abilinski

Alex Bilinski Log

**Beer Flavor Compounds and Detection Methods ** //Alex Bilinski //  Beer is a beverage that has a deep history and many trusted traditions, both in its manufacture and consumption. Beer processing is influenced by the culture of many different peoples. Across many continents some form of beer is produced using very similar processes. These many processes, although similar, provide beer with many distinct flavors and aromas. In order to characterize beer flavor it is necessary to determine the chemical compounds responsible. This task is not often a simple one due to beer’s complex nature, but many techniques have been used to develop appropriate methods of detection.

The origins of beer can be traced as far back as the fourth century B.C. (9). Ancient Sumerians were one of the first peoples to engage in the brewing process. Early clay pottery from this era was found containing calcium oxalate, a known precipitant formed during the brewing stages (9). This chemical evidence was a clue that led researchers to determine beer was a major part of these ancient times. It is also evident that early Egyptians had their own form of beer. It is often believed that beer and bread were the two most important nutritional contributors to these peoples (9). It is even believed that beer played a major role in changing the lifestyle of these times from a nomadic hunter-gathering to one based on farming and agriculture.

Brewing itself can be broken down into different stages. Starting with the barley grain, beer undergoes malting. Malting is the process by which the barley is steeped in water, allowed to germinate, and then heated for a period of time. During steeping the water content of the barley is raised and the cell walls of the barley grain are broken down. Insoluble proteins are also removed in order to allow fermentation enzymes access to the starchy endosperm. During germination the moist grain is held at 16C for three to four days. During this time enzymes are produced. Most importantly alpha-amylase and beta-amylase, which can attack the alpha-1,4-glucosidic and 1,6-glucosidic bonds of the starch molecule. Kilning then takes place in order to stop these enzymes from further activity, and make a stable product for storage until further brewing.

The next step in the brewing process is mashing. The dried malt is mixed with hot, 65C, water. This temperature is chosen since it is the temperature at which the starch will gelatinize (9). This makes the starch much more likely to undergo attack from the enzymes in the malt. After the enzymes convert the starch to soluble sugars, a sweet syrupy liquid called wort is produced. This “sweet wort” contains mostly maltose and glucose, as well as some larger dextrins. The sugars are ready to be fermented in this form. At this point hops are added and the wort is boiled. Boiling the wort has many effects. First it sterilizes the system. It also causes proteins to coagulate so that they can be filtered off. Perhaps the most important aspect as far as flavor is concerned of this boiling is the extraction of alpha-acids from the hops into the wort. Further reactions of these acids provide the characteristic bitter flavor of beer.

Fermentation is now ready to take place. Yeast is added to the bitter wort and the soluble sugars are converted to carbon dioxide and ethanol. Amino acids from the wort are also taken up by the yeast in order to aid in cell growth of the yeast itself. Along with carbon dioxide and ethanol, many flavor active compounds are also produced during fermentation. These compounds will differ based upon the strain of yeast used, hence the reason for such a variety of beer flavors. Many brewers have been using the same strain of yeast for decades in order to provide consistent flavor (10).

The final step of beer production is maturation. The “green beer” produced from fermentation still contains many unwanted flavor compounds. These volatile compounds need to be removed before the beer can be shipped to consumers. There are two major ways in which these compounds are removed during maturation, both of which use yeast. Leftover yeast from the fermentation stage has the ability to produce a small amount of carbon dioxide. This extra carbon dioxide can act as a carrier for the unwanted volatile compounds and purge the beer of off-flavors. The other method by which yeast removes unwanted flavor compounds is through chemical reactions. The yeast can act as a catalyst to reduce carbonyl compounds and render them irrelevant as far as flavor is concerned. Once maturation is complete the beer will have its characteristic flavors and aromas.

Although, by definition, the general process of making beer is the same no matter where it is made, the individual nuances of each brewer can cause significant variations in flavor. Obviously the main flavors that are thought of when discussing beer are alcohol, bitter, and cereal flavors. There is also the effect of carbonation on the mouth feel of beer (9). These are important; however, there are many other flavor active compounds in beer.

One of the areas in brewing that can vary highly is the source of the yeast used during fermentation. Each source of yeast has its own reactivity and produces different products during fermentation. Some of the most important types of products formed by the yeast fermentation are higher alcohols (i.e. higher molecular weight than ethanol). These compounds include such alcohols as 2-methyl-1-propanol and 3-methyl-1-butanol (10). The pathway by which these alcohols are formed is shown in figure 1. The type of yeast, and thus type of beer, dictates the formation of these alcohols. Smaller strains, typically found in ale brewing, produce more alcohols than larger strains used in other types of beers (10).



//Figure 1: Higher Alcohol Production in Beer from Yeast. (10) //

 Alcohols typically give strong warming “alcoholic” notes in beer. They can also give some varied flavor profiles depending on chemical nature of the compound present. Ethanol, the most abundant alcohol in beer at 20,000 to 80,000 mg/L, gives very strong alcoholic/solvent like notes (10). This differs from 3-methylbutanol (amyl alcohol), which is present at 30 to 70 mg/L. This alcohol also gives some alcoholic notes, but also gives vinous and banana like flavor notes as well. Amyl alcohol also affects the drinkability of the beer itself. As the concentration of this alcohol increases, the beer is perceived to be “heavier” than beer with low concentration of amyl alcohol (14). The production of these higher alcohols is very important to the flavor of beer. Not only do the alcohols themselves provide flavor, they are then converted to their subsequent esters which are very flavor active compounds.

Esters are well known in foods, as well as in general chemistry, as having distinct odors and flavors. In beer this is no different. As previously mentioned, esters are a byproduct of fermentation caused by the reaction of an alcohol with a carboxylic acid. The general reaction for the formation of esters is shown in figure 2. In beer, esterification during yeast fermentation follows a similar pathway as the higher alcohols. However carboxylic acids formed from the pyruvate molecule react with these alcohols to form the esters (6). There are two main esters that brewers are typically concerned with. These are ethyl acetate and iso-amyl acetate which is a mixture of 2- and 3-methylbutyl acetates (10). The structures for these compounds are shown in figure 3. 

//Figure 2: Esterification Reaction of a Carboxylic Acid and an Alcohol. Esters //

Ethyl acetate can be found in beer at levels of 10 to 60 mg/L. It also has a flavor threshold of 30mg/L (10). This compound typically gives flavors that are either solvent-like or sweet. Isoamyl acetate on the other hand is present at levels of 0.5 to 5.0 mg/L with a flavor threshold of 1 mg/L (10). This ester gives notes that are banana like or solvent like. The fact that these compounds are typically found at a range of levels that includes the sensory threshold for each compound means that the overall beer flavor is highly influenced by such materials. It is easy to tell from this information that the formation and detection of esters in beer is very important to obtaining consistent products.



​//Figure 3: Structures of Ethyl Acetate(top,// Chemspider) //and Isoamyl Acetate (bottom,// Chemspider).

A study from the //Journal of Enzyme and Microbial Technology// (6) discusses the kinetics of the formation of the two aforementioned esters in beer. A simple kinetic model was developed to predict the formation of these esters both in laboratory and in industry. This was done by measuring the amount of each species formed using either gas chromatography (GC) with a flame ionization detector (FID), or high performance liquid chromatography (HPLC). From this data it was concluded that the formation of these compounds in beer could be predicted quite accurately with the kinetic model establish during the study (6). The evolution of the esters was dependent upon which species participated in the formation reaction. For ethyl acetate these compounds were the fermentable sugars. For iso-amyl acetate the compound was iso-amyl alcohol (6).

Another type of compounds that form during the fermentation reactions is vicinal diketones (VDKs). VDKs are typically attributed to providing off flavors to beer products and brewers usually attempt to remove them. Some products, such as ales from the United Kingdom, accept the appearance of these compounds and their flavor characteristics (10). Being that these compounds are created during yeast fermentation their formation stems from pyruvate, which is similar to esters and higher alcohols (10).

Two important VDKs, and their pathways of formation, are shown in figure 4. These are 2,3-butanedione (diacetyl) and 2,3-pentanedione. These two diones are typically found at relatively low levels in beer. They show levels of 0.01-0.04 mg/L and 0.01-0.15 mg/L, respectively (10). The reduction of diacetyl forms 2,3-butanediol, which is much less flavor active. The sensory threshold of 2,3-butanediol is 4500 mg/L while diacetyl has a threshold of 0.07-0.15 mg/L. Many brewing companies use diacetyl as a marker for completion of brewing (14), but they are also very interested in removing diacetyl altogether due to its high flavor activity. There are numerous methods that have been developed to either eliminate this VDK, or try to avoid the production of diacetyl by converting the initial pyruvate compounds directly to acetoin (a precursor to 2,3-butanediol). These methods include adding enzymes and adjusting fermentation temperatures (10).

//Figure 4: Structure and Formation Pathway of Diacetyl and 2,3-pentanedione. (10)//

The detection of the VDKs is possible through the use of gas chromatography/mass spectrometry (GC/MS). Although this detection method is very simple to carry out, the preparation of the beer samples themselves can become quite tedious. Due to the high amounts of non-volatile compounds in beer, as well as the high concentration of certain compounds such as ethanol and water, direct injection into the GC/MS is not ideal. Typically some form of extraction of the volatiles of interest is necessary. This can be done using headspace analysis, or, as one method (14) describes, solid phase micro-extraction (SPME) or single drop micro-extraction (SDME) are much more effective.

In SPME the sample solution is extracted by fibers coated with a specific combination of materials that will allow the analytes of interest to be retained when they come in contact with the solution. Another article describes the use of SPME in beer (5). In this method a SPME device with carboxen/polydimethylsiloxane coated fibers was used. The selection of this type of device is typically based on the polarity of the substance of interest. This SPME method in beer, as well as wine and other spirits, showed great ability to pre-concentrate samples for detection via GC (6). In SDME a microdroplet is fixed in or above a flowing aqueous sample solution. The analytes of interest are extracted from the sample solution into the microdroplet (14). Headspace analysis may also be used in conjunction with these two methods to extract the specifically volatile compounds from the sample.

Possibly the most important flavor note of any beer is bitterness. This is by far the most distinguishing characteristic of beer throughout any culture or time period (9). The distinctive bitter notes of beer come from the hop plant (Humulus lupulus L.), which is a very essential ingredient in brewing (7). Early use of hops was as a preservative (12), but now it is added for its known flavor benefit. The bitterness does not come from the hop cones (hop cones are the form added during brewing) themselves, but rather from the alpha and beta acids as well as the prenylflavonoid xanthohumol (7). The structures of these compounds are illustrated in figure 5.





//Figure 5: Structures for the iso-alpha acids iso-humulone(top, [|Chemspider]), iso-cohumulone(middle, [|Chemspider]), and iso-adhumulone(bottom, [|Chemspider])//

 Hops are added during wort boiling to cause isomerization reactions to occur. These reactions are mainly used to convert the alpha acids to iso-alpha acids which are very flavor active and give the bitter character to beer (12). There are six major iso-alpha acids produced from the three alpha acids. Each of the alpha acids has a cis and trans isomer. The formation of these iso-alpha acids is shown in figure 6. The yield of these reactions is typically very low at 50-60% maximum, and all of this yield does not carry over to the final beer product (12).



//Figure 6: Alpha acid isomerization (12).//

Unfortunately, as with any chemical compound, iso-alpha acids do have some negative qualities. These compounds undergo photo oxidative degradation that leads to off flavors in the beer. Specifically isohumulones undergo this photoreaction due to light (280 – 320 nm) striking the beer matrix in the presence of riboflavin. The riboflavin acts as natural photosensitizer in beer (11). The process by which this occurs is detailed in Figure 7. This reaction leads to the formation of the 3-methylbut-2-enyl radical. This radical is the main precursor to the compound 3-methyl-2-butene-1-ol, which can, with addition of a source of sulfur, react to form the molecule responsible for light struck flavor in beer (11,21). This compound is 3-methyl-2-buten-1-thiol (MBT), and it has a characteristic foxy/skunky flavor profile. 

// Figure 7: Proposed photo oxidative reaction of iso-alpha acids in beer (11). //

In order to quantify and study the previously mentioned flavor compounds in beer, it is first necessary to develop a method of detection. There are many ways to detect chemical compounds with fairly good precision and accuracy. Some methods have used high performance liquid chromatography to separate and study individual compounds in beer. Others use GC or GC-MS to classify the flavor compounds. All of these detection methods are valid, but often it is dependent upon the compound of interest to determine which method is best. Since most of these volatile compounds are in very low concentrations, and are mixed in with large amounts of comparably non-volatile substances, it is necessary to develop a proper method of extraction.

There have been many studies and research articles initiated to examine different compounds in beer and how these could be detected. One study previously mentioned used SPME and SDME to examine compounds in beer such as vicinal diketones. One article in the //Journal of Chromatography A// (24) compares SDME with SPME as well as headspace analysis. In this article the author used SPME with headspace analysis and SDME with headspace analysis to extract different sulfur compounds that were added to beer at known concentrations. In this manner the methods were able to be statistically compared and analyzed to determine which was the most effective. The results showed that headspace SDME worked just as well, if not better in some cases, than conventional headspace SPME. Benefits of SDME were also explored, such as lower cost and less solvent waste. However SDME was found to be a more labor intensive method than SPME (24). This article is a good example of some of the different techniques that could be used to examine beer for compounds such as those from hop acids, or light struck effects.

An important area in any food product is storage performance. How well a product maintains its initial flavor, odor, and texture is very important to consumer acceptance. One well known problem with beer is its quickness to become stale. Without proper temperature control beer becomes stale very quickly (10). A study conducted by Callemien et al was aimed at targeting and identifying specific compounds responsible for these types of off flavors in beer. Historically trans-2-nonenal was pin pointed as a stale beer flavor and odorant (4). The study identified a new compound with similar strength as trans-2-nonenal which was 4-vinylsyringol (4). This was done using phenol extraction, GC cold trapping, and mass spectrometry.

For the detection of MBT, the light struck beer compound, many methods have been studied. Methods based on GC-MS or LC-MS are effective at identifying this compound, but do require extensive sample preparation and extraction (16). Other methods have been developed to identify this compound, as well as similar compounds, which make use of other analytical techniques. Some of these techniques include GC-olefactometry, aroma extraction dilution analysis, and GC-atomic emission detection. A method developed by Bailly et al used the GC-olefactometry and aroma extraction techniques to identify thiols such as MBT in wines. This technique gave a very accurate method for characterizing the aroma of wines based on the compounds involved (2).

Beer has been a staple of society for centuries. It has been the crux of many cultures and has lasted the test of time due to its popularity, physiological effects, and societal impacts (9). Its strong flavor characteristics are often polarizing, but distinctive to say the least. The compounds involved in both the flavor and aroma of beer are relatively simple, but the quantity of different compounds makes beer a very complex product. The complex nature of beer and numerous varieties thereof, make detection of flavor compounds very difficult. The different methods described in this research article are both similar and different at the same time. The subtle nuance of the beer matrix itself causes the methods of detection to be very unique, even using similar analytical techniques. There is much room to build upon these methods and learn even more about the flavor compounds in beer.

**References ** 1)<span style="font-family: 'Times New Roman'; font-size-adjust: none; font-size: 7pt; font-stretch: normal; font-style: normal; font-variant: normal; font-weight: normal; line-height: normal;"> Akiyama, M. et. al. //J. Food Science,// **2007**, 72(7), pp C388-C396Akiyama

<span style="font-family: 'Times New Roman','serif'; font-size: 12pt; line-height: 115%;">2)<span style="font-family: 'Times New Roman'; font-size-adjust: none; font-size: 7pt; font-stretch: normal; font-style: normal; font-variant: normal; font-weight: normal; line-height: normal;"> Bailly, Sabine et. al. //J. Agric. Food Chem.//, **2006**, //54// (19), pp 7227–7234Bailly

<span style="font-family: 'Times New Roman','serif'; font-size: 12pt; line-height: 115%;">3)<span style="font-family: 'Times New Roman'; font-size-adjust: none; font-size: 7pt; font-stretch: normal; font-style: normal; font-variant: normal; font-weight: normal; line-height: normal;"> Burns, Colin S. et. al. //Chem. Eur. J.// **2001**, 21(7), pp 4553-4561 (DOI = 10.1002/1521-3765(20011105)7:21<4553::AID-CHEM4553>3.0.CO;2-0) <Wiki would not let me put this in a link for some reason, it truncated the doi number. It works if you copy the number in your browser after the following - http://dx.doi.org/ - AB>
 * [just use this link http://www3.interscience.wiley.com/journal/85514669/abstract JCB]**

<span style="font-family: 'Times New Roman','serif'; font-size: 12pt; line-height: 115%;">4)<span style="font-family: 'Times New Roman'; font-size-adjust: none; font-size: 7pt; font-stretch: normal; font-style: normal; font-variant: normal; font-weight: normal; line-height: normal;"> Callemien, Delphine et. al. //J. Agric. Food Chem.//, **2006**, //54// (4), pp 1409–1413Callemien

<span style="font-family: 'Times New Roman','serif'; font-size: 12pt; line-height: 115%;">5)<span style="font-family: 'Times New Roman'; font-size-adjust: none; font-size: 7pt; font-stretch: normal; font-style: normal; font-variant: normal; font-weight: normal; line-height: normal;"> Campillo, Natalia et. al. //J. Chromatograph A.// **2009,** 1216(39), pp 6735-6740Campillo

<span style="font-family: 'Times New Roman','serif'; font-size: 12pt; line-height: 115%;">6)<span style="font-family: 'Times New Roman'; font-size-adjust: none; font-size: 7pt; font-stretch: normal; font-style: normal; font-variant: normal; font-weight: normal; line-height: normal;"> Garcia, Ana et. al. //J. Enzyme and Microbial Tech.// **1994**, 16(1), pp 66-71Garcia

<span style="font-family: 'Times New Roman','serif'; font-size: 12pt; line-height: 115%;">7)<span style="font-family: 'Times New Roman'; font-size-adjust: none; font-size: 7pt; font-stretch: normal; font-style: normal; font-variant: normal; font-weight: normal; line-height: normal;"> Haseleu, Gesa et. al. //J. Food Chemistry,// **2009,** 116(1), pp 71-81Haseleu

<span style="font-family: 'Times New Roman','serif'; font-size: 12pt; line-height: 115%;">8)<span style="font-family: 'Times New Roman'; font-size-adjust: none; font-size: 7pt; font-stretch: normal; font-style: normal; font-variant: normal; font-weight: normal; line-height: normal;"> Hofmann, Thomas et. al. //J. Agric. Food Chem.//, **2001**, //49// (5), pp 2382–2386Hofmann

<span style="font-family: 'Times New Roman','serif'; font-size: 12pt; line-height: 115%;">9)<span style="font-family: 'Times New Roman'; font-size-adjust: none; font-size: 7pt; font-stretch: normal; font-style: normal; font-variant: normal; font-weight: normal; line-height: normal;"> Hornsey, Ian S. //Brewing.// RSC Paperbacks. 1999. Brewing

<span style="font-family: 'Times New Roman','serif'; font-size: 12pt; line-height: 115%;">10)<span style="font-family: 'Times New Roman'; font-size-adjust: none; font-size: 7pt; font-stretch: normal; font-style: normal; font-variant: normal; font-weight: normal; line-height: normal;"> Hughes, P.S. //Beer Quality, Safety, and Nutritional Aspects.// RSC Paperbacks. 2001. Beer

<span style="font-family: 'Times New Roman','serif'; font-size: 12pt; line-height: 115%;">11)<span style="font-family: 'Times New Roman'; font-size-adjust: none; font-size: 7pt; font-stretch: normal; font-style: normal; font-variant: normal; font-weight: normal; line-height: normal;"> Huvaere, Kevin et. al. //J. Agric. Food Chem.//, **2005**, //53// (5), pp 1489–1494Huvaere

<span style="font-family: 'Times New Roman','serif'; font-size: 12pt; line-height: 115%;">12)<span style="font-family: 'Times New Roman'; font-size-adjust: none; font-size: 7pt; font-stretch: normal; font-style: normal; font-variant: normal; font-weight: normal; line-height: normal;"> Jaskula, Barbara et. al. //J. Agric. Food Chem.//, **2008**, //56// (15), pp 6408–6415Jaskula

<span style="font-family: 'Times New Roman','serif'; font-size: 12pt; line-height: 115%;">13)<span style="font-family: 'Times New Roman'; font-size-adjust: none; font-size: 7pt; font-stretch: normal; font-style: normal; font-variant: normal; font-weight: normal; line-height: normal;"> Kishimoto, Toru et. al. //J. Agric. Food Chem.//, **2006**, //54// (23), pp 8855–8861Kishimoto

<span style="font-family: 'Times New Roman','serif'; font-size: 12pt; line-height: 115%;">14)<span style="font-family: 'Times New Roman'; font-size-adjust: none; font-size: 7pt; font-stretch: normal; font-style: normal; font-variant: normal; font-weight: normal; line-height: normal;"> Kobayashi, Michiko et. al. //J. Bioscience and Bioengineering.// **2008,** 106(4), pp 317-323Kobayashi

<span style="font-family: 'Times New Roman','serif'; font-size: 12pt; line-height: 115%;">15)<span style="font-family: 'Times New Roman'; font-size-adjust: none; font-size: 7pt; font-stretch: normal; font-style: normal; font-variant: normal; font-weight: normal; line-height: normal;"> Liu, Mingming et. al. //J. Chromatography A.// **2005,** 1065(2), pp 287-299Liu

<span style="font-family: 'Times New Roman','serif'; font-size: 12pt; line-height: 115%;">16)<span style="font-family: 'Times New Roman'; font-size-adjust: none; font-size: 7pt; font-stretch: normal; font-style: normal; font-variant: normal; font-weight: normal; line-height: normal;"> Pinho, Olivia et. al. //J. Chromatography A.// **2006,** 1121(2), pp 145-143Pinho

<span style="font-family: 'Times New Roman','serif'; font-size: 12pt; line-height: 115%;">17)<span style="font-family: 'Times New Roman'; font-size-adjust: none; font-size: 7pt; font-stretch: normal; font-style: normal; font-variant: normal; font-weight: normal; line-height: normal;"> Pozdrik, Richard et. al. //J. Agric. Food Chem.//, **2006**, //54// (17), pp 6123–6129 [|Pozdrik]

<span style="font-family: 'Times New Roman','serif'; font-size: 12pt; line-height: 115%;">18)<span style="font-family: 'Times New Roman'; font-size-adjust: none; font-size: 7pt; font-stretch: normal; font-style: normal; font-variant: normal; font-weight: normal; line-height: normal;"> Pusecker K et. al.. J. //Chromatography A//. **1999.** 836 (2), pp 245-252.Pusecker

<span style="font-family: 'Times New Roman','serif'; font-size: 12pt; line-height: 115%;">19)<span style="font-family: 'Times New Roman'; font-size-adjust: none; font-size: 7pt; font-stretch: normal; font-style: normal; font-variant: normal; font-weight: normal; line-height: normal;"> Saison, Daan et. al. //J. Chromatography A,// **2009,** 1216(26), pp 5061-5068Saison

<span style="font-family: 'Times New Roman','serif'; font-size: 12pt; line-height: 115%;">20)<span style="font-family: 'Times New Roman'; font-size-adjust: none; font-size: 7pt; font-stretch: normal; font-style: normal; font-variant: normal; font-weight: normal; line-height: normal;"> Vanderhaegan, Bart et. al. //J. Food Chemistry,// **2007,** 103(2), pp 404-412Vanderhaegen

<span style="font-family: 'Times New Roman','serif'; font-size: 12pt; line-height: 115%;">21)<span style="font-family: 'Times New Roman'; font-size-adjust: none; font-size: 7pt; font-stretch: normal; font-style: normal; font-variant: normal; font-weight: normal; line-height: normal;"> Vermeulen, C. et. al. //J. Agric. Food Chem.//, **2006**, //54// (14), pp 5061–5068Vermeulen

<span style="font-family: 'Times New Roman','serif'; font-size: 12pt; line-height: 115%;">22)<span style="font-family: 'Times New Roman'; font-size-adjust: none; font-size: 7pt; font-stretch: normal; font-style: normal; font-variant: normal; font-weight: normal; line-height: normal;"> Verstrepen, Kevin J. et. al. //J. Bioscience and Engineering,// **2003,** 96(2), pp 110-118Verstrepen

<span style="font-family: 'Times New Roman','serif'; font-size: 12pt; line-height: 115%;">23)<span style="font-family: 'Times New Roman'; font-size-adjust: none; font-size: 7pt; font-stretch: normal; font-style: normal; font-variant: normal; font-weight: normal; line-height: normal;"> Vesely, Petr et. al. //J. Agric. Food Chem.//, **2003**, //51// (24), pp 6941–6944Vesely

<span style="font-family: 'Times New Roman','serif'; font-size: 12pt; line-height: 115%;">24)<span style="font-family: 'Times New Roman'; font-size-adjust: none; font-size: 7pt; font-stretch: normal; font-style: normal; font-variant: normal; font-weight: normal; line-height: normal;"> Xiao, Qin et. al. //J. Chromatography A.// **2006,** 1125(1), pp 133-137Xiao