Review: Analysis of Fiber Dyes by Liquid Chromatography Mass Spectrometry (LC-MS) with Electrospray Ionization: Discriminating Between Dyes with Indistinguishable UV-Visible Absorption Spectra

Emily C. Lennert

Category: Chemistry

Keywords: fiber, dye, liquid chromatography, electrospray ionization, UV-visible, spectra, spectroscopy, discrimination, distinguishing, differentiation

Article to be reviewed:

1. Huang, M.; Russo, R.; Fookes, B. G.; Sigman, M. E. “Analysis of fiber dyes by liquid chromatography mass spectrometry (LC-MS) with electrospray ionization: Discriminating between dyes with indistinguishable UV-visible absorption spectra.” Journal of Forensic Sciences. 2005, 50 (3).

Additional references:

2. Lewis, L. A.; Sigman, M. E. Forensic Analysis of Dyes in Fibers Via Mass Spectrometry. In Forensic Analysis on the Cutting Edge: New Methods for Trace evidence Analysis; Blackledge, R. D., Ed.; J. Wiley & Sons: Hoboken, NJ, 2007.

Disclaimer: The opinions expressed in this review are an interpretation of the research presented in the article. These opinions are those of the summation author and do not necessarily represent the position of the University of Central Florida or of the authors of the original article.


The review article discusses an analytical method for discriminating between fiber dyes. Forensic fiber analysis uses identification and comparison of dyes to link a fiber from a known source, such as a suspect, to a fiber from an unknown source, such as a fiber recovered from a crime scene. The current standard method for fiber dye analysis is microspectrophotometry by UV-visible absorption spectrometry.1, 2 UV-visible absorption spectroscopy lacks the power to discriminate between structurally similar dyes, which may result in fibers with two different dyes being mistaken for a matching pair.1, 2 For an example of what it means for dyes to be structurally similar, refer to the dye pair structures in Table 1 within the review article.1

The review article discusses the use of mass spectrometry (MS) techniques as an alternative to UV-visible spectrometry. The authors propose that MS analysis will allow for better differentiation of structurally similar dyes that are not easily differentiated when examined by UV-visible absorption spectroscopy.1 UV-visible spectra give an analyst absorbance at each wavelength within the UV-visible range. Absorbance and wavelength are plotted against each other. The wavelength corresponds to a color within the UV and visible light range. UV- visible absorption spectra allow for two primary comparisons for an analyst to make between samples. The first comparison an analyst can make is the maximum absorption wavelength, which is the wavelength at which the sample has the greatest level of absorption. The second comparison is the absorption profile, or the trend observed in the overall spectra (see Fig. 1). If one were to compare these two spectra, it could be determined that the dyes were indistinguishable or structurally similar.


Figure 1. Simple example of UV-visible absorption spectra for a pair of two different hypothetical dyes, A and B.

MS analysis results in spectra that display the masses of the components within a sample as a mass to charge ratio (m/z), plotted against the relative intensity of the component. (See Fig. 2) This allows an analyst to compare the exact masses of the components within a sample. Structurally similar dyes that differ in molecular formula should therefore be easily distinguished by mass.



The study examined seven pairs of structurally similar dyes. Each dye was separated by high performance liquid chromatography (HPLC) prior to detection, either by UV-visible absorption spectrometry or by mass spectrometry (MS). The study found that each of the seven dye pairs were not able to be differentiated by UV-visible absorption spectroscopy. Each dye pair had either identical or similar maximum absorption wavelengths, as well as similar absorption profiles. However, the study found that each dye pair was easily differentiated using MS data.

One example from this study is the dye pair Red 1 and Red 13. These dyes had similar absorption profiles and maximum absorption wavelengths, with the maximum wavelengths differing by 14 nm. Although the wavelengths differed, the authors explained that differentiation may still be difficult in an actual fiber, since the shape of the fiber and presence of non-dye components (i.e. the fiber’s polymer) could complicate the absorption spectra. UV-visible absorption spectroscopic analysis may be performed on a fiber directly, without HPLC; however, extraction of the dye from the fiber is required for MS analysis, which would result in a liquid sample similar to the dye samples which were examined in this study. The dye pair was more easily distinguished through MS analysis, with distinctly different mass spectra patterns. The same trend was seen in each of the seven dye pairs.

Finally, real world samples were examined. Ten red cotton items which were visually similar were selected. Fibers were taken from each of the ten items and paired to create five pairs. Simple microscopic analysis did not allow for differentiation of any of the dye pairs. After analysis by UV-visible absorption spectrophotometry, 3 pairs of fibers remained indistinguishable. Of those 3 remaining pairs, one pair was differentiable from one another during the extraction process, due to the extraction behavior of the dyes. The remaining two pairs were distinguished by ESI-MS analysis.

For further information on fiber dye analysis, refer to “Forensic Analysis of Dyes in Fibers Via Mass Spectrometry” in Forensic Analysis on the Cutting Edge: New Methods for Trace evidence Analysis.2

Scientific Highlights:

  • 7 pairs of structurally similar dyes were examined.
  • Each dye was separated by HPLC, then examined by two different methods: UV-visible absorption spectroscopy and ESI-MS.
  • After spectral data was gathered or each dye by each analysis method, the dye pairs were examined to determine discriminatory ability for each method.
  • ESI-MS easily distinguished between dyes that were seen as either identical or very similar by UV-visible absorption spectroscopy.
  • When tested on real world samples, ESI-MS maintained higher discriminatory power compared to UV- visible absorption spectroscopy.Relevance: UV-visible absorption spectroscopy, although a common method for fiber dye analysis, is not a sufficient method for differentiation of fiber dyes. This may result in incorrect “matches” of fiber dyes that are, in fact, different dyes. Mass spectrometry prevents the accidental matching of non-matching dyes.

Potential conclusions:

  • UV-visible absorption spectroscopy is insufficient for the differentiation of fiber dyes.
  • Mass spectrometry provides a more robust method for fiber dye analysis and allows for differentiation of previously indistinguishable dyes.