Trace Analysis of Precious Drops

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GERSTEL GmbH & Co. KG · Eberhard-Gerstel-Platz 1 · 45473 Mülheim an der Ruhr / Germany · Phone +49 208 76503-0 · E-Mail: gerstel @ gerstel.com

Modern Wine Analysis Trace analysis of precious drops

Research Searching for the pepper in Shiraz

Off flavors in wine Watch your sulfurs!

Research The Twister sniffs out the aftertaste Modern wine analysis Content

Modern wine analysis It’s not all about adulteration 2 Archaeology meets chemistry Peeking into Pharaoh’s wine glass 3 It’s not all about Research The Twister sniffs out the aftertaste 6 Pesticide analysis EZ adulteration Searching for the pepper in Shiraz 9

Lab on the slopes Prof. Dr. Manfred Grossmann Flavor from trash cans 12 Advanced Flavor and Fragrance Analysis An extra GC dimension at your finger tips 14 Degustation ine has been a permanent fixture in the microbiology and wine analysis. Consequently, Tasting a wine beats reading about it… 16 Whistory of mankind for more than 7,000 modern wine analysis is not just about years. The grape juice fermentation product is enlisting the help of traditional tools such as Off Flavors in Wine part I: Corky today justly described as a cultural heritage. spectroscopy, chromatography, photometry Efficient and sensitive determination of TCA and other off-flavors 17 Surely wine producers have always worked and electrochemistry; we must also rely on hard to improve the already high quality of their biochemical and molecular biology methods. Off flavors in wine part II: product, making sure that it stays fresh longer It would be a mistake to believe, however, Sulfur related flavors – reductive notes or improving the flavor and taste to make it that simply detecting genes and determining Watch your sulfurs! 20 more appealing. Equally, through the ages, and regulating expression rates is the road Twister extraction wine producers have regularly been spiking to fruitful knowledge. In the age of “-omics”, Two is company 24 wine with low cost ingredients to lower the Genomics, Transcriptomics and Metabolomics, Extraction techniques cost while making the wine appear of better detecting genes and determining and regulating HIT it - targeting VOCs and SVOCs 29 quality than it is. A Phoenician tombstone their expression rates is no longer sufficient. Literature dating back to 1,000 A.C. states: You shall A comprehensive determination of the Suggested reading… 32 not perform magic on your neighbor’s wine. metabolites formed during cellular activities on Fatal side effects experienced from using lead the vine and during fermentation is required; worldwide sugar as a sweetener gave rise to serious wine for the wine, these are the primary quality SPECIAL EDITION

GERSTEL GmbH & Co. KG · Eberhard-Gerstel-Platz 1 · 45473 Mülheim an der Ruhr / Germany · Phone +49 208 76503-0 · E-Mail: gerstel @ gerstel.com analysis as of the 17th century. However, those determining flavor compounds. Meanwhile Modern Wine Analysis Trace analysis of who claim that wine analysis is just a means of oenologists world-wide are also learning to precious drops detecting illegal techniques or additives are not weave a systematic web using various highly

Research delving deep enough into the matter. Modern different individual analyses and leading the Searching for the pepper in Shiraz

Off flavors in wine Watch your sulfurs! wine science makes use of a wide range of way to a Systems Biology approach. This is

Research The Twister sniffs out the aftertaste instrumental analysis techniques and methods. a comprehensive research approach that the These enable the scientist to determine Geisenheim Research Center among others the origin of the key taste and flavor has put into practice in order to safeguard a compounds in the grapes and to follow the millennia old cultural heritage and to benefit Imprint underlying metabolism processes every step the consumer. of the way during the various technical and Published by microbiological stages in the wine making Prof. Dr. Manfred GERSTEL GmbH & Co. KG process. Nevertheless, wine chemists are Grossmann is Vice- Eberhard-Gerstel-Platz 1 often faced with challenges that require new Chairman of the 45473 Mülheim an der Ruhr, Germany approaches and new solutions. In recent years Institute of Oenol- Editorial Director it was increasingly found, that compounds ogy and Beverage Guido Deußing which are odor active in the final wine are Research and Chair- man of the Depart- ScienceCommunication initially not detected in sensory tests. Neuss, Germany ment of Biology This is due to the fact that they are bound [email protected] and Microbiology, to short chain saccharides or peptides during one of the oldest Translation and editing their formation in the grape vines. The side Kaj Petersen research centres in the German speak- chains are subsequently split off by the yeasts [email protected] ing countries. Professor Grossman is a during alcoholic fermentation, but hydrolysis member of the Board of Wine Research, Scientific advisory board is not the only thing that is happening. consulting the German Federal Ministry Eike Kleine-Benne, Ph.D. Through additional biochemical modifications of Nutrition, Agriculture and Consumer [email protected] performed by the yeast during the fermentation Protection; Consultant for the Federal Oliver Lerch, Ph.D. process, new and modified flavor compounds Ministry of Education and Research as [email protected] well as external reviewer for the Univer- 2018 are created that give rise to different taste Malte Reimold, Ph.D. / sity of Stellenbosch. (South Africa). Since and flavor impressions. This means that the [email protected] 1996 Professor Grossmann has been Contact micro-organisms have significant influence on German delegate to the expert commit- [email protected] the wine flavor. A full and clear picture of the tee „Microbiology of Wine“ of the Inter- Design sensory aspects of a wine is only bestowed national Organization of Vine and Wine Paura Design, Hagen, Germany upon those that succeed in playing the entire (OIV) and has been vice-chairman of this range of instruments available for both wine committee since 2004. www.paura.de ISSN 1619-0076 · 05

2 GERSTEL Solutions Worldwide Wine Special and his contemporaries imbibed when “communicating” with the wine gods. The uncovered amphorae have been completely dry and empty; the wine evaporated an eternity ago. Not until chemists were called upon to inspect the grave goods more closely did hard facts begin to emerge. In the amphorae found in the grave of Tutankhamen, malvidine-3-

Polyphenols are universally praised for their positive health effects, in large part ascribed to antioxidant and radical scavenging properties. One might also turn to grapes, raisins, black currants, cranberries or elderberries for a less stimulating source. Most chemical research on polyphenols is reportedly performed on wine.

glucoside was identified among the remains (Armen Mirzoian et al., „Analytical Chemistry“, Vol. 76, No. 6, March 15, 2004). This compound is one of the more stable Archaeology meets chemistry anthocyanins, the group of compounds that lends a warm red hue to the class of wines known as red wines. The 18 year old Pharaoh, Peeking into Pharaoh’s in other words, had been given amphorae of red wine to accompany him, possibly wine * that he had favored during his short life. As glass an aside, anthocyanins form the main group of flavonoids that, along with phenols, make up the class polyphenols, which are thought * Ancient Egyptian sign for „Wine“ to have positive health effects. Equally scientifically intriguing was the Details on viticulture in ancient Egypt are quite well understood by search for wine residues in 700 wine jugs modern-day archaeologists. But what exactly was in Pharaoh’s glass found in Abydos, Egypt. The jugs had been dated to 3,150 B.C., around 1,800 years prior when he savored the gift of the wine gods – and was it just imbibed for to the birth of Tutankhamen. They were relaxation and merriment or was it taken as a stimulating aphrodisiac found in what was probably the tomb of the first Egyptian Pharaoh, Scorpion, from the or maybe prescribed by his physician to cure or alleviate pharaohnic first dynasty. Initial research had revealed that ailments? Answers to these questions have eluded us for ages. When the Abydos jugs had contained around 4,000 Liters (1,000 Gallons) of wine from the Valley archaeologists recently consulted analytical chemists armed with of Jordan, about 600 km (400 miles) away. thermal desorption GC/MS systems, information began to trickle out, The project described here was offering insight into ingredients used in ancient Egyptian wine. performed by scientists from the Museum of the University of Pennsylvania (MASCA)

ine from ancient Egypt is thought to “wine”, archaeologists were able to determine W have been honey-sweet – though now that grapes were being grown and wine it is just bone-dry. What was once refreshing, produced as early as 3,000 B.C. in the Nile stimulating and thirst-quenching has mostly Delta (Lower Egypt). At that point a thriving evaporated; only dust and residues remain in wine-producing industry controlled by the the 3-5 millennia old amphorae that were rulers had already taken root. Vines were found in the tombs of those ancient rulers planted in pits filled with fertile Nile river and demigods, the Pharaohs. On their way silt. Given sufficient irrigation, vines could to the netherworld, they were given gold and be grown successfully in oases. ample riches along with food and amphorae filled with precious wine. One amphora Sacrifices to the gods was marked: “Year 5. Wine of the House of Tutankhamen, Ruler of-the Southern On, the Western River. By the chief Vintner Khaa.’’ Archaeologists have found evidence that wine (Source BBC). was well appreciated for festive occasions in Some tombs are embellished with wall ancient Egypt. The only drop of bitterness To look into the soul of a wine that no longer has paintings depicting scenes from ancient in the chalice was that many an outstanding a body, the scientists had no other option but to Egyptian vineyards (cf. picture on p. 22). droplet was reserved for the gods and donated grind clay from the inside of the amphorae and From such graphical renderings, as well as as sacrifice. We have until now relied only jugs and extract wine-related substances from the Text: Guido Deußing Text: granulate. from a separate hieroglyph for the word on speculation as to what Tutankhamen

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in Philadelphia, PA, and from the Beverage Alcohol Laboratory in Beltsville, MD, part of the U.S. Alcohol and Tobacco, Tax and Trade Bureau (TTB). The scope of the project was to determine volatile and semi- volatile compounds in the wine residues, but not necessarily to determine the origins of the wine. In order to have a historically differentiated reference, a wine amphora from the Nubian town of Djebel Adda, dating back to the year 400 A.D., was analyzed as well. To look into the soul of a wine that no longer has a body, the scientists had no choice but to grind the ancient pottery and extract the oenological residues from the resulting powder using acidic or alkaline solutions. The extracts were filtered and analyzed using chromatographic techniques. The following provides an overview of the methods used Mural in the tomb of Nebamun, soldier in the army of Pharaoh Thutmose IV (Photo: archive). for analysis. The results: In both samples, the scientists identified a range of terpenoids, esters and alcohols as well as various volatile compounds and L-tartaric acid. This was definitive proof that the amphorae and jugs had contained wine. Further, the identified compounds indicated that resin and herbs had been added to the wines, making them a kind of ancient day Retsina wine, possibly similar to what is produced, and mainly served to tourists, in Greece today. The project provided facts that support the theory of a preference for wines enriched with resin and herbs at the court of the Pharaohs, covering the entire period from the beginning of the ancient Egyptian High Culture (Abydos find) until the latter parts (Djebel Adda find). The herbs may have been added mainly to produce a sought after taste or they could have been added for medicinal purposes. Herbs and tree resin were part of the ancient Egyptian pharmacopoeia as we have learned from 13 ancient papyri with

Section of „Papyrus Ebers“ (1,500 B.C.) a 20 meter long list of medicinal recipes, and thereby the most comprehensive documentation of medi- cal knowledge in ancient Egypt known to man. The content mainly deals with internal diseases and SPME Total Ion Chromatogram (top) of the Abydos sample and enlarged (12.00-13.60 min) their treatment. The papyrus was acquired for the Selected Ion Chromatograms (Middle), shown along with mass spectra and library mass spectra University of Leipzig, Germany in 1872 by Georg of select compounds (below). Ebers (1837-1898), Professor of Egyptology.

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Analytical Conditions

Solid Phase Micro-Extraction (SPME) A 50/30 µm DVB/CAR/PDMS fiber was used. The fiber was immersed in a sodium chloride solution containing the sample powder inside the sample vial for 40 min. at a temperature of 80 °C. The concentrated analytes were desorbed from the SPME fiber in the GC inlet for 3 minutes at 250°C. The SPME process was automated Multiple reaction monitoring (MRM) LC/MS/ Selected Ion Chromatogram of the peak at using the GERSTEL MultiPurpose Sampler MS chromatogram traces of an L-tartaric acid 23.13 min; retention time and mass spectrum (MPS). standard (top) based on the m/z 149 and match those of vanillin. 87 molecular fragments. The middle and Gas Chromatography / Mass bottom traces are from the aqueous extracts Spectrometry (GC/MS) of the samples from Abydos and Djebel Adda respectively. A GC/MS system consisting of a 6890 GC and a 5973 MSD, both from Agilent Technologies, was used. Separation was RT (min) Compound Abydos Djebel Adda Possible origin achieved using a HP 5MS column, 30 m 3.75 1-Hexanol x Wine x 0.25 mm ID x 0.25 µm film thickness. 5.81 Benzaldehyde x x Wine Analyte transfer was performed in splitless 5.62 Camphene x Pine mode, the MSD was set to scan mode from 5.99 Heptanol x Wine m/z = 40 to m/z = 400. GC oven program 6.27 Phenol x Mint was started at 60 °C and programmed to 6.48 Menthene x Mint 240°C at 3 °C/min. Carrier: Helium at 1.2 7.68 p-Cymol x x Pine, Rosemary 7.82 Limonene x Mint, Pine ml/min constant flow. Compounds were 7.97 Benzyl alcohol x Wine identified using mass spectral libraries and 9.27 1-Octanol x Mint, Wine Kovats Retention Indices, calculated from a 9.96 Fenchone x Rosemary, Fennel, Sage series of n-alkanes from C5 to C22. 10.09 2-Nonanone x 10.91 Phenethyl alcohol x Wine 10.92 Fenchol x x Pine Thermal Desorption 12.17 Camphene x x Pine, Mint, Wild Fennel, Sage, Mugwort, Rosemary Residues from amphorae and jugs were 12.53 g-Heptalactone x also desorbed, or thermally extracted, 13.05 Borneol x x Pine, Rosemary, Mint, Oregano 13.31 1-Nonanol x Mint using a Thermal Desorption System (TDS) 13.42 L-Menthol x Mint from GERSTEL. The desorption tempera- 14.12 a-Terpineol x Pine, Mint, Wine ture was programmed from a 50 °C start- 14.46 Ethyloctanoate x Wine ing temperature to 250 °C at a rate of 50 15.63 Cuminaldehyde x Rosemary °C/min. 16.41 Carvone x Mint, Yarrow, Wild Fennel, Sage, Mugwort 17.57 Ethyl Salicylate x Wine Liquid Chromatography – Tandem 17.67 Decanol x Mint Mass Spectrometry (LC/MS/MS) 18.55 Thymol x Mint, Wild Fennel, Sage, Basil, Thyme 23.11 Ethyl Decanoate x Wine A Waters Acquity UPLC and a MicroMass 23.13 Vanillin x Rosemary, Thyme Quattro Premier XE Triple Quadropole 25.53 Geranyl Acetone x Rosemary mass spectrometer were used. LC param- 35.34 Farnesol x Pine eters: UPLC BEH C18 column. Isocratic 37.76 Benzyl Benzoate x Pine flow at 0.20 mL/min, 98 % H2O: 2 % 38.85 Ethyl Palmitate x x Wine ACN, 0.1 % Formic acid. MS/MS: Electron 45.76 Ethyl Stearate x x Wine Spray Ionization (ESI), Cap. 4.50 KV, CV 20 46.28 Manoyl Oxide x Pine V, CE 16 V. 47.65 Biformene x Pine 56.24 Methyl Dehydroabietate x Pine Compounds in the jugs from Abydos and amphorae from Djebel Adda identified using SPME- GC/MS and Thermal Desorption GC/MS. information on medicine and various recipes. Among these are the “Papyrus Smith” (2,500 B.C.), the “Papyrus Ebers” (1,500 B.C.) or Empirical work, religion and authorized barley, a type of wheat called emmer and the “Papyrus Hearst” (1,500 B.C.), all named magic often went hand in hand. A word on date juice, beer was counted as a staple food after the people by whom they were later alcohol content of the wine in ancient Egypt: on the same level as bread. Brewing beer was purchased. In “Papyrus Smith”, diseases were Alcohol plays a useful role as an extraction of course also a way of preserving drinking clearly divided into incurable and curable solvent for, and carrier of, active compounds water and keeping it from being infested afflictions; for the latter group, systematic in herbal medicine. The intoxicating role is with undesirable microorganisms. Those instructions for treatment were listed. of course equally well recognized and this ancient Egyptians who could afford it often Knowledge about anatomy and physiology seems to have been a cherished side-effect preferred to drink wine when they wanted to (e.g. functions of organs) was, however, very to what the doctor ordered. Beer, not wine, have a good old time. Almost four thousand limited, which means that physicians at the was the national beverage in ancient Egypt, years ago, an Egyptian teacher lamented that time were quickly out of options for effective often used in religious ceremonies and as a one of his students was leading a debauched treatment. In many cases patients were meal-time beverage. Legend has it that Osiris, and alcoholized life. “Oh if only you would diagnosed as being possessed by demons; the god of the underworld, taught humans recognize that wine is a horror, if only you prayers or redemptive magic was prescribed. how to brew beer. Prepared from malted would forget the chalice”.

GERSTEL Solutions Worldwide Wine Special 5 Research The Twister sniffs out the aftertaste

What do wine and mouthwash have in common? Both leave an aftertaste. How aftertaste develops has not yet been clearly determined, but it is well known that flavors and odors play an essential part; in fact, a more correct term would be “afterflavor”. A scientist from the German Research Laboratoy for Food Chemistry sheds some light on the secrets of how we taste.

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of expert tasting. Still it remains unclear a discernable sensory impulse is released exactly how the odorous substances reach through the sensory bulb to our brains. the olfactory sensing apparatus. If modern Proof that this is how the process works was imaging tools are used, however, it becomes provided by real-time breath analysis using obvious that this is a precisely controlled Proton Transfer Reaction Mass Spectrometry Open palate while food is inserted into the mouth with a spoon (video-fluoroscopic process, which permits a flavor transfer only (PTR-MS). snap-shot). at certain times. After taking a sip of wine, a test person If there is food in the oral cavity and lips exhales through the nose and then closes the and jaws are closed, the palate and tongue lips and tongue-palate barrier. In the online base form a barrier that actually seals the oral PTR-MS measurement, a distinct initial sing a method based on Stir Bar Sorptive cavity and prevents us from unintentionally peak is seen. The signal is due to the presence UExtraction (SBSE), the German swallowing food or literally getting it down the of ethyl acetate formed by reaction between Research Laboratory for Food Chemistry has wrong pipe. A small experiment demonstrates ethanol and acetic acid. This highly volatile succeeded for the first time in determining the incredible efficiency of this natural lock compound is easily sensed at relatively low minute amounts of odor-causing substances inside the mouth. Take in the mouth at defined time intervals after a small sip of coffee or the consumption of food. The method can wine and leave it in your be used to determine aftertaste and it has mouth. Now close your helped explain how aftertaste develops. lips. In doing so, keep your There are different ways to approach a good lower jaw completely still. wine. Connoisseurs always swear by their What is your perception? own personal method. Certain principles and Now start to chew. Does details, however, simply follow from instinct anything change in your and from human anatomy. Minor tricks of perception? And then the trade and some experience can then help swallow. What is your even amateurs to a more qualified assessment perception now? and maybe to more enjoyment. To sum it „As a general rule, up: wine tasting is not just for specialists. taste impressions like Even the ancient Romans knew that a wine sour, bitter, sweet or salty should be judged not only by its taste, but by can be detected during its color and flavor as well. Sounds simple, the first part of this and it actually is – at least the part about the experiment, but no typical taste. The flavor evaluation, the palate test, flavor notes like toasty, still holds many secrets. Simple descriptors fruity or flowery“, explains such as sweet sour, salty bitter, creamy, spicy Dr. Andrea Büttner, a or toasty cannot fully describe the complex scientist at the German GC System with MPS and TDU for fully automated analysis of up to interactions that provide the full experience Research Laboratory for 196 GERSTEL Twisters. of tasting food and drink. Whether food or Food Chemistry. During drink has a short or long lasting taste, and chewing, namely the whether it turns out to be enjoyable, depends second part of the experiment, the natural concentrations and often identified as a nail mainly on to what extent and for how long barrier in the oral cavity opens for a short time polish smell. In wine, ethyl acetate occurs characteristic flavors find their way from the and small amounts of aroma components naturally. In this experiment, ethyl acetate oral cavity via the throat to the olfactory reach the olfactory bulb via the throat. is used as a marker to detect the transfer sensors in the nose. This process is referred “However the real perception of the of volatile wine flavor compounds from the to as retronasal odor perception. In contrast, wine or coffee aroma does not occur until oral cavity to the nose cavity. The initial ethyl when flavors are sensed in combination immediately after swallowing, with the first acetate peak during wine intake lasts only 2.5 with inhalation through the nose, it is called breath.” seconds. Even though the test person keeps orthonasal odor perception. An aroma impulse, by the way, can the wine in the mouth for some time, only also be registered when we place food into isoprene, a metabolite which is released into our mouth. Andrea Büttner: „This is an the atmosphere via our breath, is detected, but Learning from Mother Nature: important protective mechanism provided by no ethyl acetate. Following the ways of human tasting Mother Nature. Through it we receive a first impression on whether the substance in our But how do the flavor compounds that are mouth is fit to eat or not.” The phenomenon You can’t smell with your mouth full released from foods or beverages in our mouth has physiological reasons: as soon as we open reach their destination, the olfactory sensors? the jaw, the tongue-palate barrier opens up Though the experienced connoisseur seldom To adequately answer this question it is not for a short time. After closing mouth and jaw swallows during wine tasting, he or she is able enough to watch a connoisseur during wine again, excess air is released from the mouth to give a comprehensive retronasal evaluation tasting. You can reach some conclusions about cavity into the nose via the throat cavity; flavor of a wine. Using a few tricks of the trade, the person from the smacking, slurping and substances from the food are thus transferred mother nature and her physiological miracle guttural sounds emanating during the process to the olfactory sensors in the nose, where of sensory perception is outwitted: Text: Guido Deußing Text:

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To avoid contact, and thus a direct extraction of substances from the oral mucosa, a special sampling vessel for the Twister has been developed. BOSS provides unobstructed access of saliva and gas phase to the Twister, thereby ensuring an efficient extraction of flavor compounds.

By skillfully opening and closing be determined through sensory perception or stopper and placed inside the oral cavity of the mouth and the palate barrier, and by by using analytical instrumentation such as the test-person for a defined period of time. combining these motions with a special gas chromatography (GC) with olfactory The perforated vial walls ensure unobstructed breathing technique, flavors are pumped detection or MS detection. The duration of the contact of the Twister with saliva and the oral from the oral cavity through the throat “aftertaste” depends on several factors, among cavity gas phase, the test person moves saliva cavity and on into the nose. This process them the extent to which flavor compounds are around the vial during the extraction phase. is often accompanied by sounds that may metabolized by the saliva, resulting in reduced Sampling using the BOSS procedure seem less than appetizing to the uninitiated. sensory perception and a displacement is performed as follows: a subject tastes a The process can be examined and traced of the aroma profile. Thioles, such as food sample and subsequently places the via PTR-MS. Incidentally, experienced 2-furfurylthiol, that adds a typical roasted perforated glass vial with the Twister into wine tasters never bite off more than they coffee flavor, or 4-mercapto-4-methyl-2- his or her mouth. The vial is moved around can chew, that is, they never take too much pentanone, characteristic for Sauvignon- in the saliva inside the oral cavity. The lips wine into their mouth when tasting. Apart Blanc wines, are efficiently metabolized by are kept sealed and breathing only takes from the risk of choking, this would lead to saliva. Earthy pea or bell pepper type notes, place via the nose. After a specified period a reduction of the vapor phase volume in the however, are saliva persistent. of time, the glass capsule is removed from mouth, effectively reducing both the transfer Flavor persistence was determined the mouth and the twister taken out and of flavor compounds into the vapor phase and based on sensory evaluation in parallel with dabbed dry on a lint-free paper. The extracted the subsequent transfer of the vapor phase direct saliva analysis using the Buccal Odor compounds in the PDMS are determined to the nose cavity. If, on the other hand, the Screening System (BOSS). BOSS is a novel by thermal desorption in combination with amount of wine taken into the oral cavity is analysis method based on Stir Bar Sorptive GC separation and olfactory detection. In too small, dilution of the sample by saliva Extraction (SBSE) using the GERSTEL her work, Dr. Andrea Büttner established a and a too large volume of vapor phase can Twister. The Twister is a glass-coated clear correlation between the concentrations significantly impact the sensory perception. magnetic stir bar with an outer coating of of extracted flavor compounds and the flavor The volume needs to be just right. polydimethylsiloxane (PDMS) that can perception of the subject. With BOSS it Back to the experiments: As soon as extract organic compounds such as flavors was possible for the first time to detect even the test person has swallowed the wine and or off-odors from aqueous and other liquid small amounts of flavor compounds in the taken a breath, there is another clear ethyl samples. Direct contact between the Twister oral cavity at any given point in time after acetate signal. Even after the wine has been and the oral mucous membranes could lead consumption of a beverage or food sample. swallowed, volatile flavor compounds from to extraction of adsorbed compounds that do In other words, for quite a while after the wine are detected in the breath. Even not contribute to the sensory perception. The a food has been swallowed, traces of flavor though there is no more wine in the mouth, Twister is kept isolated from the membrane compounds can be detected that have been volatile compounds, that are typical of the surfaces by placing it inside a small, perforated absorbed by oral mucous. These can produce a aroma for the tasted wine, are released and can glass vial. The vial is sealed with a glass flavor impression over a period of time, known as the aftertaste. This finding is especially of interest to food manufacturers. Customer acceptance and the success of any given product in the marketplace depends heavily on whether consumers enjoy the taste and outh aftertaste. In addition, Dr. Büttner considers m Isoprene

the Ethyl acetate the BOSS method a promising, extremely to helpful instrument for diagnosing halitosis, as well as for examining the effectiveness of toothpastes and mouthwashes. oduction 6.00E-009 tongue/palate barrier closed It can be safely said that the BOSS intr tongue/palate barrier open swallowing system can neither replace wine tasting nor 5.00E-009 Real-time PTR-MS monitoring of can it provide specific conclusions about 4.00E-009 volatile compounds individual properties of wines and other that pass the olfac- products. It is simply a tool to describe the ent (A) 3.00E-009 tory center and are aftertaste of a given product. Dr. Büttner adds: exhaled through the 2.00E-009 „Wine tasting is and will remain a subjective Ion curr nose when drinking matter, incidentally, one that is more strongly wine. 1.00E-009 influenced by the mood and the environment in which it is performed than most people 0.00E-000 15 35 55 75 95 115 135 155 realize.“ time (sec)

8 GERSTEL Solutions Worldwide Wine Special Vineyard in One Tree Hill near Adelaide, Southern Australia. Searching for the pepper in Shiraz

Rotundone is a key flavor compound with a distinct black pepper note. Rotundone occurs naturally in pepper, but also in wines such as Australian Shiraz and others from around the world, for example in the Austrian Green Veltliner. Australian wine scientists are searching for clues as to how the peppery compound is formed in the hope that this knowledge would enable them to optimize both growth conditions for the Shiraz grape and the ensuing wine-making process. Finding answers requires an efficient analysis method. Researchers turned to Membrane Assisted Solvent Extraction (MASE) combined with heart-cut GC/MS for more efficient determination of rotundone in wine and grapes.

hen it comes to wine, tasting beats reading reviews. But phy with mass selective detection (GC/MS). This is the tool Wa little reading could offer a good starting point when used by Tracey E. Siebert and Sheridan R. Barter from the you set out to differentiate noble drops from decent every- Australian Wine Research Institute in Glen Osmond (Ade- day wines or even inferior ones. When mingling with real laide), Australia. The scientists conduct basic research; they or would-be connoisseurs, the following vocabulary samples are not trying to outperform sommeliers. could help you fit in: A wine scientist is called an oenologist; Siebert‘s and Barter‘s goal is to find out how wine fla- wine expert in a classy restaurant: Sommelier; and while vor compounds are formed in order to find ways to opti- merry wine drinkers enjoy wine tasting, highbrow mize growth conditions for best taste and bouquet. The tasting is referred to as a degustation. But even Australian scientists had set their sights experts can find themselves at wit’s end when sen- on rotundone, a bicyclic sesquiterpene sory impressions do not solve the puzzles containing a conjugated ketone group. they face and hard facts are required to With its characteristic black pep- ensure best possible product quality per note, rotundone is considered a and product acceptance. key flavor compound in Australian When faced with a wine with Shiraz wine. Shiraz is the num- its hundreds of volatile com- ber one grape grown in Austra- pounds that influence the fla- lia. It is highly valued due to its vor, the technique of choice for high yield and resistance to cold the analyst is gas chromatogra- weather, and of course due to the

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GC/MS-System comparable to the one used by Siebert and Barter for automated MASE and 2D-GC/MS Rotundone determination

Structural formula of (-)-Rotundone

excellent tasting wine it produces. Shiraz vines were originally cultivated in France (Syrah). Today the wine is not only popular in Aus- tralia, but also widely grown in South Africa, Latin America, the United States, and Ca- nada.

Wine under the influence

Many factors influence quality and character of the final wine; it is generally accepted that controlling the grape maturation process is key. Knowledge of specific factors that influence, for example, how much rotundone is Critical factor for success: The Experimental formed in the vine and how growth condi- extraction tions could be optimized could help improve The grape juice/extract or wine sample was quality, taste, flavor, and ultimately product The critical factor that helps ensure analyt- added to a 20 mL autosampler vial, the MASE value. Such knowledge might enable wine ical success or failure is the extraction step, membrane bag placed inside the vial, and the producers to provide more uniform year-to- according to Siebert and Barter. year wine quality and taste. But Sieber and Searching for a suitable technique, the Barter are convinced that such insights can scientists came across automated Membrane Sample preparation: only be gained based on access to efficient Assisted Extraction (MASE). MASE was and sensitive analysis methods. developed in cooperation between the Envi- Grapes: A 100 g sample of berries was The scientists presented their work in ronmental Research Institute Leipzig-Halle gently blended and centrifuged May 2013 in Palm Springs, California, USA and GERSTEL. and the juice supernatant col- during the 37th International Symposium This type of liquid – liquid extraction is lected. The solid residue was fur- for Capillary Chromatography (ISCC) [1]. based on using a semi-permeable membrane ther extracted by adding 60 mL of Previously used analysis methods were labor as a phase boundary, which keeps particulate an ethanol/water mixture (50:50) intensive and time consuming. Large sam- matter and other matrix components out of and internal standard (IS) to the ple volumes had to be handled and extensive the extraction solvent. Further sample clean- solids and shaking for 24 hours sample preparation was required for matrix up steps such as filtration and centrifuga- before centrifuging and collecting elimination and analyte concentration since tion are no longer needed. MASE extracts – the supernatant. The supernatant Rotundone is present only at very low con- even of heavily matrix laden samples are clean and the juice initially collected centration levels. and can be injected directly to the GC/MS or were combined and topped up According to literature: “The flavor LC/MS system for analysis. Additionally, with water to a volume of 200 threshold of Rotundone is at 16 ng/L in red MASE enables extraction using sample-sol- mL. Of the final volume, a 15 mL wine and 8 ng/L in water. A limit of quanti- vent mixtures that do not normally result in aliquot was taken and used for tation (LoQ) of < 8 ng/L would be necessary phase separation. As an example, HPLC com- analysis. for juices, grapes, mash and wine. These are patible polar solvents can be used to extract Wine: A 15 mL sample of wine was taken complex aqueous samples that contain sugars, aqueous samples in which the solvents are and used directly for analysis. ethanol, anthocyanins or polyphenols.“ [2]. normally miscible. MASE extends the analytical possibilities considerably [3,4]

vial capped. Sample preparation and introduction were performed automatically by the Schematic diagram of Membrane Assisted Solvent GERSTEL MultiPurpose Sampler (MPS). Extraction (MASE): The semi-permeable mem- MASE was performed by adding 750 µL of brane insert is immersed into the sample (left). Extraction solvent is added into the MASE insert hexane into the MASE membrane bag. The and analytes diffuse through the membrane into sample was extracted for 60 min at 35 °C. A the extraction solvent while particulate matter is 100 μL aliquot of the resulting extract was kept in the sample (center). injected (Large Volume Injection - LVI) into The extract is aspirated by the syringe for introduc- a GERSTEL Cooled Injection System (CIS tion to the GC/MS (right). 4) inlet mounted in an Agilent GC 7890.

10 GERSTEL Solutions Worldwide Wine Special GERSTEL Solutions Worldwide Wine Special

Instrument method parameters The separation took place on a multidimensional heart-cut GC system, based on an Agilent Capillary Flow Technology (CFT) Deans’ GERSTEL MPS XL, CIS and CTS Switch configured with two separation columns. A CryoTrap Sys- LVI: 100 µL at 0.58 µL/sec tem (CTS, GERSTEL) was connected between the first and second He carrier gas flow: 100 mL/min dimension columns for analyte focusing prior to the analytical sepa- CIS: Glass wool packed liner ration. An FID monitor detector was used on Column 1 and an Agi- CIS: 20 °C, solvent vent, 20 psi (0.12 min); lent 5975C MSD (Agilent Technologies) was used in Selected Ion splitless, 47.6 psi, 12 °C/sec to 240 °C (2.5 min); split, 12 °C/sec to 275 °C (2 min) Column 1: VF-35ms (30 m x 0.25 mm x 0.25 µm), Main FID-Chromatogramm and heart cut MSD chromatogram showing a medium polarity phase; He 47.6 psi clearly defined rotundone peak. The method developed by Siebert and Column 2: VF-200ms (30 m x 0.25 mm x 0.25 µm), Barter was used to determine rotundone in Shiraz grapes and in wine unique selectivity with dipole-dipole following MASE analyte extraction. interactions He 37.7 psi Column 3: Deactivated fused silica (0.70 m x 0.1 mm) GC Oven: 80°C (1 min), 5°C/min to 210°C, cool 15°C/min to 130°C (2 min), 10°C/min to 1st Dimension FID chromatogram 280°C (10 min) Heart-cut: 26.25 to 26.75 min CTS: -20°C (34 min), 20°C/sec to 300°C (1 min) Heart cut MSD SIM: m/z 147, 161, 163, 203, 208, 218, 223

Monitoring (SIM) mode for the analytical separation on Col- umn 2. Stable isotope dilution was performed using d5-Rotun- don as the internal standard. According to Siebert and Barter, the described method combining MASE and LVI minimizes sample preparation workload significantly. Problems with interferences were compensated using internal standards. By heart-cutting the key part of the chro- SIM chromatogram Rotundone matogram for separation onto a second column, a cleanly separated and clearly defined rotundone peak is obtained. The scientists reported the analysis method to be accurate, precise, rug- ged, and sensitive leading to limits of quantitation in the low ppt range and high sample throughput. The statistical data speak vol- 2nd dimension separation of umes: “In the Shiraz grapes (0 to 2000 ng/L) as well as in wine coeluting (0 to 500 ng/L), the limits of quantitation (LOQs) were 5 ng/L. 2 compounds The linear correlation (R ) was >0,999 and the reproducibility expressed as standard deviation was below 3 % (n=6) both for low and high concentration values – a very good result”, the wine scientists reported. They went on to say that “the described method will allow more detailed research on the formation of rotundone in the grape. This includes the maturation processes involved as well as whether yeast plays a role by influencing the extraction Rotundone in red wine: calibration and repeatability process. Such knowledge will make it more likely that we will be able to describe how rotundone is synthesized in the grape and ultimately control the rotundone concentration in the wine.” Fur- 3.0 ther investigations are planned and a scientific publication of the research results is scheduled to appear in the near future.

2.5 Calibration 0 to 500 ng/L Low reps 20 ng/L High reps 200 ng/L Literature

) 2.0 L / U ( [1] Siebert, T. E. and Barter, S. R.: Determination of the potent flavour o i t

a compound rotundone in grapes and wine using MDGC-MS and R 1.5 e membrane assisted solvent extraction, Posterpräsentation, 37th s y = 2.7898x + 0.0323 International Symposium for Capillary Chromatography (2013) R² = 0.9996

es po n Palm Springs, USA. R 1.0 [2] Wood, C.; Siebert, T. E.; Parker, M. et al.: From Wine to Pepper: Rotundone, an Obscure Sesquiterpene, Is a Potent Spicy Aroma Compound, J. Agric. Food Chem. 56 (2008) 3738-3744 0.5 [3] Schellin, M. and Popp, P.: Membrane-assisted solvent extraction of polychlorinated biphenyls in river water and other matrices combined with large volume injection–gas chromatography–mass 0.0 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 spectrometric detection, J. Chromatogr. A. 1020 (2003) 153–160 Concentration Ratio (U/L) [4] www.gerstel.com/en/automated-membrane-extraction-mase. htm

GERSTEL Solutions Worldwide Wine Special 11 Lab on the slopes Flavor from trash cans

To perfect the composition of a wine, scientists start by separating the score of flavors into individual notes. In their quest for a bouquet of fragrant high notes, scientists at the Research Institute Geisenheim rely on some rather unusual methods.

n a German vineyard, scientists of the complicated wine system and to influence California or South Africa, would result in IResearch Institute of Geisenheim practice the development of plants and grapes by significantly less flavor compounds being an unusual, but very sophisticated type of changing various parameters. formed. The wine would never develop the viticulture using hundreds of trash cans. The “In the trash cans, we investigate the characteristic fruity freshness. Red wines, on Geisenheim wine researchers are systematically development of the vines by specifically the other hand, have difficulties in Germany, pursuing the lofty goal of creating perfectly varying the intensity of sunlight, the supply even though the hot summer of 2003 balanced wine. They must follow an intricate of nutrients and the precipitation”, says Prof. prompted renewed discussion on whether it path so that factors which could influence Löhnertz. „We now strongly suspect that would be possible to cultivate a strong, full- the taste or cause off-odor is addressed in the climatic stress is the cause of UTA.“ bodied red wine in northern regions as well. vineyard, not in the cellar. A plant is stressed by factors such as strong UV radiation, high temperatures like Quality at the expense of quantity those experienced in Europe in the summer Searching for off-odors of 2003, and by a shortage of nutrients in the

soil. The undesirable flavor that can originate The soil type in which a vine grows in part

A white wine contains 800 to 1000 different from stress conditions is formed by young determines the flavor of a wine. But, by what taste and flavor components, a red wine even wine just a few months after fermentation. In mechanism does this happen? To answer this more. In their laboratories, the Geisenheim small doses, 1-AAP is a pleasant flavor with question, winegrowers must be familiar with scientists try to break down the flavor into a fruity note. Increase the dose, however, and the wine’s flavor compounds, and how these its individual components. Most recently, the unpleasant “mothball” odor comes to the are formed based on both soil conditions they identified „atypical aging flavor“ forefront. The “aging” flavor is not only found and microclimate. Once these substances (UTA), which is caused by the presence of in German vineyards, this research will apply are determined, and the mechanism on how 2-aminoacetophenone (2-AAP). Wines to all wines that have problems with UTA. they are generated is known, their formation containing 2-AAP are flat and appear can be directly influenced. Additionally, if a excessively aged with a distinct fragrance winegrower sets out to produce top quality Not every grape variety is right for note of mothballs. It has been due to the wines, he must reduce the crop size. 2-AAP that some Riesling wines didn’t pass every vineyard The idea behind this is simple: The flavor the official quality inspection: „We want to compounds that are generated by the vines find out why lately, in some German white From a scientific point of view, wine quality are shared between fewer grapes and so they wines such as Riesling, Kerner and Müller- depends mainly on two initial factors: climate are available in higher concentrations; in Thurgau an off-odor develops with increasing and soil conditions. Thus, winegrowers have turn, the flavor of each grape becomes more age” explains Prof. Otmar Löhnertz. to carefully consider which grape varieties are pronounced. The expert oenologist explains that the best suited for their growing area. Planting „We are trying to optimize the fruit trash cans are used to better observe the a Riesling in a warm climate zone, like flavor“, says Prof. Löhnertz. When leaves are

12 GERSTEL Solutions Worldwide Wine Special GERSTEL Solutions Worldwide Wine Special removed from the Riesling vines, for example, there: Currently, fruity, moderately sparkling effects of growing conditions, sunlight, the grapes are exposed to more sunlight wines are popular. These can be produced and soil quality, susceptibility to diseases, which helps produce larger amounts of the by growing wine under cooler conditions at fungi and pests. „When it comes to the compounds that are responsible for the fruity higher elevations or by fermenting the grape really important decisions, I use my gut flavor. However, mistakes and pitfalls must be juice at lower temperatures. feeling: You cannot produce a good wine avoided in many parts of the process. Many wine growers consider wine design without a healthy dose of creativity“, says Otmar Löhnertz mentions that that follows such market requirements a Rowald Hepp, who grows only Riesling in winegrowers often seek his advice on how potential problem. What limits should we his vineyard “Schloss Vollrads”, according to address risks to product quality. „We are impose on wine design? The limits of most to the magazine ‘Capital’ among the top especially contacted by winegrowers that lack in the industry were certainly crossed when one hundred in the world. „To create wine the chemical analysis capabilities in their employees of a winery added bell pepper with individual personality, you need to go cellars.” flavor to the wine. To most wine professionals, beyond science and call on your intuition“, adulterating wine with flavors foreign to the says Carlos Moro, a winegrower from Spain. species is nothing short of sacrilege. Some For amateurs, the basics may be Analyzing flavors is like decomposing companies have contemplated diluting strong comprehensible: Taste and flavor is a musical score into single notes. red wines with water since customers are less controlled by maturing the wine in a barrel interested in wines with high alcohol levels. In under the influence of yeast. The details

The Geisenheim Research Institute maintains Geisenheim, stricter rules apply concerning of how to influence the fermentation to a number of laboratories dedicated to flavor the limits for designer wines. „If this trend provide top wines, however, is still more of analysis. Researchers inject flavor extracts of continues, we will approach a situation where an art than a science, understood only by wines into high resolution gas chromatography only synthetic mass products are available”, master wine makers, each with his or her systems, trying to determine every nuance of says Klaus Schaller, head of the research well kept secrets. the extract by separating it into hundreds of institute. The thought is clearly not appealing If wine growers are forced to conform individual components. It is like decomposing to the seasoned oenologist. to mass market tastes, “the growers and a large musical score into single notes: high or wine makers that thrive on authenticity low, dominant or subtle. The only difference will not survive in the long run”, warns A seasoned gut feeling and intuition is that this performance is not rendered by Klaus Schaller. Just a single evening spent musicians, but by chemists, who sniff out the to hit that special note in good company sampling a wide variety flavor note and assign a sensory attribute to of wines served in proper glasses can give each molecule. In doing so, it is possible to Then what is the noble art of wine making? you an impression of the word “authentic”, link chemical analysis with olfactory sensing First of all, it requires expertise on the and why it is worth preserving. in an ideal way, enabling sensory wine analysis. The effluent from the GC column is split so that it arrives simultaneously at the nose and at another detector, usually a mass Olfactometry spectrometer since it is capable of specific compound identification. The compounds are presented to the nose through the use More than 2,000 GERSTEL ODPs of an Olfactory Detector Port (ODP), and trained personnel determine the identity and intensity of the flavor compounds both sold – Sniffing out odors of which are recorded as part of the analytical data file.

When you need to pin down odor causing Wine flavors dictated by the market compounds, standard analysis methods quickly reach their limits. Only the parallel use of analytical instrumentation and the human GC/ODP analysis can help to understand olfactory senses provides real answers. The the flavor composition of good wines, but GERSTEL Olfactory Detection Port (ODP) can of course also be used when products are makes it happen! More than one thousand recreated “synthetically”, helping to create users have opted for the ODP connected to flavors with popular appeal for mass market their GC/MS system – not even counting consumption. According to the Geisenheim the more recently introduced ODP 3 with experts, international mass markets seem to heated mixing chamber. The ODP enables require less personality than ever. Instead, sensory detection of odors by the human global trends are set by wine gurus like the nose simultaneously with analytical detec- French oenologists Michel Rolland, who is tion by any GC detector, including MSD, FID, a dominating figure in wine circles on every and FPD. Voice recognition software allows continent and by the American Robert the sensory analyst to describe odors and Parker. A winery, whose product receives a fragrances in real time – these voice descrip- positive review in Parker’s Wine Advocate tors are recorded and converted to editable magazine, can pop the Champagne corks text files. For each GC/MS run a complete ponent, and the olfactogram is recorded and celebrate. Such is the critic’s influence report is generated, including a chromato- with this intensity information. The ODP is on the market. Hence, many wine makers gram superimposed with an annotated an effective tool for obtaining simultaneous comply with Parker’s preferences. In this olfactogram. Text of the descriptors spoken sensory and analytical information to deter- by the analyst is placed above each olfac- case, profit rules the market and dictates taste; mine flavors and odors in foods, beverages, togram peak. The analyst can assign any of fragrances, and other complex samples and science becomes the tool to help tailor the four intensity levels to each eluting com- to help identify sources of odors. wine to meet the trend. Slightly more black currant flavor here, a little more freshness

GERSTEL Solutions Worldwide Wine Special 13 GERSTEL Solutions Worldwide Wine Special

Advanced Flavor and Fragrance Analysis An extra GC dimension at your finger tips

Gas chromatography (GC) experts rely on sharp peaks and baseline resolution to provide accurate answers. To perform chromatographic analysis of real-world samples, analysts often must deal with either complex sample types such as essential oils and petroleum fractions, or complex matrices like biological fluids, foods, sludge, or polymers. Once the sample has been prepared for analysis, separation of all the individual compounds present by means of a single chromatographic separation can be challenging due to the compounds having different ranges of polarity, boiling point, solubility, MW, and concentration. It is therefore necessary to use innovative yet robust techniques that go beyond using a single chromatographic dimension to achieve compound separation. This sounds simple enough, but until now it hasn’t been, since techniques that require a second dimension (column) require a lot of additional hardware, including an extra GC. Not anymore: The patented GERSTEL Selectable 1D/2D-GC/MS System enables the best of both worlds. The system can be used for routine single dimensional GC/MS analysis, and with the click of a mouse, can be switched to perform two-dimensional separation when needed for more complex matrices. This allows interesting sections of the chromatogram to be collected and concentrated from multiple runs to better separate and isolate trace compounds. This can, for example, be used for trouble shooting when off-odors are detected in a product. All this is performed using just one GC/MS system.

ood analysis is certainly not a trivial matter. peaks in the chromatogram, the analyst needs in the laboratory, such solutions didn’t always FThe typical matrix is complex, often to have a good tool kit at her or his disposal. In provide the best return on investment (ROI). requiring several sample preparation steps this case, selectable multidimensional GC can GERSTEL now offers a solution that and extensive sample clean-up. However, even be the technique that cuts through the thicket can be used for routine analysis as well as for well prepared samples can produce forests of and provides clear, reliable answers whenever special challenges. The patented GERSTEL overlapping peaks making it a case of not one-dimensional GC does not. Selectable 1D/2D-GC/MS is a flexible being able to see the trees for the forest. If Until now, multi-dimensional GC system, based on a single standard GC/MS a case of unresolved peaks is clearly at hand, required the use of a dedicated system with instrument. It is both a routine analysis system or if an odor detected by using an Olfactory two GCs coupled to each other. Due to the and a complex problem solving system that Detection Port (ODP) doesn’t match the extra cost, and to the often limited utilization offers heart-cutting and two-dimensional

SBSE – 1D/2D GC-O/MS determination of odor-active compounds in an alcoholic beverage

1D GC-O/MS analysis Heart-cutting and Backflushing Simultaneous MS and GC-O/MS can help locate the region of odor-active com- The system can be configured to perfom 2D GC-O/MS analy- olfactory detection is pounds within a complex Chromatogram, but insufficient sis without any hardware or column connection changes. After possible in both 1D and resolution may still prevent reliable compound identification. A heart-cutting, the 1st dimensional column can be backflushed. 2D GC analysis modes. heart-cut of the region of interest followed by separation on a An additional cryo-trap device is available if necessary. second column (second dimensional separation) provides the resolution needed to accurately identify individual compounds.

14 GERSTEL Solutions Worldwide Wine Special Selectable 1D/2D GC-Olfactrometry (O)/MS System

1D GC-O/MS analysis 1D GC-O/MS is operated using LTM- GC Column 1. At the same time, carrier separation on demand. Because of this dual gas is supplied to the LTM-GC Column functionality, when questions arise regarding 2 by the PCM. a section of the standard one-dimensional chromatogram, this section in question can be transferred to a 2nd dimension, i.e. a GC column with different polarity to further increase separation. Both columns are installed in the same GC and are heated Heart-cutting independently using Low Thermal Mass User selected peaks eluted from (LTM) technology.The process of cutting a LTM-GC Column 1 are transferred onto section of a chromatogram and introducing LTM-GC Column 2. The transferred it to another column is called heart-cutting. peaks are focused at the head of the The 1D/2D system can be used to determine LTM-GC Column 2 with or without analytes in either the 1st or the 2nd dimension cryo-trapping depending on the compound volatility. in a flexible manner. Neither the GC run, nor analyte detection is interrupted during the run. Detection of the analytes that were transferred to the 2nd column follows using the same detector(s) used for the 1st dimension: MSD, Olfactory Detection Port (ODP), PFPD etc. etc. Should lower detection limits be required for the analyte in question, the system enables heart-cutting from multiple repeat injection, 2D GC-O/MS analysis with cryofocusing of the sections that were After heart-cutting, the bulk of the cut, on a GERSTEL Cryo Trap System sample can be effectively back flushed. (CTS). The accumulated sections are then A few minutes after the heart-cut, nd the temperature program for LTM-GC transferred to the 2 dimension once there Column 2 is started. is sufficient mass on column to perform the determination.

Wide Range of Applicability would benefit from having such a system click through the GERSTEL MAESTRO available at a reasonable cost. Further, software, integrated with the Agilent Until now, Selectable 1D/2D-GC/MS detection of analytes in both dimensions ChemStation. It couldn’t be simpler. systems have mainly been sold to, and used is performed using one or more detectors for food, flavor, and fragrance analysis, but simultaneously including MSD, ODP, FID More information any application that occasionally requires a or PFPD. The complete system is efficiently www.gerstel.de/pdf/p-gc-an-2010-02.pdf 2nd dimension separation to solve a puzzle and conveniently controlled by mouse-

2D GC-O/MS analysis Library search The peak of interest was well separated, and detected A Wiley library search tentatively identified the peak using olfactometry (ODP) on the second dimension. A well as a b-Damascenone. defined mass spectrum was obtained for the detected peak.

GERSTEL Solutions Worldwide Wine Special 15 Degustation Tasting a wine beats reading about it…

When it comes to nobler or even divine drops, tasting a wine beats reading rave reviews. But a little reading could offer a good starting point when you set out to differentiate noble drops from pleasurable daily or even inferior ones. When mingling with real or would-be connoisseurs, the following vocabulary samples could help you fit in: A wine scientist is called an oenologist; wine expert in a classy restaurant: Sommelier; and while merry wine drinkers enjoy wine tasting, highbrow tasting is referred to as a degustation. In the following, a few points are listed as to how professionals explore the soul of a wine.

White and Rosé wines should be overfill the glass. The glass should be held at driving away the volatile aroma compounds. served cool, depending on the grape the bottom part of the stem, ensuring that the After the initial olfactory assessment, the variety between 7 and 13 °C (Follow wine is not heated by the hand and to keep wine is swirled in the glass and further scents the1 recommendations on the label). The perfume or soap flavors from interfering with emerge from the wine at the bottom of the wine shouldn‘t be colder or fruity notes in the olfactory wine impression. glass. The full picture is always composed the aroma could be lost or diminished. Red of both objective and subjective facets, but wine is best enjoyed at room temperature, Once in the glass, the wine should not be experts generally agree: White wines have typically in the range 16-18 °C, or the alcohol tasted immediately, a visual appreciation gooseberry, strawberry or vanilla aromas; red will overshadow the aroma and bouquet of should be gained first. The color allows Bordeaux wines can exhibit chocolate aromas. the wine. first4 conclusions to be drawn as to grape variety, maturity, quality and condition. A Getting the taste of a wine is a matter Uncork the bottle in a timely manner sweet or suave white wine generally has a for the tongue. Wine tasting therefore and let the wine breathe before tasting deeper yellow hue than most dry varieties. involves not just drinking, but rather it. Aeration will generally round or A higher viscosity indicates higher levels letting6 the wine cascade enjoyably over your soften2 the wine, activating or releasing aromas of sugar as indicated by patterns similar to tongue. The tongue registers sweet, salty and and bouquet allowing a full appreciation gothic cathedral windows when a layer of bitter impressions. The less than appetizing of what the wine brings to the table. This slurping sound generated by professional holds true for both red and white wines, but tasters occurs when air is pulled through the every wine has its own personality, requiring Step by step: oral cavity to aerate the wine and release the individual timing. A German Pinot Noir full impression to the olfactory bulbs in the readily surrenders its black currant notes after Tasting wine nose cavity. Finally, the litmus test: Swallow only 15 minutes. A Barolo from Piedmont or expectorate (discard into the spittoon). The with its characteristic tannins, however, answer is unequivocal: to track the sensation profits from breathing for 60 minutes. For like a pro... in the oral cavity, the wine must go down the a more efficient, or even hasty, aeration, the throat. You expectorate the good drops mainly wine can be decanted, i.e. transferred to a if several wines are to be tasted and the alcohol decanter and even swirled. In case of a well wine slowly flows down the inside of the glass shouldn‘t go to your head. If after tasting a aged wine, however, none of this is advisable; after swirling. Good quality is also indicated wine the taste subsides quickly, it is described the oxygen could make the wine go bad. Too by an intense clarity and shine of the wine, as a short aftertaste - if it persists, it‘s a long much agitation could do the same; the bottle which shouldn‘t be clouded by suspended finish as is to be expected, for example, from should be transported and the wine poured precipitate. Brown coloration of white and high quality wines. And if the taste disappears into the glass slowly and cautiously. red wines alike is a sign of excessive ageing only to reappear, it is called a peacock finish. and the wine should no longer be consumed. According to Wikipedia: „The aftertaste of An authentic optical impression of the a wine can be described as bitter, persistent, wine is best achieved in a clear glass. The olfactory evaluation is the initial short, sweet, smooth, or even non-existent. It should be bulgy with a narrower close encounter with a wine. The well Included in assessing the aftertaste of a wine opening.3 This shape is best suited to retain honed nose is the first and foremost is consideration of the aromas still present the aroma of the wine. According to ISO tool5 of the trade for a wine taster, a well after swallowing. High quality wines typically 3591: „The tasting glass consists of a cup (an trained olfactory sense can help distinguish have long finishes accompanied by pleasant „elongated egg“) supported by a stem resting and determine a large number of aromas and aromas“. As for the sample to sample carry on a base. The opening of the cup is narrower flavors. Aspirating the headspace from the over: Still water with little mineral content is than the convex part so as to concentrate glass and evaluating the olfactory profile is the right interlude between wines to ensure a the bouquet.“ In order to allow the aroma often done with eyes closed to enable full neutral transition. Chewing white bread, on to present itself to the nose, the glass must concentration during the fleeting encounter the other hand could influence the tasting have sufficient free volume (headspace) above with the most volatile fraction. At first, this since enzymes in the saliva convert starch the wine; better to revisit the bottle than to is done without agitating the wine to avoid to sugar.

16 GERSTEL Solutions Worldwide Wine Special Off Flavors in Wine part I: Corky Efficient and sensitive determination of TCA and other off-flavors

When your premium wine tastes corky, it is little consolation that this is not caused by the natural cork material used to produce the classic wine stopper. Corkiness points to the presence of 2,4,6-trichloroanisol (TCA), the most well-known malodorous culprit. But other chemical suspects are at large that can equally cause the unpleasant musty, moldy off-odor assault to your nose, and these may not even be coming from the cork stopper. To ensure an efficient, reliable and sensitive determination of all corkiness- related off-flavor compounds, the DLR Mosel in Germany successfully turned to GC/MS combined with Stir Bar Sorptive Extraction (SBSE).

Barking up the wrong tree – on the ou don’t have to be a sommelier or wine olfactory bulbs sets off Y expert to tell the difference between a perfect the mal-odor alarm origins of Corkiness wine and a corky wine. 2,4,6-trichloroanisol at concentration levels (TCA) has an extremely low odor threshold. as low as 0.5 ng/L”. In The usual suspect as a source of 2,4,6-TCA As little as a few nanograms per liter of air addition, several factors is the cork stopper made from the bark of is enough to detect an unpleasant musty off- could influence sensory the cork oak tree (Quercus suber). TCA is a flavor. In water and wine it is a similar story perception of the off- microbial metabolite, formed by methylation with odor thresholds at 0.3 ng/L and 1.4 ng/L flavors; among these of trichlorophenol (TCP) that may have been Horst Rudy respectively, “but that is only a theoretical are sweetness, alcohol applied to the bark as a pesticide. To suspect value”, says Horst Rudy from the Agricultural content and grape type. the cork stopper of introducing TCA to the Service Center (DLR) of the Mosel wine “Whoever wants to identify corky off-flavor wine is therefore region in Germany. “After all”, the laboratory compounds and track down their source has manager explains, “sensory perception is no choice but to use gas chromatography highly individual and very subjective: While combined with mass selective detection one consumer may sense no problem even at (GC/MS)”, Horst Rudy points out. much higher concentrations, another person’s

GERSTEL Solutions Worldwide Wine Special 17 GERSTEL Solutions Worldwide Wine Special only logical according to the wine expert, but when wine drinkers started experiencing 2,4,6-TCA Area TCA / concentration Area ISTD corkiness in wines with modern polymer- [ng/L] based stoppers experts knew that they had 1 0.38642 been barking up the wrong tree. 2 0.69888 Over the course of the ensuing 3 0.98844 research projects, it was found that various 4 1.36316 compounds, mainly halogenated anisols, would give a musty, moldy note to the wine. Calibration curve for 2,4,6-tri- These compounds could be formed from chloroanisol (TCA): Limit of detection: 0.39 ng/L; Limit of other chlorinated chemicals that are used determination: 0.79 ng/L for cleaning of wine production equipment or for treating wooden transport pallets or packaging material Until the end of the 1980’s, 2,4,6-TBA Area TBA / concentration Area ISTD pentachlorophenol (PCP) was used as a [ng/L] fungicide to protect, for example wooden 1.2 0.20709 pallets from microbial decay. Among 2.4 0.45745 others byproducts, PCP contained 3.6 0.64190 2,3,4,6-tetrachlorophenol (TCP), a 4.8 0.85998 compounds that is metabolized microbially Calibration curve for 2,4,6-tri- to 2,3,4,6-tetrachloroanisol (2,3,4,6-TeCA, bromoanisol (TBA): Limit of TeCA), which also causes corkiness in wine. detection: 0.50 ng/L; Limit of In animal tests, PCP was found to be determination: 1.0 ng/L carcinogenic. In Germany, the use of PCP has been prohibited since 1989. PCP was

Limit of Detection Odor thresholds Limits of detection and odor thresholds of the corkiness-causing compounds determined using 2,4,6-Trichloroanisole (TCA) 0.3-0.5 ng/L 1.4 - 4 ng/L SBSE-GC/MS. 2,4,6-Tribromoanisole (TBA) 0.5 ng/L 3 - 8 ng/L 2,3,4,6-Tetrachloroanisole (TeCA) 1.1 ng/L 4 - 24 ng/L 2,4,6-Trichlorophenol (TCP) 1.4 ng/L 4000 ng/L 2,4,6-Tribromophenol (TBP) 1.6 ng/L Pentachlorophenol (PCA) 0.9 ng/L 4000 ng/L

Chemical analysis and sensory evalu- substituted by 2,4,6-tribromophenol (TBP), a combined fungicide and flame retardant, which ation – complementary techniques SIM chromatogram is often used to protect cardboard packaging, of 1.0 ng/L TCA polymer materials, paints and coatings. As it When the DLR Mosel is asked to determine turns out, microorganisms metabolize TBP the cause of a musty and moldy off flavor in a to 2,4,6-tribromoanisol (2,4,6-TBA), a wine, sensory evaluation is only the first step compound given the sensory attributes musty, in the process. “While corky off-flavors are earthy, and chemical with a smell of solvent. typically determined quite reliably”, Horst “TBA is a corkiness causing compound of the Rudy says, “TCA concentrations at or below first order”, Horst Rudy points out. the odor threshold often lead to a subtle and indefinable change in the wine flavor, not perceived as a corky flavor note”. In such cases, 1 File TCA [ng/L] chemical analysis is needed in order to prove Day 1 1511041 6.1 that the wine is under the influence of TCA. 1511042 4.9 To snoop out the source of the contamination, 1511043 4.7 all aspects of the wine production and bottling SIM chromatogram of 2.4 ng/L TBA 1511044 4.5 process, as well as the entire production site Day 2 1511045 8.5 environment, must be carefully investigated. 1511046 6.4 Horst Rudy and his team deploy passive 1511047 5.4 samplers based on Bentonite clay to pick up Day 3 1511048 7.4 TCA traces. “Passive samplers are easy to work 1511049 6.1 with and they deliver valuable information such as a distribution profile enabling us to 15110410 5.3 more accurately localize the source”, says Mr. Standard deviations under real laboratory condi- Rudy. As a general rule, the DLR does not 2 tions. A 1.5 L sample of water used to wash cork restrict its GC/MS investigations to off- material was homogenized and ten separate flavors that are perceived as corky. Among the aliquots were extracted using SBSE (GERSTEL Corkiness-related off flavor compounds found targeted compounds are: 2,4,6-trichloroanisol Twister). GC/MS analyses were performed over in wine: 2,4,6-trichloroanisole (TCA [1]) and (TCA), 2,4,6-tribromoanisol (TBA), three consecutive days. Mean value: 5.9 ng/L; 2,4,6-tribromoanisole (TBA [2]). standard deviation s = 1.26 ng/L; Minimum value 2,3,4,6-tetrachloroanisol (TeCA), 4.5, maximum value 8.5 ng/L. 2,3,4,5,6-pentachloroanisol (PCA), as Text: Guido Deußing Text:

18 GERSTEL Solutions Worldwide Wine Special Step by step: How to extract odor active compounds from wine or water using the GERSTEL Twister

2 Instead of six hours per analysis, the Twister based analysis only requires 1.5 hours in order to determine the concentration of corkiness-related off flavor compounds. Multiple samples can be processed simultaneously. well as the TCA and TBA precursors Rudy. More modern analysis techniques, 2,4,6-trichlorophenol (TCP) and such as Solid Phase Micro-Extraction 2,4,6-tribromophenol (TBP), which are less (SPME) enabled the DLR to reduce the odor-active. The presence and distribution analysis time significantly, but the limits of 3 of TCP and TBP can give valuable detection achieved, for example 2.9 ng/L for information as to the source of an off-flavor. TCA, meant that this technique was only of To determine the identity and limited use. “We have to reliably determine concentration of odor agents, cork stoppers concentrations of odor-active compounds are extracted for two hours in a 10 % at their odor threshold levels”, says Horst ethanol-water mixture using sonication to Rudy, “and for this reason we started using speed up the process. The bentonite clay SBSE and the GERSTEL Twister”. SBSE used for passive sampling is extracted in is a fast and accurate extraction technique the same way. Subsequently, 100 mL of the that enables the DLR laboratories to reach a Ethanol solution is extracted for one hour detection limit of between 0.3 and 0.5 ng/L 4 by Stir Bar Sorptive Extraction (SBSE) for TCA as per the DIN 32645 method and using a GERSTEL® Twister™ (The the analysis time has been reduced from 6 bentonite should be allowed to precipitate hours to 1.5 hours per sample and multiple before sampling is performed). The Twister samples can be processed in parallel for is a glass-coated magnetic stir bar with improved productivity. an outer Polydimethylsiloxane (PDMS) SBSE is extremely easy to perform: layer. While the Twister stirs the sample, Following the extraction step, the Twister analytes are extracted and concentrated is removed from the sample, dried using a into the PDMS phase. Depending on the lint-free paper cloth and transferred to the 5 application and on the sample volume autosampler tray. Up to 196 Twisters can be available, SBSE can be up to 1,000 times desorbed and analyzed by GC/MS in a single more sensitive than SPME due to both the batch using the GERSTEL MPS and TDU significantly larger PDMS volume available directly mounted on a GC/MS system. The and to the larger sample volumes extracted. work reported in this article was performed Quantification in this work was performed using a GC 6890/MSD 5975 (Agilent using 2,4,6-trichloroanisol-D5 as internal Technologies). standard.

6 Fast and sensitive analysis More information using the GERSTEL Twister Horst Rudy, DLR Mosel, Dept. of Viticulture Place the Twister in the sample (1). and Oenology, Egbertstrasse 18-19, 54295 While stirring the sample, the Twister concentrates analytes in its PDMS The multi-stage liquid-liquid extraction Trier, Germany, Phone +49 (0)651/9776- phase (2). The Twister is removed previously used by DLR Mosel was highly 187 or -185, -186 Fax +49(0)651/9776- labor- and cost intensive. “Sometimes I from the sample (3), dried with a 193, E-mail: [email protected], www.dlr- lint-free cloth (4) and placed in the spent all day in the laboratory and still only mosel.rlp.de MPS tray (5) for automated thermal managed to analyze four samples”, states Mr. desorption in the TDU (6).

GERSTEL Solutions Worldwide Wine Special 19 Off flavors in wine part II: Sulfur related flavors – reductive notes Watch your sulfurs!

The flavor of a wine is heavily influenced by sulfur-containing compounds, which, among other things, give wine the flavor typical of the grape variety used. At the same time, sulfur containing compounds are responsible for some wine flaws or wine defects that are commonly referred to as reductive notes. To determine the presence and quantity of sulfur-containing compounds for the purpose of quality control, wine experts at the Geisenheim Research Center rely on Headspace Gas Chromatography (HS-GC) combined with pulsed flame photometric detection (PFPD).

Sulfur is widely used in the wine industry. off-flavor. Sulfides in particular cause off- methanethiol and ethanethiol, which give the More precisely, sulfur dioxide (SO2) is flavors that often lead to rejection of the wine wine a cheesy or burnt rubber note. Often the used as a preservative and disinfectant since in the quality control phase [5, 6]. presence of just minute amounts of volatile it reacts with oxygen to prevent oxidation sulfur compounds will mask characteristic reactions that could have undesirable effects notes or fruity notes of a wine making it Sulfur-containing compounds on the flavor and color of the wine. In appear dull and less expressive of its type. cause wine faults addition, SO2 inhibits growth of bacteria and wild yeasts, resulting in faster and cleaner How a wine arrives at fermentation during which important sulfur- Until only a few decades ago, the off-flavor containing compounds are formed, albeit in often described as “rotten eggs” was thought its (off-)flavor… significantly reduced amounts. Volatile sulfur- to be caused by a too high concentration of containing compounds, especially thiols hydrogen sulfide (H2S). In the meantime Apart from chemical, photochemical (R-SH), are highly odor-active compounds a plethora of compounds have been found and thermally induced reactions during that lend characteristic notes to many foods. that can cause or contribute to this off- preparation and storage of wine, a multitude Among these are onion, garlic, leek, cabbage, flavor. The flavor notes of individual sulfur of enzymatic reactions take place, which could asparagus, tropical fruits (such as maracuja compounds, as well as their respective cause a reductive note. Research has shown that and pineapple), grape fruit, black currant, concentrations, are decisive factors in the especially the nitrogen and sulfur metabolism gooseberries as well as cooked or fried meat, final sensory impression and in the olfactory of yeast has great impact on the formation of cheese, and coffee. intensity of the flavor profile. To describe a sulfur containing flavor compounds. Sulfate, Thanks to new extraction and enrichment reductive note, descriptors such as reductive, naturally present in grape juice, is taken up techniques and more selective and sensitive stuffy, unclean, bacony, or cheesy are used. by the yeast and reduced to sulfide. Reduced analysis techniques, a wide range of sulfur Consumers tend to associate faulty wines Sulfur (S2-) is used by the yeast to synthesize containing compounds have also been with overly ripe tropical fruit, black currant, sulfur-containing amino acids e.g. methionine determined in wine in recent years. Some of burnt rubber, bread crust, yeast extract, or a and cysteine as well as peptides and proteins these contribute significantly to the typical dull moldy smell. The descriptor reductive has used to form enzymes. To synthesize sulfur flavor of grape varieties. 4-Mercapto-4- gained acceptance internationally even for less containing amino acids, the yeast must not only methylpentan-2-on (4-MMP), for example, intensive sulfur caused off-flavors. Growing reduce the sulfate, it must also take up nitrogen is a key compound in Sauvignon Blanc flavor the vines under conditions that reduce the in the form of ammonia or amino acids as well [2]. Thiols, formerly known as mercaptans, amount of oxygen taken up by the plants can as sugar used as an energy source. If there is a are often blessed with an extremely low odor help reduce oxidation of those thiols typical of nitrogen deficiency, the sulfide formed in the threshold. Concentrations in the microgram the grape variety helping to stabilize the levels yeast cannot be converted to sulfur-containing or even nanogram per liter range can be of off odor causing compounds such as H2S, amino acids since the required building blocks sufficient to cause a noticeable and decisive

20 GERSTEL Solutions Worldwide Wine Special S-Compound Abbre- Retention viation time

Hydrogen sulfide H2S 3.09 Methanethiol MeSH 5.58 Ethanethiol EtSH 9.02 Dimethylsulfide DMS 9.75

Carbon disulfide CS2 10.56 Methylisopropylsulfide1 MiPS 15.11 Thioacetic MeSAc 15.55 acid-S-methylester Dimethyldisulfid DMDS 16.94 Thioacetic acid-S-ethylester EtSAc 17.45 Butylmethylsulfide2 BMS 18.60 Ethylmethyldisulfide MeSSEt 19.11 Diethyldisulfide DEDS 20.93

1 Internal Standard 1 for calibration and quantitation 2 internal Standard 2 for control (peak area ratio of the two standards)

Table of compounds analyzed by HS-GC-PFPD in Wine. The determination of volatile sulfur compounds is critical for wine quality assessment. In addition to sensory evaluation, a reductive wine fault can be determined unambiguously using Analysis of faulty wines with five replicates of each. The main known causes of reductive notes are a lack analytical instrumentation. of yeast nutrients during fermentation or very reductive winemaking conditions.

Proben- H S MeSH EtSH DMS CS2 MeSAc DMDS EtSAc DEDS Wein Nr. 2 nummer (μgL-1) (μg-1) (μgL-1) (μgL-1) (μgL-1) (μgL-1) (μgL-1) (μgL-1) (μgL-1) 060107 A 6.1 2.2 5 2.8 5.2 41.1 0.6 53.3 5.1 B 6.1 2.1 5.1 2.7 5.2 41.6 0.6 53.9 5 C 5.9 2.2 5 2.8 5.3 41.6 0.6 53.1 5.1 D 6.1 2.2 4.9 2.8 5.2 42.5 0.6 53.8 5.2 E 6 2.2 5.1 2.6 5 41.2 0.6 52.7 5.2 x (μgL-1) 6.04 2.18 5.02 2.74 5.18 41.6 0.6 53.36 5.12 s 0.089 0.045 0.084 0.089 0.110 0.552 0.000 0.498 0.084 VK in % 1.481 2.051 1.667 3.264 2.115 1.328 0.000 0.933 1.634

060113 A 363.8 12 57.9 7.3 90.1 74.8 n. d. 38.1 0.6 B 362.3 12.2 56.6 7.5 89.3 74.3 n.d. 37.8 0.5 C 361.7 12.4 56.9 7.2 89.9 74.6 n.d. 37.1 0.6 D 370.2 11.9 56.5 7.3 90.8 75.4 n.d. 38.3 0.6 E 362.5 12.1 57.6 7.3 91.1 73.8 n.d. 37.8 0.6 x (μgL-1) 364.1 12.12 57.1 7.32 90.24 74.58 37.82 0.58 s 3.495 0.192 0.620 0.110 0.720 0.593 0.455 0.045 VK in % 0.960 1.587 1.087 1.497 0.798 0.796 1.203 7.711

060119 A 98.5 6 18.9 9.6 95.6 81 0.9 44.8 3.2 B 99.6 6 19.2 9.6 98.8 81 0.9 43.2 3.1 C 98.3 6.2 19.2 9.8 96.2 81.7 0.9 43.5 3.1 D 97.3 6.1 18.9 9.7 95.2 81.7 0.8 44 3.2 E 90 6.2 18.6 9.7 98.4 82.3 0.9 45.5 3.8 x (μgL-1) 96.74 6.1 18.96 9.68 96.84 81.54 0.88 44.2 3.28 s 3.855 0.100 0.251 0.084 1.652 0.550 0.045 0.946 0.295 VK in % 3.985 1.639 1.324 0.864 1.706 0.675 5.082 2.140 8.993

060201 A 167.9 15.1 83.4 4.5 16.7 101.2 n.d. 37.6 0.4 B 166.6 15 84.2 4.6 16.4 100.9 n.d. 37.1 0.4 C 168.9 15 83.8 4.4 16.3 99.9 n.d. 37.9 0.5 D 167.9 15.2 84.3 4.8 17 100.1 n.d. 37.2 0.4 E 169.2 15 82.3 4.2 16.7 99.6 n.d. 37.1 0.5 x (μgL-1) 168.1 15.06 83.6 4.5 16.62 100.34 37.38 0.44 s 1.022 0.089 0.809 0.224 0.277 0.680 0.356 0.055 VK in % 0.608 0.594 0.968 4.969 1.670 0.678 0.953 12.448

A reductive wine fault that has been determined by a sensory panel can be confirmed and assessed or quan- x - Median s – Standard deviation tified using HS-GC-PFPD. The picture shows the analysis system used by the Geisenheim Research Center for CV – Coefficient of Variation the determination of reductive notes. n. d. – not detected (below the limit of detection)

GERSTEL Solutions Worldwide Wine Special 21 Wine with fault No.

MiPS BMS internal internal standard standard Analysis A

Analysis B

Analysis C

Analysis D

Instrumental analysis doesn’t replace sensory evaluation, but it delivers facts that can be used for unambiguous assessment of wine quality.

Analysis E

are not available in sufficient quantities [1]. Five replicate chromatograms of a wine with reductive note wine fault. Under normal conditions, the yeast forms only as much sulfide as is needed. If there is a nutrient deficiency, however, the sulfide synthesis cannot be fully controlled and excess Wine with fault sulfides are formed, which are partly released

MiPS BMS No. internal internal into the wine as hydrogen sulfide, and partly standard standard reacted to form highly odor active compounds such as methanethiol, ethanethiol and their oxidation products dimethyldisulfide and diethyldisulfide. An elevated level of hydrogen sulfide also leads to enhanced synthesis of thioacetic acid esters, most notably thioacetic acid-S-methylester and thioacetic acid-S-

MiPS BMS internal internal ethylester. Both these compounds have odor standard standard Wine without fault No. thresholds in wine above 40 μg/ L, which means that they only contribute to wine faults at significantly enhanced levels. During storage and maturation of the wine, hydrolysis of thioacetic acid esters and other fermentation esters takes place until a chemical equilibrium has been established. The rate and the degree to which hydrolysis takes place depends on the Stacked view of chromatograms resulting from a wine with reductive note wine fault (top) pH of the wine and the storage temperature. and without (bottom trace). During hydrolysis, the very volatile and highly odor active compounds methanethiol and ethanethiol are released, both of which have an odor threshold around 1 to 2 μg/L. This means that the release of even the smallest amounts of thiols during bottling and storage can result Calibration curve for in a reductive note or a wine fault [5, 6]. diethyldisulfide (DEDS). Quality Control based on HS-GC with PFPD Detection

In recent years, yeast problems and reductive notes have increasingly been experienced Calibration curve for internationally. Stress-causing physiological methanethiol (MeSH). impact on vines during the growth season was thought to be responsible for these effects, for example, caused by a lack of water and nitrogen. If nitrogen is not

22 GERSTEL Solutions Worldwide Wine Special GERSTEL Solutions Worldwide Wine Special

Sample Preparation Storage temperature for samples prior to sample preparation: 4 °C be influenced or treatment started before a 10 mL Headspace vials purged with Argon prior to sample loading problem gets out of hand to prevent or correct Sample volume: 5 mL wine flaws or faults. The analysis method Internal standards: Methyl-isopropylsulfide (6 μg S/L) and presented here is in use both for wine quality butylmethylsulfide (6 μg S/L) assessment and in support of various research projects. In addition to the studies on sulfur Reagents: 2,6-Di-t-butyl-4-methyl-phenol, 4 mg/L metabolism of yeast and lactic acid bacteria NaCl, 1.7 g/5 mL during wine production we have, for example, EDTA, 0.2 g/L studied the sulfur metabolism of Lactobacillus SO -binding agent: Propanal, 500 mg/L 2 casei during cheese production in cooperation with the working group of Dr. Stefan Irmler Headspace (HS) Conditions [3, 4]. Headspace Autosampler: GERSTEL MultiPurpose Sampler (MPS) Injection volume: 1000 μL Syringe temperature: 63 °C; Authors Sample equilibration temperature: 60 °C Equilibration time: 45 min. Prof. Dr. Doris Rauhut and Mgr.-Ing. Beata Beisert Dept. of Microbiology and Biochemistry GERSTEL Cooled Injection System (CIS) Geisenheim Research Center Glass liner, deactivated, filled with quartz wool Von-Lade-Strasse 1 65366 Geisenheim, Germany Temperature program: - 100 °C (0 min) → 12 °C/s → 40 °C (1 min) E-mail: [email protected] → 12 °C/s → 180 °C (8 min) Split ratio: 10:1

GC 6890 (Agilent Technologies) Literature GC Column: SPB-1 Sulfur, 30 m x 0.32 mm I. D. x 4 μm 1 | Bell, S.-J. und Henschke, P.A. (2005), „Implica- film thickness, Supelco tions of nitrogen nutrition for grapes, fermentation Temperature: 29 °C (7 min) → 10 °C/min → 180 °C (10.5 min) and wine”; Australian Journal on Grape and Wine Carrier gas: Helium (He) Research, Vol. 11, 3, 242-295 Linear carrier gas velocity: 20 cm/s at 60 °C 2 | Darriet, P., Tominaga, T., Lavigne, V., Boidron, J., Dubourdieu, D. (1995), „Identification of a powerful aromatic compound of Vitis vinifera L. PFPD 5380 (OI Analytical) var. Sauvignon wines: 4-mercapto-4-methylpentan-2-one“; Flavour and Fragrance Journal 10, Detector temperature: 250 °C 385-392 Combustion gas settings: Air 420 kPa; Hydrogen: 420 kPa 3 | Irmler, S., Raboud, S., Beisert, B., Rauhut, D., Berthoud, H. (2008), „Cloning and characterization of two Lactobacillus casei genes encoding References for the analysis of volatile sulfur-containing compounds in wine, materials, methods and a cystathionine lyase“; Appl. Environ Microb 74: reagents: [7, 8, 9] and modified [3, 4]. 99-106 4 | Irmler, S., Schäfer, H., Beisert, B., Rauhut, D., Berthoud, H., (2009); „Identification and characterization of a strain-dependent cystathionine b/g- lyase in Lactobacillus casei potentially involved in present in the proper form and in adequate wine samples without any pre-treatment. cysteine biosynthesis“; FEMS Microbiol Lett 295, amount in the grapes, the grape juice will The introduction takes place directly 67-76 Appl. Environ. Microb. 74: 99-106 not have sufficient nitrogen for a successful from the headspace vials based on very 5 | Rauhut, D. (2003); „Impact of volatile sulfur fermentation [1, 5]. In order to monitor the small sample volumes. A large number compounds on wine quality. In: Sulfur Transport and Assimilation in Plants“; Edited by Davidian, ripening of grapes on the vines, control the of sulfur-containing compounds can be J.-C., Grill, D., De Kok, L. J., Stulen, I., Hawkesford, fermentation process, and ultimately arrive determined over a wide concentration M. J., Schnug, E. and Rennenberg, H., Backhuys at the best possible wine quality, a sensitive range. Quantitation was based on using Publishers, Leiden, Netherlands, 121-131 analysis technique and a suitable enrichment methyl-isopropyl sulfide and butyl methyl 6 | Rauhut, D. (2009); „Usage and Formation of technology for labile compounds are needed. sulfide as internal standards, both at a Sulphur Compounds. In: Biology of Microorgan- isms on Grapes, in Must and in Wine”; König H, This is the only way to ensure correct and concentration of 6 μg Sulfur/L. Unden G, Fröhlich J (eds.), Springer-Verlag Berlin, reliable determination of sulfur-containing Heidelberg, 181-207 compounds in wine. Excellent results in terms An ounce of prevention… early 7 | Rauhut, D., Beisert, B., Berres, M., Gawron- of sensitivity, selectivity, and productivity Scibek, M., Kürbel, H. (2005) „Pulse flame photo- were achieved using gas chromatography analysis provides the basis for metric detection: an innovative technique to anal- (Agilent Technologies GC 6890) combined good wine quality yse volatile sulfur compounds in wine and other beverages.“ State of the art in flavour chemistry with Automated Headspace Sampling and biology. Hofmann, T., Rothe, M., Schieberle, (GERSTEL MultiPurpose Sampler MPS Our investigations have shown that P. (eds.) Deutsche Forschungsanstalt für Lebens- in HS mode) and pulsed flame photometric monitoring of undesirable sulfur compounds mittelchemie, Garching, Germany, pp. 363-368 detection (O.I. Analytical PFPD 5380). This in the early stages of grape ripening and 8 | Rauhut, D., Beisert, B., Berres, M., Gawron-Sci- combination of instruments, in particular the fermentation allows the producer to take bek, M., Kürbel, H. (2006); „Poster presentation th automation of headspace sampling with large counter-measures and achieve good results at the 29 International symposium on capillary chromatography”; (Riva, Italy, May-June, 2006). volume introduction to the GC, enables both even if such compounds have started to simple and highly efficient analysis of the be formed. The yeast metabolism can

GERSTEL Solutions Worldwide Wine Special 23 Twister extraction Two is company

A novel Ethylene Glycol- (EG) and Silicone based combined sorbent phase has been developed for stir bar sorptive extraction (SBSE) using the GERSTEL Twister® with the aim of improving recovery of analytes across a wide polarity range. In this article, the performance of EG-Silicone and polydimethylsiloxane (PDMS) Twisters is investigated to determine their usefulness in generating qualitative flavor profiles of beverages such as whisky, white wine, and multivitamin juice.

tir bar sorptive extraction (SBSE) is based on principles similar Experimental Sto solid phase micro-extraction (SPME). Both techniques rely on partitioning of analytes between a sorbent phase and a liquid sample Samples: Scotch whisky (40 % EtOH v/v); white wine, sauvi- phase, resulting in extraction and concentration of the analytes in the gnon blanc (13 % EtOH v/v) and multivitamin juice. sorbent phase depending on the partitioning coefficient. Following Instrumentation. The TD-GC/MS analysis was performed using sample extraction, the coated stir-bar is thermally desorbed in a flow a Thermal Desorption Unit (TDU) combined with a MultiPurpose of carrier gas, releasing and transferring the analytes to a GC/MS Sampler (MPS) and a Cooled Injection System (CIS 4) programmed system. The most widely used Twister phase is polydimethylsiloxane temperature vaporization (PTV) type inlet (all GERSTEL). An (PDMS), which is non-polar. It has been reported that the extraction Agilent 6890N gas chromatograph with a 5975B inert XL (triple efficiency of the PDMS based Twister can be up to 250 times higher axis) mass selective detector (MSD) was used. The entire analysis than for PDMS based SPME fibers [1] due to the much larger sorbent system was operated under MAESTRO software control integrated phase volume, improved phase ratio and improved phase contact with Agilent ChemStation software using one integrated method and during extraction, all of which enable more efficient extraction and one integrated sequence table. extraction of larger volumes. Successful applications of SBSE include The more polar EG-Silicone Twister does retain some water during extraction and analysis of VOCs, SVOCs, PAHs, pesticides, and off- extraction of aqueous samples, but excess water can be eliminated prior odors in water; drugs of abuse such as Tetrahydrocannabinol (THC), to GC/MS analysis by operating the TDU in solvent vent mode. In barbiturates and benzodiazepines; phthalates and various metabolites this mode, water is evaporated at low initial temperature, for example in biological fluids; flavor compounds, preservatives, thrichloroanisole, at 30-40 °C and ambient pressure (0 kPa) for a pre-determined time pesticides, and fungicides in food and beverages [2,3]. For polar before the temperature ramp for the thermal desorption starts. As a compounds with an octanol-water partition coefficient (Ko/w) lower result, the introduction of water into the GC/MS system is avoided or than 10,000 (logKo/w) < 4), it has been found that recoveries gradually significantly reduced. An alternative way to reduce water background decrease with decreasing Ko/w when using PDMS based Twisters. is to leave the Twisters exposed to a dry atmosphere for approximately Among the more hydrophilic solutes are, for example, polar pesticides, 15 minutes. alcohols, esters, and phenolic compounds. Although recoveries could In this work, we used the TDU solvent vent mode for water removal successfully be improved for many polar pesticides by adding 30 % since it is an automated process, which delivers more reproducible and NaCl (w/w) into the water sample, the salting-out technique does not reliable results. Extraction of aqueous samples using an EG-Silicone necessarily help for all polar compounds and there has increasingly been Twister is performed in exactly the same way as with a PDMS Twister. demand for a Twister with a more polar phase. A new Twister with Aqueous sample was transferred into a 10 mL headspace vial. The a more polar phase is now available from GERSTEL: The Ethylene Twister was added and the vial was sealed with a screw cap. The Glycol (EG) Silicone Twister. This new Twister extracts several extraction was performed at room temperature for 60 min while classes of polar compounds more efficiently than the PDMS Twister stirring at 1000 rpm on a multiple position magnetic stirrer. After due to its polar nature. In addition, the EG-Silicone Twister, since the extraction had been completed, the Twister was removed from it is silicone based, will also efficiently extract non-polar compounds. the sample with a magnetic rod and briefly rinsed with HPLC grade

24 GERSTEL Solutions Worldwide Wine Special GERSTEL Solutions Worldwide Wine Special

Analysis conditions TDU: 40 mL/min solvent vent (0.5 min) EG-Silicone Twister: 40 °C (0.5 min); 120 °C/min; 220 °C (5 min) PDMS Twister: 40 °C (0.5 min); 120 °C/min; 270 °C (5 min) PTV: split 1:10 -100 °C (0.5 min); 12 °C/s; 300 °C (5 min) Polar separation Column: 15 m ZB-FFAP (Phenomenex) di = 0.25 mm df = 0.25 μm Pneumatics: He, constant flow = 1.4 mL/min Oven: 50 °C (2 min); 5 °C/min; 60 °C; 10 °C/min; 165 °C; 20 °C/min, 250 °C (5 min) Non-polar separation Column: 30 m ZB-5 (Phenomenex) di = 0.25 mm df = 0.25 μm Pneumatics: He, constant flow = 1.2 mL/min Oven: 60 °C (2 min); 5 °C/min; 200 °C; 10°C/min; 300 °C (5 min) water. After carefully drying it with a lint-free tissue, the Twister was stored in a 1.5 mL vial. The Twister was finally placed in a TDU glass liner and the liner stored on an MPS sample tray for GC/MS analysis.

Scotch Whisky

The EG-Silicone Twister is especially well suited for extraction of polar compounds which form hydrogen bonds as hydrogen donors, for example, phenols and similar substances. In figure 1, two chromatograms from extractions of a whisky using different Twisters are shown. The EG-Silicone Twister extraction provided the best recovery for phenols, ethyl esters and fatty acids from whisky. It is clearly seen that the EG-Silicone Twister extracts more compounds, and in greater amount. In table 1, peak areas are listed for the annotated compounds shown in the chromatograms. The peak areas that result from the EG-Silicone Twister extraction are an order of magnitude higher Figure 1. Whisky extraction chromatograms obtained using EG-Silicone than the compound peaks obtained using the PDMS Twister for and PDMS Twisters, non-polar column separation. 5 mL whisky almost all compounds. sample (20 % EtOH (v/v), 1:1 dilution with water), 1000 rpm for 1 hour Due to its polydimethylsiloxane basis, the EG-Silicone Twister at room temperature. Peak identification: 1. Phenol; 2. Ethyl hexanoate; 3. o-Cresol; 4. p-Cresol; 5. Phenethyl alcohol; 6. o-Ethylphenol; also has high affinity for non-polar analytes like long carbon-chain 7. 2,4- Xylenol; 8. Ethyl octanoate; 9. Octanoic acid; 10. Ethyl decanoate; ethyl esters and acids. When comparing the chromatograms from the 11. Decanoic acid; 12. Ethyl dodecanoate; 13. Dodecanoic acid. EG-Silicone- and PDMS Twister extractions, it becomes clear that the EG-Silicone Twister extraction (top chromatogram) results in the same number of peaks in the region after 25 minutes, but the peaks Table 1. Peak Areas of marked peaks obtained from extractions using EG- are significantly larger and recoveries significantly better. Silicone and PDMS Twisters. Table 2 shows the extraction efficiency (recovery in %) for selected Peak Compounds Extracted Ion Peak Areas whisky components: phenol, o-cresol, cis-whisky lactone and eugenol, No. [m/z] EG-Silicone PDMS obtained with the two types of Twister from spiked water samples. The 1 Phenol 94 1.1E+07 6.0E+04 highest recovery for phenol and o-cresol was obtained using an EG- 2 Ethyl hexanoate 88 1.8E+06 7.6E+06 Silicone Twister: 5.7 % and 9.8 %, respectively. The PDMS Twister 3 o-Cresol 108 1.5E+07 1.8E+05 gave high extraction efficiency for non-polar compounds like lactone 4 p-Cresol 108 1.1E+07 1.6E+05 and eugenol: 24.3 % and 32.9 %, respectively. 5 Phenethyl alcohol 91 2.4E+07 1.6E+06 These results prove that the EG-Silicone Twister extracts phenolic 6 o-Ethylphenol 107 9.3E+06 2.1E+05 substances very efficiently and that it is also highly suitable for many 7 2,4-Xylenol 107 1.7E+07 4.3E+05 non-polar compounds. As is clearly seen in table 3, a significantly larger 8 Ethyl octanoate 88 1.5E+08 1.4E+08 number of phenols and aromatic compounds were extracted from the 10 Ethyl decanoate 88 2.3E+08 2.3E+08 whisky sample with the EG-Silicone Twister than with the PDMS 12 Ethyl dodecanoate 88 1.1E+08 7.0E+07

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Table2. Recovery in % for selected whisky standard substances obtained with the EG- Twister. The total number of two Twisters. For more polar alcohol-terpenes Silicone and PDMS Twisters. extracted compounds is 126 for linalool, 4-terpineol, alpha-terpineol, the EG-Silicone Twister and and nerolidol, the EG-Silicone Whisky Log EG- PDMS 92 for the PDMS Twister. For Twister does provide better standards Ko/w Silicone the other compounds classes, recovery than the PDMS Phenol 1.46 5.7 3.0 both Twisters extract a similar Twister; conversely, o-Cresol 1.95 9.8 1.5 number of compounds, but the for monoterpenes cis-Whisky 2.00 6.1 24.3 EG-Silicone Twister generally like alpha-pinene, lactone gives better recovery. beta-myrcene, delta- Eugenol 2.27 29.5 32.9 In order to achieve better 3-carene and d-limonene the separation of polar compounds PDMS Twister gives better Table 3. Whisky compounds extracted from the whisky sample, recovery. by SBSE using the PDMS and EG-Silicone a ZB-FFAP column was Twister respectively. subsequently used. The resulting White Wine chromatogram is shown in (Sauvignon Blanc) Compound class PDMS EG- figure 2, the whisky profile was Silicone obtained based on extraction EG-Silicone- and PDMS Twisters were used to Phenols and aromatic compounds 14 40 with an EG-Silicone Twister. extract a broad range of volatile compounds and generate a flavor Fusel alcohols 10 10 Table 4 lists the proposed profile of a white wine. Subsequently, the extraction results for the Fatty acids 11 11 compound names identified two Twister types were compared. PDMS Twisters can be added Aliphatic acid ethyl esters 15 15 with the mass spectral database directly to the wine sample without modifying the sample. Prior to Other esters 22 22 (Wiley 6N). All identified peaks extraction with EG-Silicone Twister, the wine sample needed to be Lactones 1 2 have a hit quality higher than 80. neutralized to pH 3.6 in order to avoid break-down of the Twister Acrolein derivates 7 7 The plausibility of the phase. Chromatograms were obtained using both ZB-5 (non-polar) Terpenes and norisoprenoids 6 7 identification was checked and ZB-FFAP (polar) columns, the tentatively identified wine Miscellaneous 6 12 against literature to ensure that compounds are listed in Table 7. Except for the oven programs and Total 92 126 the reported compounds were column flow rates used, all conditions for TDU, CIS, and MSD were known to be present in whisky. the same for all analyses performed. Using the polar column, the A stacked view comparison of chromatograms from extractions peaks for acids, phenols and other polar compounds show a better peak using different Twisters is shown in figure 4. It can be seen that the shape. Many important whisky compounds (vanillin, ethyl vanillate, etc.), which were covered by broad co-eluting acid peaks when using Table 4. Tentatively identified compounds found in Scotch whisky by Twister the ZB-5 non-polar column, were now well separated and could easily extraction and GC/MS analysis using a ZB-FFAP Column. be identified. Peak Proposed Peak Proposed Peak Proposed No. Identity No. Identity No. Identity Multivitamin Juice 1 Ethyl octanoate 9 trans Whisky lactone 17 Capric acid 2 Ethyl nonanoate 10 o-Cresol 18 Farnesol Extraction of multivitamin juice or of other fruit juices is often 3 Ethyl decanoate 11 p-Ethylguaiacol 19 Lauric acid negatively influenced by fruit pulp, which blocks analyte access to 4 1-Decanol 12 d-Nerolidol 20 Vanillin the extraction phase and/or hinders phase separation following the 5 Phenethyl acetate 13 Octanoic acid 21 Ethyl vanillate extraction. In contrast, the presence of fruit pulp has no effect on the 6 Ethyl dodecanoate 14 o-Ethylphenol 22 Myristic acid SBSE extraction process for multivitamin juice. A 10 mL sample was 7 Guaiacol 15 2,4-Xylenol directly dispensed into a 10 mL vial, the Twister was added and the 8 Phenethyl alcohol 16 p-Ethylphenol sample stirred for 1 hour at 1000 rpm. Both EG-Silicone- and PDMS Twisters were used for the extraction. As can be seen in the chromatograms in figure 3, the EG-Silicone Twister extracts more compounds than the PDMS Twister and with better recovery. The peaks obtained using the EG-Silicone Twister are significantly larger. In the chromatogram obtained with EG-Silicone Twister, 39 peaks were clearly identified. Nine compounds were not at all found or identified using the PDMS Twister: formic acid, acetic acid, furfural, furfural alcohol, 2-hydroxycyclopent-2-enone, 3-methyl-2,5-furandione, 5-methyl-2-furfural, 2,3-dihydro-3,5- dihydroxy-6-methyl-4H-pyran-4-one and Hydroxymethylfurfurole (HMF). Most of these compounds are furfurals and derivatives of furan. Moreover, the peaks for these nine compounds were very large using EG-Silicone Twister extraction, for example furfural (No. 3) and HMF (No. 21). Some important terpenes in the multivitamin juice were extracted by both Twisters, these are listed in table 6. Eight terpenes were selected and their peaks integrated based on extracted ion chromatograms (EICs). The EIC masses used and the resulting peak areas are also listed in table 6. It can be seen that EG- Silicone- and PDMS Twisters have similar extraction efficies for the terpenes judging by the very similar peak areas obtained using the Figure 2. SBSE-TD-GC/MS chromatogram, polar column separation, resulting from an EG-Silicone Twister extraction of a 5 mL whisky sample diluted 1:1 with water (20 % EtOH v/v). Sample extracted for one hour at room temperature and 1000 rpm.

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EG-Silicone Twister extracts a larger number of individual substances (30 tentatively identified peaks) from wine than the PDMS Twister. Substances like furfural, cis- and trans-4-hydroxymethyl-2-methyl- 1,3-dioxolane, glycerin, malic acid, methyl 2,3-dihydroxybenzoate are only found in the EG-Silicone Twister based chromatograms. The EG-Silicone Twister extracts acids much more efficiently from wine than the PDMS Twister, see peaks No. 11, 17, 21, 22, and 24 as well as alcohols like 2,3-butanediol (No. 3), 1-hexanol (No. 6), and Phenethyl alcohol (No.15). PDMS Twisters, conversely, extract larger amounts of esters compared to EG-Silicone Twister (see No. 4, 7, 12, 13, 18, 25). To achieve better resolution and separation of polar compounds extracted from the wine, a polar column was also used. As can be seen in figure 5, the polar column produced sharp acid peaks and enabled the separation of several key polar compounds that were covered by big co-eluting ester peaks in the chromatogram produced on the non-polar column. Although a different column was used, the quantitative results and determined compound identities obtained from EG-Silicone- and PDMS Twister extractions were in good agreement. Some polar acids, alcohols, as well as other polar compounds could be extracted only using

Table 5. Identified compounds found in multivitamin juice by Twister extraction and GC/MS analysis using a ZB-5 Column. Peak Proposed Identity Peak Proposed Identity Peak Proposed Identity the EG-Silicone Twister. Additionally, 5-methyl-2-furfural (No. 11) No. No. No. and Hydroxymethylfurfurole (HMF) (No. 24 ), p-Hydroxyphenethyl 1 Formic acid 14 gamma-Terpinene 27 Nerolidol alcohol (No. 27) and ethyl 3-(4-hydroxyphenyl)-propenoate (Z or 2 Acetic acid 15 alpha-Terpinolene 28 Methoxyeugenol E) (No. 29) were found only when combining EG-Silicone Twister 3 Furfural 16 Linalool 29 alpha-Cubebene extraction with separation on a polar column (Table 8). 4 Furfural alcohol 17 Apple oil 30 Myristic acid 5 Isoamyl acetate 18 2,3-Dihydro-3,5- 31 Nootkatone dihydroxy-6-methyl- 4H-pyran-4-one Table 6. Peak area responses of Terpenes resulting from Twister extractions. 6 2-Hydroxycyclopent- 19 4-Terpineol 32 8-Hydroxy-6- Peak No. Compounds Extracted Ion Peak Areas 2-en-one methoxy [m/z] EG-Silicone PDMS 7 alpha-Pinene 20 alpha-Terpineol 33 9-Hexadecenoic acid 7 alpha-Pinene 93 1.7E+05 3.9E+05 8 3-Methyl-2,5- 21 Hydroxymethyl- 34 Palmitic acid 10 beta-Myrcene 93 1.4E+06 2.6E+06 Furandione furfurole (HMF) 11 delta-3-Carene 93 2.8E+06 4.7E+06 9 5-Methyl-2-furfural 22 Eugenol 35 Limetin 12 D-Limonene 68 1.3E+07 1.8E+07 10 beta-Myrcene 23 trans-Caryophyllene 36 Xanthotoxin 16 Linalool 71 1.5E+06 4.3E+05 11 delta-3-Carene 24 alpha-Humulene 37 Linoleic acid 19 4-Terpineol 71 4.3E+05 2.7E+05 12 D-Limonene 25 Valencene 38 Isopimpinellin 20 alpha-Terpineol 59 1.5E+06 4.3E+05 13 Isoamylbutyrate 26 Elemicin 39 Squalene 27 Nerolidol 69 5.2E+05 4.8E+05

Figure 3. Multivitamin juice chromatogram obtained from EG-Silicone- Figure 4. Sauvignon Blanc chromatogram profiles obtained from EG-Silicone- and PDMS Twisters, non-polar column separation. 10 mL sample, and PDMS Twister extractions of 5 mL samples for one hour at 1000 rpm, 1000 rpm, 1 hour, room temperature. non-polar column separation.

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CONCLUSION Table 7. Tentatively identified compounds extracted from white wine using different Twisters and separated on a ZB-5 GC Column. The novel EG-Silicone Peak Proposed Identity Peak Proposed Identity Peak Proposed Identity Twister presented in this No. No. No. work enables higher ex- 1 2-Methyl-butanol 11 Hexanoic acid 21 Nonanoic acid traction efficiency than 2 3-Methyl-butanol 12 Ethyl hexanoate 22 Malic acid the traditional PDMS 3 2,3-Butanediol 13 1-Hexyl acetate 23 Methyl 2,3-dihydroxy- Twister for polar com- 4 Ethyl butanoate 14 Glycerine 24 Capric acid pounds in samples like 5 Furfural 15 Phenethyl alcohol 25 Ethyl decanoate whisky, multivitamin 6 1-Hexanol 16 2,3-Dihydro-3,5- 26 p-Hydroxyphenethyl juice and white wine. dihydroxy-6-methyl- alcohol For compounds like 4H-pyran-4-one phenols, furans, alco- 7 Isoamyl acetate 17 Octanoic acid 27 2,4-Di-tert-butylphenol hols, and acids, use of 8 trans-4-Hydroxymethyl- 18 Ethyl octanoate 28 Methyl 2,5-dihydroxy EG-Silicone Twister re- 2-methyl-1,3-dioxolane benzoate sults in the best extraction 9 cis-4-Hydroxymethyl- 19 Phenethyl acetate 29 Lauric acid efficiency. For non-polar 2-methyl-1,3-dioxolane compounds such as terpenes and ethyl esters etc., 10 Citraconic anhydride 20 Ethyl dl-malate 30 Ethyl laurate EG-Silicone Twisters, due Table 8. Tentatively identified compounds extracted from white wine using to their dimethylsiloxane different Twisters and separated on a ZB-FFAP Column. base, provide extraction efficiencies similar to those achieved using PDMS Twisters. By using both the EG-Silicone- and the PDMS Peak Proposed Identity Peak Proposed Identity Peak Proposed Identity Twister in a sequential SBSE process, an overall analyte profile of No. No. No. non-polar and polar organic compounds in a sample can be obtained. 1 1-Hexyl acetate 11 5-Methyl-2-furfural 21 Glycerine The pH value of the sample is a critical point for the EG-Silicone 2 1-Hexanol 12 Phenethyl acetate 22 2,3-Dihydrobenzofuran Twister. In water-based standards, the optimum pH range was found 3 Ethyl octanoate 13 Hexanoic acid 23 Lauric acid to be from 3.5 to 10.0, for wine samples from 3.6 to 7.0. Like the 4 Acetic acid 14 Phenethyl alcohol 24 Hydroxymethyl- PDMS Twister, the extraction using the EG-Silicone Twister is easy furfurole (HMF) to perform. Only a few instrumental parameters have to be adjusted. 5 Furfural 15 Ethyl dl-malate 25 Malic acid Additionally, operating the TDU in solvent vent mode is important 6 trans-4-Hydroxy- 16 Octanoic acid 26 Myristic acid when desorbing EG-Silicone Twisters in order to remove excess methyl-2-methyl- water that is retained due to its polar nature. This is needed in order 1,3-dioxolane to eliminate water from the GC/MS system. 7 2,3-Butanediol 17 Nonanoic acid 27 p-Hydroxyphenethyl alcohol 8 Ethyl decanoate 18 2,3-Dihydro-3,5- 28 Palmitic acid dihydroxy-6-methyl- 4H-pyran-4-one 9 cis-4-Hydroxymethyl- 19 Decanoic acid 29 Ethyl 3-(4-hydroxy 2-methyl-1,3-dioxolane phenyl)-propenoate (Z or E) 10 Clorius 20 2,4-Di-tert-butylphenol

Acknowledgment The authors would like to thank Dr. Kevin MacNamara, Irish Distillers, Pernod-Ricard for his kind support.

References

1. Frank David, Bart Tienpont,Pat Sandra, Stir-bar sorptive extraction of trace organic compounds from aqueous matrices, LCGC North America, 21: 21-27 (2003) 2. Kevin Mac Namara, Michelle Lee, Albert Robbat Jr., J.Chromatogr. Figure 5. Sauvignon blanc chromatogram profiles obtained from EG-Silicone- A 1217 (2010) 136 and PDMS Twister extractions of 5 mL samples for one hour at 1000 rpm, 3. Kevin Mac Namara, Dagamara Dabrowska, Meike Baden, polar column separation. Norbert Helle, LC/GC Chromatography, Sep. 2011

Further information Authors

www.gerstel.com - Applications: AppNote 3/2011 Yunyun Nie, Eike Kleine-Benne GERSTEL GmbH & Co. KG, Eberhard- (www.gerstel.com/pdf/p-gc-an-2011-03.pdf) Gerstel-Platz 1, D-45473 Mülheim an der Ruhr, Germany

28 GERSTEL Solutions Worldwide Wine Special GERSTEL Solutions Worldwide Wine Special

Extraction techniques HIT it – targeting VOCs and SVOCs

Application experts from GERSTEL K.K. in Japan pondered how volatile compounds could be determined even more efficiently using Headspace techniques. The answer was only a mouse-click away.

eadspace gas chromatography (HS- a one-step sampling technique: An aliquot transfer to the GC system. For SPME, high HGC) is frequently used for the deter- of the gas phase in equilibrium with a solid selectivity can be achieved through choice of mination of flavor compounds in food. HS or liquid sample inside a closed vial is taken the optimal fiber coating, but the small vol- sampling techniques can be divided into the and introduced to the GC inlet. Since only ume of solid phase on the fiber may limit the following broad categories: a fraction of the headspace can be injected, sensitivity of the analysis. the technique may not achieve adequate sen- In this study, we describe a new enrich- • Static headspace (SHS), sitivity. For this reason, pre-concentration of ment method for SHS and HS-SPME using • Dynamic headspace (DHS), the analytes found in the headspace has been the MultiPurpose Sampler (MPS) and a • Headspace solid phase micro studied. DHS and HS-SPME, on the other Thermal Desorption Unit (TDU) combined extraction (HS-SPME) hand, are two-step sampling techniques that with a programmable temperature vaporizer A futher technique, headspace sorptive extrac- incorporate analyte concentration on an (PTV) inlet, the Cooled Injection System tion (HSSE) using the GERSTEL Twister, is adsorbent trap or on an SPME fiber, respec- (CIS) all from GERSTEL. The new enrich- not addressed in this work. SHS is typically tively, followed by thermal desorption and ment method is called Hot Injection and

Total ion chromatograms of canned coffee by SHS analysis: (a) Conventional SHS with S/SL inlet (injection volume 1 mL, Split 1:1); (b) HIT-HS with CIS 4 Initial Temp. -50 ºC (injection volume 1 mL, Split 1:1); (c) HIT-HS with CIS 4 Initial Temp. 10 ºC, optimized for medium- to high boilers (injection volume 2.5 mL x4, Split 1:1).

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Selectable: Headspace or SPME fibre

The key player is the GERSTEL MultiPurpose Sampler (MPS), which can perform both Head space and SPME analysis. Thermal The TDU is heated to 250 Desorption Unit (TDU) Agitator °C, which means that the PTV-type inlet: Cooled injected analytes remain Injection System (CIS) in the gas phase, without condens- ing in the TDU. THe analytes are transferred to – and focused in the Cooled Injection System (CIS) inlet. If needed, multiple headspace injections can be performed and the combined analytes concentrated and subsequently transferred to the GC column for highly sensitive GC/ MS determination.

Trapping (HIT): A HS syringe or SPME earity, and repeatability was demonstrated fiber is inserted into the TDU, which is kept with aqueous samples such as drinking water heated at 250 ºC to prevent cold spots where and beverages. analytes might be adsorbed. The desorbed analytes are trapped in the Tenax TA packed Canned Coffee by HS and HIT-HS CIS liner at temperatures between -50 and 30 ºC, enabling the use of multiple headspace injections for improved limits of detec- The figure on page 14 shows total ion chro- tion (LODs). Trapped analytes can be intro- matograms (TICs) of canned coffee ob- duced to the GC column in splitless mode tained by conventional HS using S/SL inlet for maximum sensitivity. The performance of (TIC a) and that of HIT-HS (TIC b, c). The GERSTEL MPS configured for HIT-SPME in combination with the TDU. the HIT technique in terms of LODs, lin- In TIC b, as injection was done with CIS

SIM chromatograms showing 3 off-flavor compounds in water (1 ng/L and blank).

SIM chromatogram of water spiked with 10 ng/L of 2-MIB, TCA and geosmin. Concentrating the analytes through multiple injections for each GC/MS run enables clear and unambiguous determination.

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MAESTRO and HIT-HS:To improve the sensitivity and lower the limits of detection significantly for your Headspace analysis, simply increase the number of injections per GC/MS run: On the MAESTRO method page, just type in the number of injections (concentration steps) and off you go. This is the most convenient way by far to automate analyte concentration and sample introduction for HS-GC/MS.

4 initial temp. at -50 ºC, very volatile compounds (e.g. acetalde- hyde) were detected. In TIC c, 2.5 mL x4 was introduced into the TDU-CIS 4 system for further concentration. The CIS 4 initial temperature was set to 10 ºC, enabling proper water management. Using four 2.5 mL HS injections resulted in enhanced sensitivity. Furthermore, lower vapor pressure compounds (e.g. furfuryl methyl disulfide) could be determined.

Off-flavors in Water by HIT-HS

To maximize the sensitivity, we chose four 2.5 mL injections as optimum for the determination of 2-MIB, TCA, and geosmin in water. The method resulted in good linearity with a correlation coefficient (R2) above 0.9950 for 7-level calibration curves between 1 and 200 ng/L. SIM chromatograms of fortified natural water spiked at 1 ng/L are shown on page 15. The repeatability of this method was determined by analysis of natural water spiked at 1 ng/L. The relative standard deviation (RSD) for each compound was in the range from 4.2 to 8.6 % (n=7). The limits of detection (LODs) were calculated as 3 times the standard deviation estimat- ed from replicate determinations (n=7) at the lowest concentration determined for each compound. LODs were 0.12 ng/L for 2-MIB, Source 0.36 ng/L for TCA and 0.30 ng/L for geosmin, respectively. Jun Tsunokawa, Kikuo Sasamoto and No- buo Ochiai, GERSTEL K.K., Tokyo: “Hot In- Conclusion jection and Trapping Using SHS/SPME and a Thermal Desorption System for GC/MS- The HIT technique provides significantly enhanced sensitiv- Analysis”. Poster presentation at PITTCON ity for HS analysis of flavor and off-flavor compounds. Also, 2012. HIT resulted in good linearity and repeat-ability for the determination of off-flavor compounds in water, achieving very low LODs at sub-ng/L levels.

HIT-HS-GC/MS System based on the GERSTEL MPS.

GERSTEL Solutions Worldwide Wine Special 31 Literature Suggested reading…

Recent journal papers, a selection:

Dual solid-phase and stir bar sorptive extraction combined with gas Flavor, Fragrance, and Odor Analysis, chromatography-mass spectrometry analysis provides a suitable tool for assaying limonene-derived mint aroma compounds in red wine Second Edition M. Picard, C. Franc, G. de Revel, S. Marchand Anal. Chim. Acta 1001 (2018) 168-178

Determination of ppq-levels of alkylmethoxypyrazines in wine by here are many advantages to stir bar sorptive extraction (SBSE) stir bar sorptive extraction combined with multidimensional gas Tfor isolating and concentrating flavor active chemicals from foods. chromatography-mass spectrometry These include simplicity, wide application range, efficient analyte Y. Wen, I. Ontañon, V. Ferreira, R. Lopez concentration, and generally the absence of masking solvent https://doi.org/10.1016/j.foodchem.2018.02.089 peaks. Written from a practical, problem- solving perspective, the second edition of Flavor, Effect of a grapevine shoot waste extract on red wine aromatic Fragrance, and Odor Analysis highlights this properties powerful technique and emphasizes the range M. J. Ruiz-Moreno, R. Raposo, B. Puertas, F. J. Cuevas, F. Chinnici, J. M. Moreno-Rojas, E. C.-Villar of applications available. Topics discussed https://doi.org/10.1002/jsfa.9104 include: Sequential SBSE, a novel extraction Changes in sparkling wine aroma during the second fermentation procedure · A simplified method for switching under CO2 pressure in sealed bottle from one-dimensional to two-dimensional GC/ R. Martínez-García, T. García-Martínez, A. Puig-Pujol, J. C. Mauricio, MS · How to improve analytical sensitivity and J. Moreno recovery of phenolic compounds with aqueous J. Food Chem. 237(2017)1030-1040 acylation prior to SBSE GC-MS · Analyzing and combating off- https://doi.org/10.1016/j.foodchem.2017.06.066 flavors caused by metabolites from microorganisms · A technique for Toasted vine-shoot chips as enological additive measuring synergy effects between odorants · The identification of the C. Cebrián-Tarancón, R. Sánchez-Gómez, M. R. Salinas, G. L. Alonso, characterizing aroma-active compounds of tropical fruits with high J. Oliva. A. Zalacain economic potential · The parameters utilized during the production of J. Food Chem. 263 (2018) 96-103 aqueous formulations rich in pyrazines · How spectral deconvolution https://doi.org/10.1016/j.foodchem.2018.04.105 can be used to speciate the subtle differences in essential oil content

and track key ingredients through the manufacturing process Characterization of the key aroma compounds in Chinese Syrah wine The final chapter summarizes chemical identities of characterizing by gas chromatography-olfactometry-mass spectrometry and aroma reconstitution studies aroma chemicals in fruits, vegetables, nuts, herbs and spices, and P. Zhao, J. Gao, M. Qian, H. Li savory and dairy flavors. It also provides a brief compendium of the Molecules 2017, 22(7), 1045; characterization of off-flavors and taints that are reported in foods. https://doi.org/10.3390/molecules22071045 With contributions from a distinguished panel of international experts, this volume provides chemists and researchers with the latest The contribution of wine-derived monoterpene glycosides to techniques for analyzing and enhancing food flavor and fragrance. retronasal odour during tasting M. Parker, C. A. Black, A. Barker, W. Pearson, Y. Hayasaka, I. L. Francis J. Food Chem. 232 (2017) 413-424 https://doi.org/10.1016/j.foodchem.2017.03.163 More information

Optimization and validation of an automated DHS–TD–GC–MS Ray Marsili (Edi.), Flavor, Fragrance, and Odor Analysis, method for the determination of aromatic esters in sweet wines Second Edition, 280 Pages, ISBN-10: 1439846731 A. Marquez, M. P. Serratosa, J. Merida, L. Zea, L. Moyano and ISBN-13: 978-1439846735. Talanta 123 (2014) 32-38 https://doi.org/10.1016/j.talanta.2014.01.052

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