Volume 4 • Issue 1 • 1000157J Anal Bioanal TechniquesISSN:2155-9872 JABT, an open access journal

Open AccessResearch Article

Joshi et al., J Anal Bioanal Techniques 2013, 4:1 DOI: 10.4172/2155-9872.1000157

Keywords: Chromatographic analysis; Red pigments; Lipsticks;Lipstick samples; TLC; Solvent system; hRf value

IntroductionIn forensic science, comparative examinations are usually based

on the physical or chemical nature of a substance, or both. Lipsticks are composed of waxes, oils, organic dyes [1], and inorganic pigments [2,3]. Color matching can do identifying the lipstick responsible for leaving a smear. This color analysis may be used to identify the lipstick found at the crime scene [4].

The colors of lipstick are often due to a mixture of several pigment compounds. These pigments can be separated using thin layer chromatography. Depending on the type of pigment, the mobile phase will vary. Lipsticks are soluble in toluene, so toluene serves as the mobile phase. After separation, the chromatogram is complete and illustrates the different pigments that make up a particular color of lipstick [2,3,5-10].

Traces of Lipsticks, cosmetics, nail polish, or other smears could be found left on drinking cups, glasses, cigarette butts, and tissue papers and may all be significant forensic evidence [11] in the investigation of a crime, especially in cases such as a sexual assault or a homicide [12]. This physical evidence may be found on clothing, parts of the body, a tissue, or cigarette [5,13]. By comparing the composition of a lipstick smear with that of a victim, forensic scientists can demonstrate indirect proof of contact or a relationship between victim and suspect. Also, it is sometimes possible to extract saliva DNA from the print and may link a suspect to a crime scene [2].

Various methods of forensic lipstick analysis were reported [6,8,14-17]. Small amount of lipstick (approximately 10 µg) could lead to good comparisons in TLC [3]. Thin Layer Chromatography (TLC) is a widely-used chromatography technique used to separate chemical compounds. Thin-layer chromatography incorporates a solid stationary phase and a moving liquid phase to cause a separation of the constituents of a mixture. Although simple test may be run by simply allowing a solvent to pick up a piece of porous paper, a more revealing test requires the preparation of a plate. Because most compounds are colorless, no separation will be noticed after development unless the materials are visualized. This is may be done by:

• Exposing to UV light

• Exposing to fluorescent dyes

• Exposing to iodine

• Spraying with a reagent

These procedures may be used alone or in conjunction to make thecomponents of a sample visible. The distance a component has traveled up a plate can be assigned a numerical value known as the Rf value. Rf is defined as the distance traveled by the component divided by the distance traveled by the solvent. In the example to the right, the solvent was allowed to travel 10 cm up the plate before the plate was removed from the chamber and dried.

Materials and MethodsJar with lid, lipstick samples (Branded and Local), TLC plates,

mobile phase/solvent phase, clean cotton piece, sterilized scissor, fine capillaries, ruler, pencil, todine pellets, titer plates, graduated cylinder, gloves, protective mask and thirty samples of lip impressions bearing lipstick were obtained on cloth using various brands and types of lipsticks, which were available from volunteers [5,13,18-20].

Thin-Layer Chromatography (TLC)

A thin-layer plate is prepared by coating a glass plate with a thin film of a granular material. Commonly, silica gel or aluminum oxide or cellulose immobilized onto a flat, inert carrier sheet is used, but paper may suffice in simpler experiments. These serve as the solid stationary phase. If the sample to be analyzed is a solid, it must first be dissolved in a suitable solvent. A few micro liters of the solution are then applied

*Corresponding author: Kapil Verma, M.Sc Forensic Science, Amity Institute of Forensic Sciences (AIFS), B-Block, Lower Ground Floor, Amity University, Uttar Pradesh, Sec-125, Noida-201303, Uttar Pradesh, India, E-mail: forensic.kapilalert@gmail.com

Received November 22, 2012; Accepted December 17, 2012; Published December 24, 2012

Citation: Joshi B, Verma K, Singh J (2013) A Comparison of Red Pigments in Different Lipsticks Using Thin Layer Chromatography (TLC). J Anal Bioanal Techniques 4: 157. doi:10.4172/2155-9872.1000157

Copyright: © 2013 Joshi B, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

A Comparison of Red Pigments in Different Lipsticks Using Thin Layer Chromatography (TLC)Bhawana Joshi1, Kapil Verma1* and Jyoti Singh2

1Forensic Science, Amity Institute of Forensic Sciences (AIFS), Amity University, India2Amity Institute of Forensic Sciences (AIFS), Amity University, India

AbstractThe main aim of present work is chromatographic analysis of red pigment in different well known and local

brands of lipsticks. Lipstick samples of different brands of similar color were selected for this study. Coloring agent was analyzed by thin layer chromatography (TLC). Using different solvent systems [Toluene/Benzene] (4:12), Toluene/Benzene/Cyclohexane (4:12:4), Toluene/Benzene/Diethyl ether (4:4). It is hypothesized that through thin layer chromatography analysis of the red pigment in these different brands will provide no characteristic data to distinguish among lipstick sources. There is no significant difference in the hRf values among the local and well known brands of lipsticks which can be used as unique feature.

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ISSN: 2155-9872

Volume 4 • Issue 1 • 1000157J Anal Bioanal TechniquesISSN:2155-9872 JABT, an open access journal

Citation: Joshi B, Verma K, Singh J (2013) A Comparison of Red Pigments in Different Lipsticks Using Thin Layer Chromatography (TLC). J Anal Bioanal Techniques 4: 157. doi:10.4172/2155-9872.1000157

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to the lower edge of the plate. A liquid sample may be applied directly to the solid state in the same manner. The plate or paper is then placed upright into a closed chamber that contains a selected solvent.

The solvent will slowly begin to pick up the plate by capillary action. It is the rising solvent that serves as the moving phase in thin-layer chromatography. As it moves past the sample spot, the components of the sample will become distributed between the stationary solid phase and the moving liquid phase. Those components with the greatest affinity for the moving phase will travel up the plate at a faster speed as compared to those that have greater affinity for the stationary phase.

Thin-Layer Chromatography (TLC) is a method use to separate components from each other in a mixture. It is normally experimented on glass, aluminum foil or plastic which is coated by some kind of absorbent material (ex: silica gel, aluminum oxide). It takes the Rf values of each color component and compares them; Rf values are experiments that depend on the polarity of the substance on the paper chromatography [21];

1. A piece of cotton cloth was washed in a detergent solution, immersed in a hot water, and dried.

2. This cloth was cut into small pieces (2 cm×2 cm) and lipstick smears were rubbed onto these pieces.

3. The stained areas from the cloth pieces were cut and transferred into serially marked small bowls.

4. These samples were mixed with an extracting solution and each bowl was then shaken for about 5-10 minutes to remove the stain from the cloth piece.

5. Cloth piece is taken out, and the extract was used for further examination with TLC.

6. Obtain jar with lid, piece of filter paper and a TLC plate. Handle TLC plate by the edges only, avoid touching the white silica layer.

7. With a pencil and ruler, gently draw a line across the short side of the TLC plate about 1.5 cm from the bottom of the plate. At even intervals label the top of the plate with the letters: 1B, 2B, 3B, 4B, 5B, 6B for six different well known brands of red lipsticks on a single TLC plate. Similarly 1L, 2L, 3L, 4L, 5L, 6L for six different local brands of red lipsticks.

8. Using a capillary, place a dot of each lipstick sample along the bottom penciled line directly under the corresponding label on the top of the plate. The dots should be about 0.2 cm in diameter and dark enough to be clearly visible.

9. Using a pipette dispenses mobile phase/solvent in the ratio (4:12:4) into the jar. The mobile phase/solvent should be about 0.5 cm deep.

10. Carefully insert the TLC plate into the jar, sample end down. The lipstick dots must be above the mobile phase/solvent. Secure the lid.

11. Allow the mobile phase/solvent to rise to within one cm of the top of the plate (5-10 minutes). Watch–do not allow the mobile phase to rise to the very top of the plate. Remove the TLC plate and mark the solvent front with a pencil.

12. Measure the distance the mobile phase/solvent moved in cm (the distance from the spotted pencil line to the solvent front end). Also measure the distance in cm each component of the lipsticks moved

from the spotted pencil line. Some lipsticks have only two or three components, and some have more. Enter these measurements on the Data Table.

13. Determine the Rf for each lipstick component of all lipstick samples. Enter those values on the Data Table. To calculate the Rf value, divide the distance traveled by each lipstick component by the distance traveled by the solvent.

fDistance traveled by one lipstickcomponent from the spotted pencil lineR

Distance the solvent moved from thespotted pencil=

Samples are run for both the samples of branded and local lipstick. Use hood if possible. Self life is about one month.

Steps followed are shown in figures 1-11.

ResultsThe data collected from the chromatograms were recorded, and

Figure 1: Sample of different lipstick for study.

Figure 2: Samples rubbed on a clean cotton piece Bowls contains the extracted sample.

Figure 3: Picture showing TLC plate.

Figure 4: Extract preparation in Toluene (Bowls contains theextracted sample).

Volume 4 • Issue 1 • 1000157J Anal Bioanal TechniquesISSN:2155-9872 JABT, an open access journal

Citation: Joshi B, Verma K, Singh J (2013) A Comparison of Red Pigments in Different Lipsticks Using Thin Layer Chromatography (TLC). J Anal Bioanal Techniques 4: 157. doi:10.4172/2155-9872.1000157

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the Rf values were calculated using distance solute travelled/distance solvent travelled (dsu/dsv). All of these values are shown in the table. The Rf values of the red dyes in the local brands of lipsticks are given in table 1 and in the well known brands are given in table 2.

For local brands

Table 1 shows the result obtained from the local brands of lipsticks. In which sample marked as 1L shows six pigments having

Figure 5: Extract preparation in Toluene (Bowls contains the extracted sample).

Figure 6: TLC plate inside the jar containing developing solvent.

Solvent System:- Toluene /Acetone

Figure 7: Jar containing iodine pellets.

Solvent System:- Toluene/Benzene/Diethyl ether

Figure 8: Jar containing iodine pellets.

Figure 9: Chromatogram appeared after interaction with iodine fumes (Spots seen for local brand of lipstick when viewed under Iodine fumes).

Figure 10: Chromatogram of local lipsticks (Spots seen for local brand of lipstick when viewed under Iodine fumes).

Solvent System: Toluene/Benzene/Cyclohexane

Figure 11: Chromatogram of branded lipsticks (Spots seen for Branded brand of Lipstick when viewed under Iodine Fumes).

Column 3 Column 4Lipstick Samples

No. of Spots

Distance Lipstick Components Moved (cm)

DistanceMobile Phase Moved (cm)

hRf Values for Each Colored Component

1L 1 0.7 10 72 1.2 10 123 2.7 10 274 3.7 10 375 4.7 10 476 7.6 10 76

2L 1 1.3 10 132 2.7 10 273 3.7 10 374 7.6 10 76

3L 1 0.3 10 32 1.3 10 133 2.5 10 254 4.2 10 425 7.9 10 79

4L 1 0.3 10 32 1.3 10 133 3.7 10 374 7.9 10 79

5L 1 1.3 10 132 3.6 10 363 7.8 10 78

6L 1 0.3 10 32 1.3 10 133 2.9 10 294 3.6 10 365 8 10 80

Table 1: Rf values of the red dyes in the local brands of lipsticks.

Volume 4 • Issue 1 • 1000157J Anal Bioanal TechniquesISSN:2155-9872 JABT, an open access journal

Citation: Joshi B, Verma K, Singh J (2013) A Comparison of Red Pigments in Different Lipsticks Using Thin Layer Chromatography (TLC). J Anal Bioanal Techniques 4: 157. doi:10.4172/2155-9872.1000157

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hRf values (7,12,27,37,47,76), sample 2L shows four pigments having hRf values (13,27,37,76), sample 3L shows five pigments having hRf values (3,13,25,42,79), sample 4L shows four pigments having hRf values (3,13,37,79), sample 5L shows three pigments having hRf values (13,36,78) and sample 6L shows five pigments having hRf values (3,13,29,36,80). This shows that there is no significant difference in the hRf values among the different local brands of lipsticks under study.

For well known brands

( )( )f

Distance traveled by one lipstickcomponent from the origin column 3R

Distance the solvent moved fromthe origin column 4=

Table 2 shows the result obtained from the local brands of lipsticks. In which sample marked as 1B shows one pigment having hRf value (4), sample 2B shows Two pigments having hRf values (3.5,13), sample 3B shows two pigments having hRf values (25,15.5), sample 4B shows two pigments having hRf values (2,13), sample 5B shows two pigments having hRf values (2.5,15) and sample 6B shows two pigments having hRf values (2.5,15). This shows that there is no significant difference in the hRf values among the different well known brands of lipsticks under study.

Discussion and ConclusionThe conclusion is that through thin layer chromatography analysis

Column 3 Column 4Lipstick Samples

No. of Spots

Distance Lipstick Components Moved (cm)

DistanceMobile Phase Moved (cm)

Rf Values for Each Colored Component

1B 1 0.4 10 42B 1 0.35 10 3.5

2 1.3 10 133B 1 0.25 10 25

2 1.55 10 15.54B 1 0.2 10 2

2 1.3 10 135B 1 0.25 10 2.5

2 1.5 10 156B 1 0.25 10 2.5

2 1.5 10 15

Table 2: Rf values of the red dyes in the well known brands of lipsticks.

Sample No. Name & Number Brand Color1L biros 14 local pink2L biros 8 local rosy red3L matte lip color 293 local red4L aver matte 147 local light red5L iffy super soft 15 local red6L iffy matte soft 17 local maroon

Table 3: LOCAL brand lipstick examined.

Sample No. Solvent System1 Toluene/Benzene (4:12)2 Toluene/Benzene/Cyclohexane (4:12:4)3 Toluene/Benzene/Diethyl ether (4:12:2)

Table 4: Mobile or Solvent phase.

Sample No. Name & Number Brand Color1B bridal dream 104 revlon spunky red2B cherry sparkles 57 revlon bright red3B red flame 13 revlon bright red4B red spice street wear light red5B crystal shine street wear dark red6B color burst lipstick plum revlon maroon

Table 5: REVLON brand lipstick examined.

of red dye concentrations in these different (well known) and (local) brands of lipsticks, there is no characteristic data to distinguish among the lipstick sources, and therefore the concentrations of the red dye is not be a unique identifier for the lipstick sources. The concentration of red pigment differs among these “well known” and “local” brands within a small constant sample of red lipsticks.

The chromatogram of local lipsticks (Table 3) shows that majority of the local brands contains more than 2 dyes pigments given in table 4. While branded lipsticks contains only 1 or 2 dyes pigments given in table 5. There is no significant difference in the hRf values among the local and well known brands of lipsticks which can be used as unique feature.

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