbons

PAHs are inevitably formed during the smoke generation process, regardless of the pyrolysis
technology. Therefore, their determination is of paramount importance to fulll the legislative
requirements.

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The major problems associated with the sampling of PAHs in food are
Only trace amounts available
Coextraction of PAH-like materials
Chen [30] and imko [31] give a comprehensive overview. From the analytical point of view, meat
is regarded as a problematic matrix, and the sampling procedure is an important step in the entire
analysis sequence. imko compiled a comprehensive list of methods used for extracting PAHs
from meat products. Sample preparation includes three basic steps: saponication, extraction, and
cleanup. Typical procedures are as follows:
Saponication with mixture of ethanol, water, and KOH, extraction with cyclohexane,
preseparation by solid-phase extraction (SPE) on isolute aminopropyl and C18 columns
Saponication in methanolic KOH, liquidliquid extraction (methanolwatercyclohexane
and N, N-Dimethylforamamide [DMF]watercyclohexane), precleaning on silica gel, and
gel permeation chromatography (GPC) on Sephadex LH 20
Saponication with a mixture of methanol, water, and KOH, partition with DMF,
precleaning on Kieselgel 60
Recently, Jira [32] presented a practical method described in detail for the extraction of PAHs
from smoked meat products. The procedure is as follows:
1. Accelerated solvent extraction (ASE). About 68 g of the homogenized meat product is
levigated with the same amount of the drying material polyacrylic acid and partial sodium
salt-graft-polyethylene oxide. The resulting material is placed into a 33-mL cell with micro
glass ber lters at the outlet of the extraction cell. Then 500 L of the 13C-PAH standard
mixture is added as IS. The extraction was performed with an ASE 200 (Dionex, Sunnyvale,
CA) using n-hexane at 100C and 100 bar with a static time of 10 min. The ush volume
was 60%, and the purge time was 120 s. After two static cycles, the solvent is evaporated
with a nitrogen steam, with the sample placed in a water bath at 40C.
2. GPC. The evaporated ASE-extract is dissolved in 4.5 mL cyclohexane/ethylacetate (50:50
v/v) and ltered through a polytetrauoroethylene (PTFE) lter with a pore size of 1 m.
The GPC column is lled with Bio-Beads S-X3 (lling height 42 cm). Samples are eluted at
a ow rate of 5 mL/min, with the same solvent used for sample dissolution. Dump time was
036 min; collection time was 3665 min. Solvent is removed with a rotary evaporator, and
the residual solvent is removed in a nitrogen stream.
3. Column chromatography with silica gel. The dried GPC eluate is dissolved in 1 mL n-hexane.
The silica gel column (7 mm inner diameter) is lled with 2.5 g silica gel (15% deactivated
with water). Samples are eluted with 30 mL n-hexane/dichloromethane (80:20 v/v). Solvent
is removed with a rotary evaporator, and the eluate is dried in a nitrogen stream.
4. SPE. For complex matrices such as liquid smoke, the dried eluate from the silica gel
column is dissolved in 1 mL n-hexane and applied onto a conditioned cyano (CN) SPE
cartridge (500/1000 mg bed material). The SPE cartridge is rst rinsed with 2 mL n-hexane,
and the eluate is discarded. The PAH fraction is eluted with 5 mL acetonitrile/toluene
(75:25 v/v). Solvent is removed with a rotary evaporator, and the eluate is dried in a nitrogen
stream. The sample is now ready for GC-MS analysis.

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6.3 Separation and Detection of Smoke Components

6.3.1

Polycyclic Aromatic Hydrocarbons

Separation of the PAHs can be accomplished by GC separation followed by detection with a

mass spectrometer or after separation with high-performance liquid chromatography (HPLC) and
detection with a uorescence detector (FLD).

6.3.1.1

Gas Chromatography

Several methods for PAH analysis in smoke or smoke avorings have been described in the literature [15,16,3336]. However, none of these methods have been validated or cover all 15 priority
E.U.-PAHs [10]. In the E.U., the maximum allowed concentration of benzo[a]pyrene (BaP) in
food treated with smoke avors was set to 0.03 g/kg in 1998, and in November 2003, a new
regulation was adopted by the European Council and Parliament (No. 2065/2003) to control
smoke avors at the point of production [37]. The regulation denes the aqueous part as primary
smoke condensate (PSC) and the puried tar extract of condensed wood smoke as primary tar
extract. Both fractions are considered to be primary products to be used for the manufacture of
derived smoke avors. For these primary products, the European regulation established maximum
permitted concentrations of 10 g/kg for BaP and 20 g/kg for benzo[a]anthracene. Furthermore,
the regulation determines that only validated analytical methods can be used for the verication
of these limits. To support the implementation of legislation, the method described here was developed and validated in a single-laboratory approach according to the IUPAC harmonized guideline
[38] to enable the quantication of the E.U.-PAHs in PSC.
The method for sample preparation recently described by Simon et al. [37] is described in the
following section. Further details on the instrumentation GC-MS conditions and data evaluation
can be found in Section 6.3.1.1.1 [37].

6.3.1.1.1 Sample Preparation

Ten grams of the liquid sample and 100 L of IS solution (50 g/L of each labeled standard) in
2-propanol were combined in a 250-mL round-bottom ask. To this mixture, 3.2 g solid potassium
hydroxide and 32 mL methanol were added. The solution was reuxed for 30 min. The sample
was extracted three times with 25 mL cyclohexane by liquid/liquid partitioning. The organic
phases were combined in a round-bottom ask and dried with anhydrous sodium sulfate. After
evaporation of the solvent under reduced pressure, the sample was reconstituted in 1 mL cyclohexane. For removal of residual interferences, the extract was passed through a silica gel SPE tube
(Supelclean, 3 mL, 0.5 g) and eluted with 7 mL cyclohexane. After evaporation of the eluate and
reconstitution of the residue with 1 mL n-hexane, 1 L of the solution was analyzed by GC-MS.
A drawback of the GC-MS method is its incapacity to resolve the two isomers benzo[j]
uoranthene and benzo[k]uoranthene.
A further detailed description is given by Jira [32], who suggested the following procedure for
GC-MS analysis:
1. Preparation for GC-MS analysis. The dried eluate of the silica gel column (or optional, the
eluate of the CN-SPE cartridge) is dissolved in 500 L of the deuterated PAH standard
mixture in isooctane and transferred to a 1-mL tapered vial. The sample is then carefully
concentrated to a volume of approximately 50 L in a nitrogen stream.

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2. GC. The GC is as follows: capillary column dimensions are 60 m 0.25 mm inner diameter, 0.25 m lm thickness; lm is cross-linked 5% phenylmethylsiloxane. Helium is used
as carrier gas; injection temperature is 300C, and injection volume is 1 L (splitless). The
following oven program is used: isothermal at 80C for 1 min, rising by 20C/min to 100C,
by 8C/min to 130C, and by 5C/min to 320C, then isothermal at 320C for 10 min.
3. MS. The MS analysis is performed with a high-resolution mass spectrometer working in the
EI positive mode, using an electron energy of 35 eV. Transfer line and ion source temperature
are kept at 280 and 250C, respectively. Selected ion monitoring is used for acquisition of
spectral data.

6.3.1.2

High-Performance Liquid Chromatography

The determination of the most toxic PAH BaP in liquid smoke by liquid chromatography was
demonstrated in 1992 [39]. Owing to the incapacity of the GC-MS method to resolve the two
isomeric PAHs benzo[j]uoranthene and benzo[k]uoranthene and the need of having a second
validated method in case of legal conicts, a second method based on HPLC with ultraviolet
adsorption and uorescence detection for the separation and determination of the 15 priority
E.U.-PAHs was developed at the Joint Research Centre of the European Commission [40].

6.3.1.2.1 Sample Preparation for High-Performance Liquid Chromatography

Ten grams of the liquid smoke condensate were reuxed for 30 min with alkaline methanol
(3.2 g of potassium hydroxide in 32 mL of methanol) to saponify interfering compounds and ionize
weak acids, for example, phenol. The analytes were extracted from the methanolic solution three
times with 25 mL of cyclohexane each. The organic phases were pooled, and the aqueous phase was
discarded. The organic phase was dried with sucient anhydrous sodium sulfate. The organic phase
was removed by rotary evaporation under reduced pressure (T = 40, p = 100 mbar) to dryness. The
sample was reconstituted with 500 L of cyclohexane and transferred onto a silica cartridge activated
with 2 mL of cyclohexane. The ask was rinsed with a second 500 L of fresh cyclohexane, which
was also transferred onto the cartridge. The rst milliliter of the eluate was discarded. The analytes
were eluted with 7 mL of cyclohexane. The eluate was now collected, and the solvent was removed
under reduced pressure as mentioned earlier. The nearly colorless sample was redissolved in 1 mL
acetonitrile by vortexing for 1 min and transferred to a capped amber 2-mL autosampler vial.

6.3.1.2.2 Analysis and Instrument Conditions

A 20-L aliquot was injected into an HPLC (1100 series, Agilent, Waldbronn, Germany) system
equipped with autosampler, quaternary pump, thermostated column oven (T = 40) and FLD
(G1321A). For the separation a Pinacle II reversed-phase column for PAHs, 250 2.1 mm,
5-m particle size (Restek GmbH, Bad Homburg, Germany) was used. The ow of the aqueous
mobile phase (acetonitrile/water) was set to 0.3 mL/min. The gradient program for the mobile
phase started with 80% acetonitrile (0 min) changing linearly to 85% (30 min) and 100%
(40 min). After 60 min, the mobile phase was reset to the initial composition within 10 min and
allowed to equilibrate for another 10 min. Total runtime of one analysis was 80 min. The analytes
were detected and quantied by monitoring the UV absorbance at 375 nm and the uorescence

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emissions simultaneously at 370, 420, 470, and 500 nm with one common excitation wavelength
of 270 nm. Details on data evaluation can be found in Ref. 40.

6.3.2 Aroma Constituents from Traditional Smoking

The analysis of smoke components is a very dicult task due to the complexity of the pyrolysis
products [12,41]. Smoke composition can be studied either using condensed smoke from traditional smoke generators or by analyzing commercial, modern LSFs.

6.3.2.1

Traditional Smoking

Basically, saw dust from beech wood is used as feedstock. whereas in the United States hickory
wood is preferred. Alder or oak may be added to beech to get a darker color. Moreover, juniper
berries, herbs, pine needles, and r cones may be used to enhance the smell and taste. Probably,
each smoke generation company has its own recipe and individual taste.
Analysis of smoke preparations from traditional smoking processes were already described by
Fujimaki et al. [42] and Kim et al. [43] in the early 1970s. A review of main smoke components
and the chemistry of smoked foods was presented by Gilbert and Knowles [44]. Very detailed
studies on the phenolic fraction of preparative smoke samples obtained from a laboratory smoke
generator were presented by Tth et al. [4547].

6.3.3 Aroma Constituents from Liquid Smoke Flavorings

6.3.3.1

Determination of Major Chemical Parameters

in Liquid Smoke Flavorings

Major chemical parameters are often needed for the description of typical chemical and physical
properties. These data are useful for an application for product authorization in the E.U. and for
the technical data sheet of a liquid smoke product. There are no public detailed data available,
as most of these methods described in the following sections are so-called house methods.
Therefore, only the basic principles are explained.

6.3.3.1.1 Water
Water in LSFs should not be determined by distillation, as many other components form an
azeotropic mixture with water. It is recommended to determine the water content in LSFs by
Karl-Fischer titration. There are ready-to-use solutions, such as Hydranal Composite 2 from Riedel
de Han. The solution for the titration consists of iodine, sulfur dioxide, pyridine, and methanol
in the ratio 1:3:10:50. The detection is based on the oxidation of sulfur dioxide with iodine.
During the reaction, water is consumed. The following chemical reactions are involved:
SO2 C5 H5 N C5 H5 N SO2
C5 H5 N I2 H2 O C5 H5 NH IOH I

basic reactions during

IOH C5 H5 N SO2 C5 H5 N C5 H5 N SO3 C5 H5 NH I

Karl Fischer titration

C5 H5 N SO3CH3OH C5 H5 N

CH3OSO3

(6.1)

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Accordingly, the gross reaction is as follows:

3C5 H5 N I2 SO2 CH3OH H2 O 2C5 H5 NHI
C5 H5 NH CH3OSO3

gross reaction of
Karl Fischer titration

(6.2)

The endpoint of the titration is determined potentiometrically by dead-stop indication.

6.3.3.1.2 pH
The pH can simply be determined with a standard pH meter.

6.3.3.1.3 Acids
Acids are measured as titratable acidity calculated as acetic acid. Liquid smoke contains a wide variety
of organic acids. The total content of organic acids can be determined potentiometrically by titration
with sodium hydroxide to pH 7. Generally, a standardized 0.1 M NaOH solution is used. The consumption of NaOH in 1 mL is equivalent to the acid number, and the reporting is as acetic acid.

6.3.3.1.4 Phenols
There are several colorimetric methods for the determination of phenols:
1. Gibbs method. The sample is mixed with Gibbs reagent (2,6-dichloro-p-benzoquinone 4chloroimine). The reagent adds to the para position of phenol derivatives to give indophenol,
a blue color.
2. Modied Gibbs method. Phenols are calculated as 2,6-dimethoxyphenol. It is determined by
means of 2,6 dibromoquinone-4-chlorimide. The reagent reacts with phenols to produce a
magenta color. The extinction is measured at 590610 nm.
3. Emersons method. Phenols also react with 4-aminoantipyrine (Emersons reagent) in alkaline
solutions to give a red color. Potassium ferricyanide is used as alkaline oxidant.

6.3.3.1.5

Carbonyls

A widely used method for total carbonyl determination is the hydroxylamine hydrochloride
method, which was used as early as 1895 and has been improved by using pyridine as the oximation
catalyst for quantitative determination of pure compounds. Hydrochloric acid is titrated potentiometrically with NaOH solution to pH 2.90. The total carbonyl content is reported as 2-butanone.
The methods for overall characterization are not very specic and thus give only approximate
results of the sample composition. Hence, the results can only be used for comparing dierent
samples.

6.3.3.1.6 Staining Index

The color-forming potential of carbonyl compounds from liquid smoke solutions with selected
amino acids can be determined with a colorimetric procedure [48]. A common amino acid is
glycin, which gives in an acidic environment a typical color that is determined at 440 nm.

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6.3.3.2

121

Determination of Single Smoke Constituents

6.3.3.2.1 Direct Determination by Gas Chromatography (Dilute and Shoot)

For the determination of single constituents, high-resolution capillary gas chromatography
is necessary. Based on the legislative requirements of the E.U., a practical method is helpful to
determine as many smoke components as possible. A reliable method, which is already used in
LSF analysis, is based on the dilute and shoot principle [6]. In this method, the complete liquid
smoke product is dissolved with acetone containing uoranthene as IS. The solution is injected into
a GC system equipped with both a mass spectrometer and a ame ionization detector. Example
chromatograms of three dierent LSF formulations are shown in Figure 6.4
Special attention should be paid to the selection of the capillary column. The liquid smoke
components have a wide range of boiling points and a wide range of dierent polarities ranging
from strong and week acids (acetic acid and phenols, respectively) to neutral compounds such as
alcohols, aldehydes, lactones, and anhydrosugars.
A suitable lm phase for the dilute and shoot method is 14% cyano-propyl-phenyl86%
dimethylpolysiloxane (GC phase number 1701). This semipolar lm has been successfully used to
separate both carbohydrate-derived [49] and lignin-derived pyrolysis products [50,51].
The column end is connected to a T-piece which splits the column ow in a ratio of 1:1. One
part goes into the MS, the other part into the FID, so that the generated mass spectra can be used
for identication and the FID signal for quantitation. A collection of mass spectra from wood
carbohydrates and lignin can be found in Refs 5255.
The sample preparation is as follows:
LSF is dissolved with acetone containing IS uoranthene (200 g/mL). The concentration of
the LSF, based on the organic part, should be approximately 5%. If necessary, lter the solution through a 0.45-m lter.
The GC conditions are as follows:
Injection. Split injection, split ratio 30:1; temperature 250C, constant ow 2 mL, 226 kPa
helium pressure
Oven. 45C constant for 4 min, heating rate 3C/min to 280C, hold for 20 min
Detector. FID, 280C, column: 60 m 0.25 mm, 0.25 m lm thickness, phase composition:
14% cyanopropyl-phenyl86% dimethylpolysiloxane (1701)
The MS conditions are as follows:
Ionization with EI at 70 eV
Ion source temperature 230C
Scan range 33400
Table 6.1 shows the complete list of constituents, which can be determined by the dilute
and shoot method. For quantication, the components should be calibrated according to the IS
method. For this purpose, the response factor of each compound versus the detector response of
the IS has to be determined. In this case, uoranthene is used as IS. The relative response factor
(RRF) is calculated as follows:
RRF

amount sample
area IS

area sample
amount IS

(6.3)

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7.50e5

5.00e5

2.50e5

20.00

40.00

60.00

min.

40.00

60.00

min.

40.00

60.00

min.

(a)

1.25e6

1.00e6

0.75e6

0.50e6

0.25e6

20.00
(b)
6.25e5

5.00e5

3.75e5

2.50e5

1.25e5

20.00
(c)

Figure 6.4 Gas chromatograms of different liquid smoke samples: (a) typical nontreated
sample, (b) product enriched in phenols, and (c) product enriched in browning compounds.

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123

Table 6.1 Chemical Constituents in Liquid Smoke Samples as Determined by the Dilute
and Shoot Method, Relative Retention Times (RRT) Based on Fluoranthene
Compound

RRT

CAS-No.

Butandione, 2,3-(diacetyl)
Propanoic acid methylester
Acetaldehyde, hydroxyAcetic acid
Acetol; (hydroxypropanone)
Ethene, 1,2-dihydroxyAcetoin; (butanone-2, 3-hydroxy-)
Propanoic acid
Ethyleneglycol
Cyclopentanone
Butanone, 1-hydroxy-2Propionaldehyde, 3-hydroxy
Furanone, 2(3H)Butanoic acid, 2-propenyl ester
Butenoic acid
Tetrahydrofuran, 2,5-dimethoxy-trans
Cyclopenten-1-one, 2Furaldehyde, 2-; (2-furfural)
Furfuryl alcohol, 2Acetyloxypropane-2-one, 1Cyclopentene-1-one, 2-methyl-2Furan, 2-acethylCyclopenten-1-one, 2-hydroxy-2Furaldehyde, 5-methyl-2Cyclopenten-1-one, 3-methyl-2Butyrolactone, gammaFuranone, 2(5H)Cyclopenten-one, dimethyl- (not 4,4- or 2,3-)
Furan-2-one, 5-methyl-(5H)Pyran-4-one, 3-hydroxy-5,6-dihydro-(4H)Cyclopenten-1-one, 2,3-dimethyl-2Cyclopenten-1-one, 2-hydroxy-3-methyl-2Furan-2-one, 2,5-dihydro-3,5-dimethylFuranone, 3-methyl-2(5H)Cyclopenten-one, trimethylCyclopentanone, dimethylFuran-2-one, 2,5-dihydro-3,5-dimethylPhenol
Cyclopenten-one, trimethylGuaiacol
Cyclopenten-one, trimethylCyclopenten-1-one, 3-ethyl-2Cyclopenten-one, trimethylCresol, oCyclopenten-1-one, 3-ethyl-2-hydroxy-2Cyclopenten-one, trimethyl-

0.084
0.086
0.105
0.115
0.136
0.147
0.159
0.174
0.193
0.198
0.201
0.204
0.215
0.228
0.237
0.243
0.245
0.247
0.278
0.283
0.287
0.299
0.328
0.349
0.359
0.361
0.367
0.369
0.378
0.386
0.398
0.399
0.400
0.400
0.405
0.409
0.409
0.423
0.432
0.435
0.438
0.441
0.448
0.458
0.460
0.464

431-03-8
554-12-1
141-46-8
64-19-7
116-09-6
513-86-0
79-09-4
107-21-1
120-92-3
5077-67-8
2082-571-2

930-30-3
98-01-1
98-00-0
592-20-1
1120-73-6
1192-62-7
620-02-0
2758-18-1
96-48-0
497-23-4
591-11-17
1121-05-7
80-71-7
22122-36-7

108-95-2
90-05-1
568-26-99
95-48-7
21835-01-8
(Continued)

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Table 6.1

(Continued)

Compound

RRT

CAS-No.

Phenol, 2,6-dimethyl(5H)-Furan-2-one, 4-methylCresol, pCresol, mGuaiacol, 3-methylLacton derivative (base mass 85)
(5H)-Furan-2-one, dimethylGuaiacol, 4-methyl
Phenol, 2-ethylAnhydrosugar (unknown)
Phenol, 2,4-dimethylPhenol, 2,5-dimethylToluene, 3,4-dimethoxy
Phenol, 2,4,6-trimethylPhenol, 2,3-dimethylPhenol, 3,5-dimethylPhenol, 4-ethylPhenol, 3-ethylGuaiacol, 3-ethylPhenol, 3,4-dimethylGuaiacol, 4-ethylPhenol, (2,3,4- or 2,4,5-)trimethylAnhydrosugar (unknown)
Anhydrosugar (unknown)
Dianhydro--D-glucopyranose, 1,4:3,6Phenol, 4-propylGuaiacol, 4-vinylGuaiacol, 4-allyl-; (eugenol)
Guaiacol, 4-propylFuraldehyde, 5-(hydroxymethyl)-2Lactone derivative
Syringol
Furanone, dihydro-4-hydroxy-2(3H)Guaiacol, 4-propenyl-; (Isoeugenol) cis
Anhydro--D-xylofuranose, 1,5Guaiacol, 4-propenyl-; (Isoeugenol) trans
Syringol, 4-methylVanillin
Hydroquinone
Syringol, 3-ethylBenzene, dihydroxy-methylSyringol, 4-ethylDeoxysugar (unknown, unspecic spectrum)
Acetoguaiacone; (Phenylethanone, 4-hydroxy-3-methoxy-)
Anhydrosugar or deoxysugar (unknown)
Deoxysugar (unknown)
Syringol, 4-vinyl-

0.469
0.478
0.482
0.483
0.485
0.487
0.495
0.506
0.509
0.510
0.514
0.515
0.522
0.528
0.537
0.539
0.541
0.542
0.549
0.557
0.560
0.568
0.570
0.573
0.583
0.596
0.597
0.612
0.613
0.620
0.627
0.630
0.639
0.646
0.673
0.678
0.684
0.688
0.692
0.702
0.716
0.725
0.726
0.735
0.741
0.743
0.758

576-26-1
106-44-5
108-39-4

93-51-6
90-00-6
105-67-9
95-87-4
494-99-5
527-60-6
526-75-0
108-68-9
123-07-9
620-17-7
95-65-8
2785-89-9

645-56-7
97-53-0
67-47-0
91-10-1
5469-16-9
97-54-1
5932-68-3
121-33-5
123-31-9

498-02-2

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Table 6.1

125

(Continued)

Compound

RRT

CAS-No.

Guaiacyl acetone
Syringol, 4-allylSyringol, 4-propylSyringol, 4-(1-propenyl)-cis
Levoclucosan; (Anhydro--D-glucopyranose)
Syringol, 4-(1-propenyl)- trans
Dihydroconiferyl alcohol
Syringaldehyde
Homosyringaldehyde
Acetosyringone
Anhydro--D-glucofuranose, 1,6- or galactofuranose, 1,6Syringyl acetone
Propiosyringone
Isomer of sinapyl alcohol
Dihydrosinapyl alcohol
Fluoranthene = IS

0.762
0.767
0.768
0.796
0.822
0.828
0.830
0.841
0.863
0.875
0.892
0.895
0.912
0.921
0.958
1000

2503-46-0
6627-88-9
627-88-9
498-07-7

134-96-3
2478-38-8

206-44-0

As not every constituent is commercially available, the response of chemically similar components
can be estimated from known constituents. Once all response factors are collected, quantitation
of the sample can start, using the following formula:
Weight (mg)

area sample RRF

amount IS (mg )
area IS

(6.4)

In general, 8595% of the chromatogram arearepresenting the volatile fractioncan be

determined using this method. It must be emphasized that the detectable part of the LSF depends
on the amount of oligomeric components left in the sample. These components are derived from
lignin and represent the pyrolytic lignin, which is an integral part of all crude pyrolytic liquids
from wood. Depending on the production process of the LSF, the pyrolytic lignin portion can be
in the range of 15 wt% for crude primary smoke products.

6.3.3.2.2 LiquidLiquid Extraction

Because of the wide polarity of the LSF components, liquidliquid extraction methods have been
used to separate chemical groups such as acids, phenolics, carbonyls, basic, and neutral fractions
[42]. Extraction techniques are useful to facilitate chromatographic separation and to get a closer
look into components with low concentrations. This technique has also been used for the separation
of other pyrolysis liquids from wood [56]. Other approaches use only a water-immiscible solvent
such as methylene chloride to analyze the organic [57] and the aqueous fraction [5860]. As a
consequence, Guilln and coworkers could separate nitrogenated as well as dimeric and trimeric
lignin-derived products. Separation and detection of the compounds is by GC-MS. Details can be
found in the corresponding literature.

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Handbook of Processed Meats and Poultry Analysis

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