LAUNDRY CHAMILA J. DENAWAKA, IAN A. FOWLIS, KATHERINE STAPLETON, JOHN R. DEAN* *Corresponding author Northumbria University, Department of Applied Sciences, Newcastle upon Tyne NE1 8ST, United Kingdom John R. Dean Gas chromatography and associated techniques in the analysis of laundry malodour KEYWORDS: malodour, laundry, gas chromatography, ion mobility spectrometry, mass spectrometry Abstract This paper reviews the use of chromatography in the identification of malodour in laundry. Two approaches are described: the first involves the use of the traditional approach of gas chromatography mass spectrometry while the second approach uses a new approach based on multi-capillary column gas chromatography ion mobility spectrometry (MCC-GC-IMS). Examples of GC-MS data representative of the volatile compounds associated with the inside of a washing machine are shown. MCC-GC-IMS has the advantages of extreme sensitivity for volatile compounds as well as the ability to detect nitrogen and sulphur containing molecules. Finally, selected examples of the application of chromatography for the analysis of malodour are presented. INTRODUCTION The science of laundry is complex and at Northumbria University we have been applying new technologies to attempt to understand the extent of the generation of malodour during human activity and also to follow the removal of the associated compounds in washing processes. This also necessitated a study of the generation of malodours in washing machines which are also a by-product of human activity. Our sense of smell is generally well developed although there is a tendency for it to degenerate in later life. It is fortunate that this is the case since the range of smells which we encounter not only may give us pleasure but in addition sometimes provide warnings of possible dangers or unpleasant environments. So what are smells and odours? Although single organic compounds frequently have characteristic odours by which we are often able to identify them, the aromas and odours associated with plants, animals and everyday life generally are not single compounds but are a mix of different sizes of molecules and of different classes of organic compounds with varying compositions. One thing that they all have in common is the fact that the compounds involved are all present in the vapour phase at ambient temperatures and pressures. After all if they were not volatile we would not smell them. A perfume, either natural or synthetic, may be made up of several hundred individual compounds. Malodours generally comprise fewer compounds. In analytical chemistry we can usually work on the basis that if we can smell something then we can analyse it using gas chromatography. Gas chromatography also has the advantage 14 that it is a high resolution technique, that is that will separate mixtures of compounds into their individual components, the resulting separation being presented as a chromatogram. Gas chromatography alone will not, however, identify the individual compounds separated in the analysis but requires a specific detection system at the exit of the gas chromatographic column, for example, mass spectrometry. GENERATION OF MALODOURS Malodour is defined as an olfactory stimulant which when detected is considered to be offensive to the individual. Common malodours are often attributed to poor personal hygiene including halitosis (1) and sweat (2) as well as damp clothing after laundry (3, 4). It is well known that bacteria emit a wide range of volatile organic compounds (VOCs). Therefore, it is not unreasonable to postulate that bacteria that have colonised washing machines could contribute to any malodour associated with both the machine itself and clothing washed in it (3). The generation of malodour on the various parts of the body for example foot, underarm, hair or mouth are mainly due to microbial transformation of odourless substances into malodour volatile compounds. In addition to microbial activities, a number of factors such as gender, genetic, environmental factors, diseases, human activities, habits, diet, aging groups and ethic group also influence body odour (2, 5). During wearing, fabrics can be contaminated by sebaceous lipids, sweat, and dead skin cells. These substrates provide the nourishment and facilitate micro-organism survival on laundries (6, 7).
Numerous factors have been identified for the formation of malodour in laundries such as humidity, drying time, chemical oxidation and metabolism of micro-organisms as well as human odour. Limited studies have been carried out to investigate the bacterial colonisation (biofilms) in washing machines (8, 9). In household washing machines, microbial survival followed by biofilm formation can occur due to soiled garments and poor water treatments. A number of microbe species have been isolated and identified from the biofilm within washing machines (3, 8, 9). Among the different parts of a washing machine, hot spots (i.e. product drawer, sump and rubber seal) of biofilm formation are identified (9). Moreover it has been shown (9) that a possible link exists between contamination of fabrics in washing machines and the bacteria identified in washing machine seals. This study identified potential VOC markers for both high levels of bacteria and malodour in washing machines as dimethyl disulphide and dimethyl trisulphide. is observed that while many components are identified (in this case by a peak number) their identification (by GC-MS) and odour (by GC-OD) is often inconclusive and non-correlated. For example, the identification of a compound via the GC-MS database does not necessarily correlate with the known odour of that compound (as determined by the human nose, using GC-OD). Bacteriological assessment of samples taken from the areas of interest in the washing machine was carried in order to assess whether there was a correlation between malodour and bacterial contamination (9). GAS CHROMATOGRAPHY MASS SPECTROMETRY Apart from olfactory assessment of odours all other methods have been chromatography based. Capillary column gas chromatography with mass spectrometry detection (GC-MS) is widely used in chemical analysis having the advantage of providing component separation on the high resolution chromatographic column and identification based upon the fragmentation pattern generated in the mass spectrometer for each individual peak as they are eluted from the column. Unfortunately, even with the high sensitivity of analytical equipment the concentration of many of the components in odour samples is too low to allow identification based upon the simple injection of a vapour sample, thus pre-concentration methods are often necessary prior to analysis: Solid phase micro extraction (SPME) is one such technique where a silicon fibre coated with an adsorbent is placed in to the headspace of the odour. The VOCs are adsorbed building up an enhanced concentration and the fibre then injected into the heated injection port of the GC from which the components are eluted into the chromatographic column for subsequent analysis. This static headspace method can be automated. Thermal desorption (TD) is an alternative approach that involves passing a stream of gas through the tube containing the sample and then into an external Tenax adsorption tube. This can be a more exhaustive method with greater pre-concentration of analytes being possible. The adsorption tube is subsequently heated and desorbed onto the GC column, hence the term thermal desorption. A further useful option with these methods is the possibility of splitting the column effluent into two; sending one part to the detector and the other to an outlet tube where a trained chemist can frequently identify compounds eluting from the column by the smell (a technique called GC-Olfactory Detection, GC-OD). Example chromatograms resulting from dynamic headspace sampling of the atmosphere in selected areas (i.e. product drawer, sump, drum and rubber seal) of a domestic washing machine, thought to give rise to malodour on washed fabrics, concentrated using Tenax adsorption tubes and thermal desorption analysis are shown in Figure 1. The complexity of the interpretation of the chromatograms obtained is illustrated in Table 1. Table 1 summarises the (potential) identification of the 53 components shown in Figure 1 after simultaneous analysis by GC-MS fitted with an olfactory detector (GC-OD). It Figure 1. GC-MS chromatograms from a washing machine: (A) product drawer, (B) sump, (C) drum and (D) rubber seal. 15
Table 1. Gas chromatography olfactory detection / mass spectrometry analysis of the atmosphere in selected areas of a washing machine (A) Product Drawer, (B) Sump, (C) Drum, and (D) Rubber Seal. Key to terms: GC-OD = gas chromatography olfactory detection; GC-MS = gas chromatography mass spectrometry; RT = retention time of volatile organic compound; No. = number of peak on chromatogram (Figure 1); * good MS library match, + tentative identification via MS library; I = olfactory intensity of identified peak termed weak (W), medium (M) and strong (S); ND = not detectable peak; unknown = a peak is present but not possible to identify from MS databases. 16
MULTI-CAPILLARY COLUMN GAS CHROMATOGRAPHY ION MOBILITY SPECTROMETRY In this section our experience with much more recent technology, multi-capillary column gas chromatography combined with ion mobility spectrometry detection, MCCGC-IMS, to the application of malodour analysis will be considered. An IMS instrument is compact, ideal for use in the field, relatively simple to operate, sensitive, generally not requiring pre-concentration methods and produces results very rapidly. IMS instruments are widely used by security operations in addition to the military and can frequently be seen in television documentaries based upon airport security and import controls. In recent years IMS has been combined with, for example, gas chromatography (GC) (10). Gas chromatography combined with ion mobility spectrometry extends the usefulness of IMS by providing the additional dimension of retention time separation of the gas chromatograph with the drift time separation of the spectrometer. Further, the IMS signal intensity, provides quantitative data in addition to the qualitative information. Thus there are two types of separation, the first is chromatographic separation and the second drift separation (Figure 2). conditions (see for example, Figure 3). Although there is no fragmentation of ions in IMS, since it is a soft ionization technique, there is normally more than one response to the presence of a compound ion, these are referred to as monomers, dimers and trimers. These dimers and trimers are not the same as the dimers and trimers in the conventional chemical sense but indicate more than one ion species to be present in a water cluster with the whole still having a single charge. [Note: monomers, dimers and trimers appear at the same retention time for a particular compound and are useful for confirmation of identity.] Detection limits for the technique are typically in the low ppb range for volatile compounds MCC-GC-IMS has an added advantage in that it will detect very small molecules containing nitrogen or sulphur which are not normally detectable by other GC detection modes. Figure 3. Two-dimensional maps of 8 VCs separated by MCC-IMS. Figure 2. 2D (A) and 3D (B) data visualisation using static headspace sampling MCC-GC-IMS: Example topographic view for formation of monomer (M) and dimer (D) for 1-butanethiol (100 ng). RIP = reactive ion peak [for further details on the interpretation of MCC-GC-IMS chromatograms and the theory behind the technique please see reference (10)] As a means of providing a rapid confirmation of identity of compounds detected on the two dimensional plot normally used, map type presentations based upon co-ordinates for known compounds of interest, i.e., retention time and drift time, have been established for each set of operating 18 Table 2. Summary of malodour studies Key to terms: HS = headspace; SPME = solid phase microextraction; GC = gas chromatography; MS = mass spectrometry; DHS = dynamic headspace; OD = olfactory detection; SHS = static headspace sampling; MCC = multicapillary; IMS = ion mobility spectrometry; TD = Thermal desorption; AED = atomic emission detector.
SUMMARY A range of gas chromatography techniques have been used to investigate malodour associated with the laundry process i.e. the washing machine itself and soil garments. As has been considered above the interpretation of the data obtained is often complex. A summary of the research that has been undertaken in the area of malodour identification using gas chromatography is shown in Table 2. While some researchers have identified some specific compounds linked to malodour e.g. dimethyl disulphide and dimethyl trisulphide (9,11) and 4-methyl-3-hexenoic acid (4,14) uncertainty is still evident. 7. 8. 9. 10. REFERENCES AND NOTES 1. 2. 3. 4. 5. 6. Hughes, F.J., McNab, R. Oral malodour. A review. Archives Oral Biol. 53 (Suppl. 1), S1 S7 (2008). Curran, A. M., Rabin, S. I., Prada, P. A., Furton, K. G. Comparison of the Volatile Organic Compounds Present in Human Odor Using SPME-GC/MS, Journal of Chemical Ecology, 31, 1607-1619 (2005). Munk, S., Johansen, C., Stahnke, L. H., Adler-Nissen, J. Microbial survival and odor in laundry, Journal of Surfactants and Detergents, 4, 385-394 (2001). Takeuchi, K., Hasegawa, Y., Ishida, H., Kashiwagi, M., Identification of novel malodour compounds in laundry, Flavour and Fragrance Journal, 27, 89-94 (2012). Haze, S., Gozu, Y., Nakamura, S., Kohno, Y., Sawano, K., Ohta, H., Yamazaki, K., 2-Nonenal newly found in human body odor tends to increase with aging, Journal of Investigative Dermatology, 116, 520-524 (2001). Munk, S., Munch, P., Stahnke, L., Adler-Nissen, J., Schieberle, P., Primary odorants of laundry soiled with sweat/sebum: Influence of lipase on the odor profile, 11. 12. 13. 14. Journal of Surfactants and Detergents, 3, 505-515 (2000). Nagoh, Y., Tobe, S., Watanabe, T., Mukaiyama, T., Analysis of odorants produced from indoor drying laundries and effects of enzyme for preventing malodor generation, Tenside Surfactants Detergents, 42, 7-12 (2005). Gattlen, J., Amberg, C., Zinn, M., Mauclaire, L., Biofilms isolated from washing machines from three continents and their tolerance to a standard detergent, Biofouling, 26, 873-882 (2010). Stapleton, K., Hill, K., Day, K., Perry, J. D., Dean, J. R., The potential impact of washing machines on laundry malodour generation, Letters in Applied Microbiology, 56, 299-306 (2013). Denawaka, C. J., Fowlis, I. A., Dean, J. R., Evaluation and application of static headspace - multicapillary column - gas chromatography - ion mobility spectrometry for complex sample analysis, Journal of Chromatography A, 1338, 136-148 (2014). Stapleton, K., Dean, J. R., A preliminary identification and determination of characteristic volatile organic compounds from cotton, polyester and terry-towel by headspace solid phase microextraction gas chromatography-mass spectrometry, Journal of Chromatography A, 1295, 147-151 (2013). Akutsu, T., Sekiguchi, K., Ohmori, T., Sakurada, K., Individual comparisons of the levels of (E)-3-methyl2-hexenoic acid, an axillary odor-related compound, Chemical Senses, 31, 557-563 (2006). Prada, P. A., Curran, A. M., Furton, K. G., The evaluation of human hand odor volatiles on various textiles: a comparison between contact and noncontact sampling methods, Journal of Forensic Sciences, 56, 866-881 (2011). Kubota, H., Mitani, A., Niwano, Y., Takeuchi, K., Tanaka, A., Yamaguchi, N., Kawamura, Y., Hitomi, J. Moraxella species are primarily responsible for generating malodor in laundry, Applied and Environmental Microbiology, 78, 3317-3324 (2012).