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Hanging by a Thread:

Enhancing the Forensic Value of Dyed Cotton Trace Evidence through
the Application of Novel Techniques in Fiber Discrimination

By: Rachel M Russo
Mentor: Barry Fookes

Abstract and Introduction


This thesis examines the capability of current techniques in fiber classification such as UV-visible microspectrophotometry (MSP) (for dye in situ and/or extracted) to discriminate between fibers from sources known to be different. When these methods fail to adequately distinguish the fibers, novel alternative techniques'such as pulsed pumped laser-induced fluorescence spectroscopy (LIF) and liquid chromatography-mass spectrometry (LC-MS)'are utilized to provide definitive forensic evidence.

The FBI Dye Extraction Classification and Chromatography Schemes: Forensic Fiber Examination Guidelines provides the methodology used by the majority of crime labs across the United States (Fong, 1984). In the case of cotton fibers'the most frequently encountered fiber form of trace evidence'the scheme fails to produce adequate evidence to establish a questioned/known match (Grieve & Wiggins, 2001). In fact, in many criminal investigations the protocols indicate a false positive association (Cheng, 1991). New methods of discriminating between dyed cotton fibers are needed to promote the evidential value of trace fibers.

The preliminary data confirm unique identification of all the fibers using these enhanced investigative tests, a task not possible by conventional analysis alone. Analysis by multiple techniques greatly enhances the probative value of trace fibers in criminal investigations by providing fiber discrimination at a higher degree of certainty. This study demonstrates the benefit of applying new techniques in the forensic investigation of fibers to reduce the chance of an incidental match. Sixty percent discrimination was achieved by employing current protocols; discrimination was improved to one-hundred percent by applying the methods outlined in this paper. The application of liquid chromatography-mass spectrometry (LC-MS) and ultraviolet fluorescence spectroscopy to the analysis of cotton fibers is shown in this paper to greatly increase their evidentiary value by providing highly specific chemical and structural information about the dyes and brighteners.

Introduction and Background

Cotton Properties

Cotton is Abundant

Cotton fibers are everywhere. According to William Goynes (1998), a renowned cotton microscopist, 'Cotton is the most important and widely used natural fiber in the world.' Cotton, of the genus Gossypium, consists of more than 43 recognized species. The three most common types'Sea Isle, Egyptian, and Pima'account for more than 80% of all fibers used in the production of textiles worldwide (Box, 2000). Moreover, cotton fibers tend to shed more easily than synthetic fibers because they are naturally short and cannot be converted into a continuous filament (Cellulose, 2003). The result of these traits is a trail of cotton fibers left behind at every location visited by an individual wearing a cotton garment.

Cotton Structure and Growth

Cotton grows in a boll consisting of a base containing over 5000 seeds each giving rise to a single seed hair. Two types of seed hairs are formed: (1) lint or linters: longer, finer fibers with a flexible cuticle, used in the production of textiles (hereby referred to as the fiber); and (2) fuzz: shorter, thicker, and less flexible, used in the production of paper products (Box, 2000). The fibers grow projecting upwards as hollow sheaths, each night depositing a new layer of cellulose on the inside of the sheath until approximately thirty layers have been laid. After the last layer has been deposited the boll bursts open and the fibers, now exposed to the environment, cease to grow (Beaudet, 1999).

The fiber itself is a hollow cylinder comprised of two walls of cellulose; a thin primary wall surrounding a thick secondary wall enclosing a nutrient-filled central lumen. The outer, primary wall protects the inner cellulose fibrils from environmental damage after the boll has burst (5). The hollow central lumen inside the cellulose cylinder creates a canal for nutrients while the fiber is growing. When exposed to sun and air, the fiber dries up, collapsing the lumen resulting in a characteristic flattened-ribbon shape. This flat-ribbon cotton fiber twists alternating between clockwise and counter-clockwise every 2 or 3 turns (Beaudet, 1999).

Cotton is more than 90% cellulose, comprised of more than 6000 monomeric subunits of poly[b-1,4-D-anhydroglucopyranose] per cellulose chain (Cellulose, 2003). The abundant hydrogen bonds between cellulose chains allow cotton to withstand high temperatures without melting; cotton fibers tend to char above 200 degrees Celsius.

The cellulose chains contain sporadic crystalline regions separated by amorphous arrangements of molecules. The degree of crystallinity affects the ability of each fiber to absorb water and dyes. The hydrogen bonding and cross-linking between chains responsible for these crystalline regions increase fiber strength and account for the ability of cotton to absorb as much as 70% of its own weight in water while maintaining structural integrity (Cotton, 2003).

Cotton Processing and Textile Manufacture

Wrinkle Resistance

When cotton is immersed in water, the water molecules penetrate between cellulose chains. The fibers swell to accommodate the extra molecules and wrinkle to release structural tension. This characteristic is undesirable to consumers and led to the development of permanent press processing. Wrinkle resistant cotton has been altered chemically to form crosslinks not broken by water. Often the substitution of formaldehyde derivatives for hydroxyl groups on the pyranose ring is the method by which a durable press is achieved. However, the heat and the acid needed to catalyze this reaction can shorten fabric life; often, synthetic fibers are added to the yarns to increase textile strength. The most common synthetic used in this manner is polyester; such fabrics are referred to as a poly-cotton blend (Cellulose, 2003).


Mercerization is a method textile manufacturers employ to decrease twists in the fibers, thereby increasing the luster of the fibers and their ability to absorb dyes by 25%. Mercerization is achieved by the application of caustic soda (sodium hydroxide) to make the fiber lumen swell, become round, and straighten out. Subsequently tension is applied to the fiber to stretch the fibers straight. When the fibers dry and the lumen collapses there are fewer twists, creating a round, smooth surface that reflects light creating a lustrous sheen (Beaudet, 1999).

Long staple cottons (Sea Island, Egyptian, and Pima) naturally have fewer twists and the highest dye adhesion characteristics, so they are the preferred breeds used in textile manufacturing. Mercerization in combination with direct dye can optimize dye absorption and dye-fastness. Fiber maturity and micronaire also influence dye absorption. Fibers in a given textile are of varying degrees of maturity and thickness at the time of harvest. Often all types of fibers are mixed to homogenize yarn quality (Mogahzy, 1998).

Current Fiber Identification Protocols

The standard protocols for fiber dye investigations adopted by the majority of crime laboratories across the United States are published in the FBI Handbook for Forensic Services (Federal Bureau of Investigation [FBI], 2004). The exact protocols can be found in Appendix B. Primarily, fibers must match in three ways to be considered from the same source: generic class, physical characteristics, and color. Generic class (whether a fiber is natural or synthetic, polyester, nylon, cotton, or wool) can be established by a variety of methods. For natural fibers polarized light microscopy is the most efficient method of discrimination (Fong, 1989). It is relatively simple for an investigator to tell a synthetic fiber from a natural fiber by visual examination. Physical characteristics can be determined by microscopic investigations. These examinations include determination of cross-sectional shape, diameter, and the presence of delustering agents. If necessary, Fourier Transform Infrared Spectroscopy (FTIR) can be employed to determine the chemical make up of the fiber. Other methods investigators use to determine the generic class of a fiber include solubility, melting point, and refractive index determination. Color can be initially determined visually and further discriminated by fluorescence microscopy and microspectrophotometry used in this investigation. Color is related to the dye applied to a fiber, thus an alternative to examining the cotton fiber structure itself is to examine the dye applied to it (Grieve & Wiggins, 2001).

The FBI protocols for dye extraction (found in Appendix C) use generalized requirements for all types of cotton. Dye classification depends on the analyst's perception of a 'good extraction.' If the extraction was good then the dye class can be deduced from the corresponding chemical used for extraction. If the extraction was not good then the analyst must attempt extraction with a different chemical on a different sample. Extraction time and temperature will affect the quality of extraction with any given solvent. As previously addressed, dye absorption is affected by cotton species, maturity, and processing treatments. The quality of extraction may be affected by the fiber itself and cannot be attributed to the dye alone. When extraction protocols rely on subjective assessment of the extent to which a dye eluted to indicate the chemical class of that dye there is extensive room for error (Grieve, Dunlop, & Haddock, 1988, 1990). This deficiency contributes to the misconception that cotton fibers are of little evidential value; the protocols simply must be revised.

Currently cotton protocols require large amounts of sample, more than what is typically available to forensic analysts. Successive reactions requiring a lot of fiber and many supplies can also become very costly for small labs (Grieve & Wiggins, 2001). However, common chromatography methods (TLC, HPLC) can be aided greatly by knowing dye class and the most efficient dye extraction solvents (Bresee, 197).

The results of a study by Cheng demonstrate red dyed cotton fibers do not meet traditional expectations when examined by current protocol (Cheng, 1991). Seven out of 41 dyed fibers exhibited atypical behavior. Cotton fibers dyed with red sulfur dye do not obey color-change trends with disodium sulfide and polyvinylpolypyrrolidone. Half of the dye classes were misclassified by one or more scorer in blind trials. Moreover, natural fiber variation caused atypical results with low reproducibility. Cheng concluded dye classification by extraction should be treated cautiously at best. The study reinforces the aforementioned need for current dye-extraction/fiber-examination protocols to be revised for the examination of cotton fibers.

Cotton's Properties Inhibit Current Protocols

Because cotton is a biological material no two cotton fibers will ever be exactly alike (unlike synthetic fibers) which creates an inherent degree of uncertainty during a questioned-known match. The variable nature of cotton fibers along with their abundant use has lead many investigators to disregard the evidentiary value of cotton fibers (Grieve, 1993). Their tendency to shed easily makes cotton ideal for Locard transfer; if investigators were able to more effectively utilize cotton fibers as evidence, it could provide the key to solving many criminal cases. Simply put, if investigators can find a way to locate these fibers at the scene and match them to the original garment they can effectively place a suspect at the scene of the crime.

Cotton Structure and Growth

The presence of the central lumen and the nature of the twists makes the determination of the refractive index and optic sign extremely difficult if not all together impossible (White, 1992). The Hertzel test cannot be applied to distinguish cotton fibers, limiting the data an analyst can gather about the optical properties of cotton.

The chemical structure of cotton also poses problems for traditional analysis. The arrangement and strength of intermolecular forces within the crystalline regions make cotton a durable fabric for consumers but prevent forensic examiners from obtaining any characteristic melting point data, which is useful in the analysis of synthetic fibers. For the same reason, cotton will not dissolve in common solvents, eliminating solubility as a means of distinguishing fibers. Water absorption interferes with common tests employed by forensic analysts. Water interferes with dye extraction and creates noise in FTIR spectra.

Additionally, naturally varying crystallinity and twists affect dye concentration even along the length of the same fiber, which makes peak normalization by area necessary to compensate for variances in absorbance intensity before meaningful comparisons can be made between microspectrophotometric absorption data. The implications of such variability, natural or induced by treatment or processing, would indicate a more refined protocol is necessary to assess dyes extracted from cotton fibers.

The Effect of Processing on Current Protocols

Wrinkle Resistance

Treatment with formaldehyde allows forensic analysts to distinguish permanent press cotton from untreated cotton using a variety of techniques, most notably Fourier Transform Infrared Spectroscopy (FTIR). The presence of polyester reinforcements in a thread can allow scientists to determine the blend ratio, which could be another distinguishing feature if a thread (as opposed to a fiber) was found at a crime scene.


Mercerization results in stretching of the fibers, which also affects their optical properties. Applying tension to fibers alters their natural refractive index, which adds another complication to forensic examinations.

However, mercerization could be used to provide more individualizing information to forensic examiners. If a fiber was known to be Pima cotton and the twist-frequency distributions of untreated and mercerized Pima were known, it could be possible to establish the likelihood of a fiber being treated. Again, more research would need to be done to establish natural distribution before any such comparison would be useful. It would theoretically be possible to compare twist frequency between a questioned and a known fiber, but the high degree of variation within a garment could make comparison questionably useful until more studies have been conducted.

Long staple cottons and short staple cottons have entirely different qualities: strength, water absorption, dye absorption, dye extraction kinetics, and frequency of twists. These variables may be of benefit to analysts if further research is conducted to establish their distribution (Beaudet, 1999). Currently, there are few fiber examiners experienced enough to visually distinguish the species of cotton that bore a fiber. It would therefore be beneficial to utilize fiber properties to establish a way of distinguishing long staple from short staple cotton using a single fiber. Sufficient information exists to discriminate long from short staple cotton in bulk samples; however, forensic scientists often deal with trace samples of only a few fibers. More research would need to be done to allow differentiation in a forensic context.

Overall, the characteristics and treatments that make fibers more valuable to consumers make analysis more difficult for forensic examiners. The only examinations listed in the current protocols that add forensic value to cotton despite the aforementioned difficulties are microspectrophotometry and dye extraction. However, the traits that make cotton dye-fast complicate dye extraction. In many cases 100% of the dye cannot be extracted, rendering studies in dye extraction kinetics of little benefit for discrimination. Dye extraction'including dye classification by extraction as commonly used under the current protocols'can still be beneficial but many problems already addressed in this paper still exist.

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