The Identification of a Lipstick Brand: A Comparison of the Red Pigment R f Values using Thin Layer Chromatography Ali Robertson and Margaret Mercer Heathwood Hall Episcopal School 11 th Grade 1
ABSTRACT Forensic evidence can be left behind at a crime scene in many different ways. Lipstick, when analyzed correctly, can be a valuable addition to any selection of forensic evidence. Essentially, all lipsticks are composed of wax, oil, alcohol, and pigments. In addition to these main ingredients, numerous other ingredients can be added to change the physical properties of the lipstick that may range from complex organic compounds to entirely natural ingredients. Thin layer chromatography (TLC) is one method that can be used to identify a lipstick by displaying the band pattern of its dyes. In TLC, the dyes and pigments in the samples of lipstick will travel with the solvent up the chromatography paper and separate according to their solubility within the solvent. It was hypothesized that three lipsticks could be identified by their red pigments alone- that they would have a pigment with a solubility unique to their brand. A solvent was prepared from Acetone, ammonium hydroxide, distilled water, and isoamyl alcohol. Four chromatograms were completed and R f values for each lipstick's banding patterns were calculated. The data was analyzed using an Analysis of Variance (ANOVA) single factor test, and the differences between each lipstick's R f values for the red pigment were found to be statistically insignificant. Thus, the hypotheses were rejected, and the null hypotheses were accepted. It was further noted that a qualitative identification of the lipsticks was possible through the presence of bands that were unique to their chromatogram. 2
INTRODUCTION Evidence can be left behind at a crime scene in many different ways as a fingerprint, an epithelial cell under a fingernail, or even a lipstick smear on an old napkin. Unfortunately, evidence like lipstick prints is often overlooked. Forensic science is defined as the use of scientific methods or techniques to help solve legal questions or a crime. Modern television can often be very misleading in portraying the role of science in solving crimes, and has contributed to society s fixation with forensic evidence in a court room. This sensation is often referred to as the CSI Effect (1). Just one key difference between an investigation on TV and a real life crime scene investigation is in the processing and analysis of trace evidence. When a TV investigator like Horatio Cane lifts a print from a crime scene, a computer quickly comes up with a conclusive match of a suspect. However, in a real life situation, fingerprint database systems only show the probability that a fingerprint may be a match, and often are calibrated to generate a list of possible matches. In actuality, the results are often studied by a human. Even then, the match isn't always conclusive (2). In 2004, for example, an FBI computerized fingerprint analysis erroneously identified an Oregon attorney as a participant in the Madrid terror bombing. He was freed after Spanish fingerprint examiners identified the print as belonging to someone else (2). Often overlooked, lipsticks, cosmetics, nail polish, or other smeared powders are chemical evidence (3), and may all be significant forensic evidence in the investigation of a crime, especially in cases such as a sexual assault or a homicide. This physical evidence may be found on clothing, parts of the body, a tissue, or cigarette. If properly identified, a smear of lipstick may even be enough evidence to convict a criminal, but more often than not, it may link a suspect to a crime scene (4). 3
Lipsticks are essentially composed of wax, oil, alcohol, and pigments. Usually, the wax used is made up of a combination of three types of wax: beeswax, candelilla wax, or the more expensive camauba (5). Waxes are organic compounds that belong to the lipid group. A lipid is hydrophobic: their fatty acid tails, made up of molecules of carbon and hydrogen, contribute to the compound s fear of water (6). Wax in the lipstick allows the other ingredients to be formed into the tube-like shape of most lipsticks. Oils, like waxes, are lipids, and are also a component of lipstick. The oils, such as mineral, caster, Lanolin, or vegetable are added to the wax, giving the lipstick its smooth texture (7). The color of lipstick is mainly due to aluminum, calcium, or barium dyes added to the lipstick in concentrations of 15% to 20% (5). Fragrances are also added, along with preservatives and antioxidants. To make lipstick, first the raw ingredients are melted separately, and then oils and solvents are ground together with the desired color pigments (5). In addition to these main compounds, a wide variety of other ingredients can also be included to make substances smoother, glossier, or shinier. The amounts of these other substances, however, vary greatly. The ingredients themselves range from complex organic compounds to entirely natural ingredients, whose proportions in the mixture determine the characteristics of the company s lipstick (5). The analysis of lipstick in criminal investigations can be a key part in linking a particular suspect to a crime scene or victim. A Scanning Electron Microscope can be used to determine the origin or identity of a sample. This kind of microscope emits a beam of electrons concentrated by magnetic lenses at a sample. Electrons that are released from the sample have a specific energy level that is analyzed by a computer, which then produces an image of the sample (8). One particular method for the analysis of lipstick is high performance liquid chromatography. HPLC uses the same principles that thin layer chromatography and column 4
chromatography both utilize. HPLC forces a solvent through a column under high pressures of up to 400 atmospheres, which allows analysis in a shorter period of time and achieves a higher degree of resolution than conventional chromatography (10). The lipstick, which would be in the mobile phase, travels through a column of tightly packed solid particles, which would be the stationary phase. Another method for lipstick analysis is thin layer chromatography. Thin layer chromatography (TLC) is used in the separation and qualitative analysis of small amounts of material. Like column chromatography, TLC is a solid liquid partitioning technique that uses a thin film of silica coated onto glass or a plastic strip (CITE). A mixture of the compounds to be separated is placed at one end of the strip and it is placed in a solvent that moves up the strip because of capillary action. This technique is often called spotting. Capillary action is the process by which liquids are drawn upward against the force of gravity due to the attraction between molecules. It can also be determined by adhesion, cohesion, and surface tension. One compound will move up the strip faster than the others because it is more soluble than the other dyes. Therefore, as the chromatogram separates, the more soluble dyes will be at the top of the chromatogram because they can be more easily dissolved in the solvent, and the less soluble dyes will be at the bottom of the chromatogram because they are not as dissolved in the solvent. A given compound will always travel a fixed distance relative to the distance the solvent front travels, under a certain set of conditions. TLC conditions include a solvent system, an adsorbent, the thickness of the adsorbent layer, and the relative amount of material spotted. This ratio of the distance the compound travels to the distance the solvent travels is called the R f value. The symbol Rf stands for retardation factor or ratio-to-front. It can be expressed through a decimal fraction: 5
R f = distance solute traveled distance solvent traveled front When measurement conditions are completely specified, the R f value will be constant for any given compound, and it will correspond to a physical property of that compound. The purpose of this study is to identify three different lipstick brands using solely their red pigment and to determine if this method can be applied for forensic use. The three lipsticks that will be used are in three different price ranges and are as follows: Chanel Allure in Audace 58, which is described as having a luminous and satiny effect (cite); Urban Decay Lipstick in Revolution, which is described as true red/full coverage cream (12); and Revlon Colorburst in True Red 090. It is hypothesized that through thin layer chromatography analysis of the red dye concentrations in these three different brands will provide characteristic data to distinguish among lipstick sources. The Chanel Audace will be distinguishable from the Urban Decay Revolution lipstick. The Urban Decay Revolution will be distinguishable from the Revlon True Red lipstick, and finally the Chanel Audace lipstick will be distinguishable from the Revlon True Red lipstick. The null hypothesis is that through thin layer chromatography analysis of the red dye concentrations in these three different brands of lipstick, there will be no characteristic data to distinguish among the lipstick sources, and therefore the concentrations of red dye will not be a unique identifier for the lipstick sources. The concentration of red pigment will differ among three brands within a small constant sample of red lipstick. 6
MATERIALS & METHOD Gloves Protective Eyewear 5 600 ml beakers Filter Paper 5 watch glasses Isoamyl alcohol Acetone Ammonium hydroxide Distilled water Graduated cylinder Whatman 250 micrometer Layer 20 x 20 cm Flexible TLC plates containing silica gel Urban Decay Revolution lipstick Revlon True Red 090 Lipstick Chanel Rouge Audace 58 Lipstick Ruler Pencil UV light 7
Prior to experimentation, the developing solvent was prepared under a fume hood, using 5 ml of isoamyl alcohol, 3.5 ml of acetone, 3 ml of distilled water, and 0.5 ml of ammonium hydroxide. This developing solvent was prepared for each of the five chromatograms. The solvent was poured into a 600 ml beaker with filter paper inside to create a moist and saturated atmosphere for the chromatogram. A watch glass was placed over the top of the beakers to prevent evaporation. To prepare the chromatograms, 6 cm x 10 cm rectangles were cut from 250 micrometer Layer 20 x 20 cm Flexible TLC plates containing silica gel. A centimeter from the bottom of the gel was measured and marked with a pencil. A small sample of each brand of lipstick was placed on this line. The chromatograms were then placed carefully into the beakers containing the solvent. After the solvent had traveled through the gel, the chromatograms were taken out of the beakers and the solvent front was measured and marked with a pencil. After drying in the fume hood, the chromatograms were observed under a UV light. Band distances were measured and recorded, and R f values were calculated. The data were analyzed used a single factor Analysis of Variance (ANOVA) Test. RESULTS The data collected from the chromatograms was recorded, and the R f values were calculated using distance solute traveled/distance solvent traveled (d su /d sv ). All of these values are shown in Appendix A, Tables 1-3. The R f values for the red dyes of the 8
lipsticks were separated and placed into Table 1 to set up for further analysis. It was further noted that two of the three lipsticks displayed an orange band. These R f values were separated, much like the red R f values, in preparation for analysis, in Table 2. Before performing statistical analysis, both the components of Table 1 and those of Table 2 were graphed to reveal correlations (Figures 1 and 2). After this initial look, an Analysis of Variance, or ANOVA, single factor test was run on both the data in Table 1, the red R f values, and the data in Table 2, the orange R f values. The results of this analysis are shown in Tables 3 and 4. In Table 3, the ANOVA test for the Red Rf values, the variances were small numbers, ranging from 0.00016 to 0.00043. The ANOVA test showed that the differences between the red dye R f values of the lipsticks were not statistically significant. Table 4 shows the ANOVA single factor test on the orange pigment R f values. Likewise, the variances are minute numbers, and the test once again showed that the difference between the R f values of the orange pigments in the Urban Decay lipstick sample and the Chanel lipstick sample are not statistically significant. Table 1. Calculated R f Values of Red Pigment in Each Lipstick Calculated R f Values - Red Pigments Revlon True Red 090 Urban Decay Revolution Chanel Audace 58 Trial One 0.22 0.21 0.22 Trial Two 0.24 0.23 0.25 Trial Three 0.24 0.24 0.27 Trial Four 0.25 0.24 0.25 Table 2. Calculated R f Values of Orange Pigment in Two Lipsticks Calculated R f Values Orange Pigments Urban Decay Chanel Trial One 0.18 0.19 Trial Two 0.2 0.2 9
Trial Three 0.21 0.21 Trial Four 0.2 0.19 Figure 1. Comparison of Red Pigment R f Values in 3 Lipsticks Figure 2. Comparison of Orange Pigment Rf Values in 2 Lipsticks Anova: Single Factor Table 3. ANOVA test for Red R f Values 10
SUMMARY Groups Count Sum Average Variance Revlon 4 0.95 0.2375 0.000158 Urban Decay 4 0.92 0.23 0.0002 Chanel 4 0.99 0.2475 0.000425 ANOVA Source of Variation SS df MS F P-value F crit Between Groups 0.000617 2 0.000308 1.180851 0.350426 4.256495 Within Groups 0.00235 9 0.000261 Total 0.002967 11 Table 4. ANOVA Single Factor Test on Orange R f Values SUMMARY Groups Count Sum Average Variance Urban Decay 4 0.79 0.1975 0.000158333 Chanel 4 0.79 0.1975 9.16667 11