Using Thin Layer Chromatography To Monitor Reactions Environmental Sciences Essay

Thin layer chromatography (TLC) is a very useful technique for monitoring reactions. It can also be used to determine the proper solvent system for performing separations using column chromatography. TLC stationary phases are usually alumina or silica. They are polar for standard experiments or non-polar for reverse phase chromatography. The mobile phase is a solvent whose polarity is chosen by the person conducting the experiment. In most laboratory work standard phase silica plates are used. Different compounds will travel different distances up the plate depending on the polarity of the components of the mixture. The more polar compounds will be more attracted to the polar silica gel and travel shorter distances on the plate. Mon-polar substances will spend more time in the mobile phase and as a result will travel larger distances on the plate. The measure of the distance a compound travels is called the retention factor (Rf ) value.

The retention factor, or Rf, is defined as the distance traveled by the compound divided by the distance traveled by the solvent.

For example, if a compound travels 2.1 cm and the solvent front travels 2.8 cm, the Rf is 0.75:

The Rf for a compound is a constant from one experiment to the next only if the chromatography conditions below are also constant:

solvent system

adsorbent

thickness of the adsorbent

amount of material spotted

temperature

Since these factors are difficult to keep constant from experiment to experiment, relative Rf values are generally considered. “Relative Rf” means that the values are reported relative to a standard, or it means that you compare the Rf values of compounds run on the same plate at the same time.

1.1 Thin Layer Chromatography

There have been a numbered of important milestones in the evolution of chromatography in the last 100 years. Each of these milestones has signalled the start of an important branch of chromatography. Some examples of these are; partition chromatography (1941), gas chromatography (1951-1952), high performance liquid chromatography (mid- 1960’s), capillary electrophoresis (1980) and capillary electrochromatography (past decade).

In all the chromatographic techniques mentioned, separation is carried out in a column. However, it is also possible to carry out separations on a planar surface. Two examples of this are paper chromatography (1944) and thin-layer chromatography (1937-1938). Thin-layer chromatography (TLC) replaced paper chromatography as the most popular, routine chromatographic technique.

TLC was first used in 1937 to 1938 by Nikolai A. Izmailov and Maria S. Shraiber at the Institute of Experimental Pharmacy of the State University of Kharkov. At the time Izmailov was the head of the institute and Shraiber was his graduate student. They were searching for a method for the rapid analysis of galenic pharmaceutical preparations (plant extracts). As classical column chromatography would have taken too much time they felt that if the absorbent would be prepared in the form of a thin-layer on a glass plate. They believed that it would behave like a column but the characterization time would be much shorter. They coated microscope slides with a suspension of various adsorbents (calcium, magnesium and aluminium oxides). They deposited one drop of the sample solution on this layer and added one drop of the same solvent used in a column to develop separation. The test was a success as the separated sample components appeared as concentric rings that fluoresced in various colours under a UV lamp. They showed that the sequence of the concentric multicoloured rings on the plate would have been identical to the sequence of coloured rings obtained on a normal chromatographic column. They called this technique spot chromatography and the result on the microscope slides ultrachromatograms.

The paper on this experiment was published in a Russian pharmaceutical journal that was practically unknown outside the then Soviet Union. Its abstract was included in a Russian review journal and through it in chemical Abstracts. It was then read by M. O’L. Crowe of the New York State Department of Health. He then adapted the technique for his own use. Crowe prepared the adsorbent layer in a Petri dish, added a drop of the sample solution in the centre and then added the developing solvent dropwise until sufficient separation was obtained.

In 1947 T.I. Williams described a further improvement of the method of Izmailov and Shraiber in his textbook on chromatography. He prepared the adsorbent-coated glass plates in the form of a sandwich. The adsorbent layer was covered by 2 glass plates and had a small hole which the sample drops could be applied through.

Meinhard and Hall made the next major step in the development of TLC at the University of Wisconsin. They used corn starch, which acted as a binder, to hold the coating on the glass plate and added a small amount of Celite powder to the adsorbent particles to improve the consistency of the layer. They called this surface chromatography. They used it to separate inorganic ions.

Modern TLC started 50 years ago with the work Department of Agriculture Fruit and Vegetable laboratory in Southern California. He investigated the flavour components of the juices of citrus fruits. However, he stated that very large volumes had to be processed because the amount of flavour material was extremely small. Another problem was in finding an analytical method for the investigation of the juice concentrate composition.

He followed the method of Meinhard and Hall that he read in Chemical Abstracts. However, instead of adding just a drop of the developing solvent he developed the plates as in paper chromatography. The plates were developed in a closed chamber and one side was dipped into the solvent. The solvent then ascended through the plate by capillary action. It carried with it the sample components and they were separated as a result. The experiments carried out were published and are considered the start of modern TLC.

Egon Stahl was responsible for TLC becoming a universally accepted technique. He was also the first to use the term thin-layer chromatography to characterize the technique. This choice of name was almost immediately accepted.

Stahl investigated various essential oils and obtained good results using adsorbent-coated glass plates. However, neither the method nor the adsorbent to be used had been optimized. Also, the adsorbents had to be modified and treated before they could be used for the coating of plates. Stahl started investigating the operational parameters and the adsorbent preparations. In the spring of 1958 his efforts were fulfilled as the necessary basic instrumentation, made by Desaga and “silica gel G according to Stahl for TLC”, made by E. Merck were both introduced at the international Achema exhibition of chemical equipment in Frankfurt. Stahl also published an article outlining the use of the system and a wide range of applications. Because of this standardized method TLC became a widely used laboratory technique. He also went on to publish a TLC handbook in 1962.

Although TLC had a wide application it was still thought to be a qualitative technique for the analysis of simple mixtures. As a result advances were directed toward improving the technique. Instrumentation which permitted more precise spotting of the sample onto the plates and the quantitative evaluation of the separated spots was developed. Faster analysis and higher separation power was also achieved. As a result of the higher performance ability the name was change to high-performance TLC (HPTLC) by R.E. Kaiser, who was instrumental in its development.

The particle size and range of the adsorbent was the main difference between TLC and HPTLC. The silica gel for TLC had broad particle sizes of 10-60µm with an average of 20µm whereas HPTLC has an average of only 5µm. the HPTLC plates were also smaller in comparison with TLC plates, 10 x 10cm and 20 x 20cm respectively. The improved method and design allowed reduction in the diameter of the starting spots. These improvements lowered the analysis time and increased the efficiency. Problems arose with flow rate which Kaiser overcame by applying pressure to the TLC plate. This in turn led to forced-flow TLC.

Due to the constant condensation-evaporation process associated with developing TLC plates in developing chambers problems can be encountered because of the changing velocity of the mobile phase. To overcome this forced-flow TLC (FFTLC) was developed by Tyihák, Mincsovics and Kalász. In this method the spotted plates (dry) are placed into a pressurized development chamber. The stationary-phase layer is tightly covered and sealed on its side by an elastic membrane and pressurized by an inert gas or water filling up the cushion above the layer. The mobile phase is delivered via a pump at a constant velocity through a slit in the membrane to the stationary phase. There are various configurations which can be handled using this method.

TLC is a very simple technique. As a result very little instrumentation is needed. Application of samples to the stationary-phase is carried out using a micropipette or syringe. The developing chambers are simple glass structures. Detection is carried out by visual inspection or made visible by spraying the plate with reagent. Also, a wide variety of precoated plates are available so coating equipment isn’t needed.

In more advanced systems the samples may be spotted by automated loading devices (dosimeters). This allows the application of small and uniform sample spots. More sophisticated developing chambers are also available (FFTLC). The plates can be scanned by densitometers and quantitatively analysed using absorbance or fluorescence measurements. Chromatograms with peaks of the individual separated spots recorded against the length of the plate are produced with such analyses. Their area is also a proportion to the amount present. More complex systems can also be created by combining TLC systems with other systems such as mass spectrometry and Fourier transform infrared.

1.2 Ink Analysis

Ink analysis is a very important forensic procedure. It can reveal useful information about questioned document. Modern inks contain many substances which are aimed at improving the ink. The most important component of the ink is the colouring material. It comes in the form of a dye, pigment or a combination of both. Dyes are soluble in the liquid body of the ink, also known as the vehicle. Pigments are finely ground multi-molecular granules that are insoluble in the vehicle. The vehicles composition affects the flowing and drying characteristics of the ink and can consist of oils, solvents and resins.

1.3 Chromatography Studies

Djozan et al developed a new and fast method for the differentiation of inks on a questioned document. They designed specific image analysis software for evaluating thin layer chromatograms. They sampled forty-one blue ballpoint pens which were purchased from their local markets in Iran (Table 1).

They first wrote a circle of diameter 5 mm uniformly by pen on a paper. One fourth of this was then punched out for extraction. They carried out extraction in 1 ml glass tubes and added 0.1 ml of methanol. This was then vigorously shaken for 1 min. the ink component was then fully dissolved in methanol. The supernatant methanoic solutions were then used to spot the TLC plates. A blank sample of paper with equal dimensions was also treated in the same way.

Table 1. List of blue ballpoint pens studied

List of blue ballpoint pens studied

1 Cello pyramid 0.7 mm fine TC ball

2 OBA

3 AIHAO

4 Bic 01

5 Cenator

6 PARKER

7 A.T.CROSS FINE

8 Pelikan STICK 918

9 Marvy SB-10 1.0 mm

10 Bic 02

11 PIANO crystal

12 My pen 2001 PENS High Quality Bluce CE

13 AIBA

14 STAEDTLER Stick 430M A IRAN

15 Reynolds Medium 048 France

16 EIFEL Elegance

17 CASPIAN STICK 2001 M

18 STABILO liner 308

19 FABER-CASTELL 1.0 mm Medium (transparent)

20 BIC 08

21 Bocheng A-100

22 SCHNEIDER TOPS 505 M Germany

23 FIBER-CASTELL 1.0 mm Medium

24 MILAN PI 1 mm

25 Reform

26 PAPER = MATE FLEXGRIP ultra MED

27 PARKER UK

28 CANDID-DINI 2853

29 STABILO-galaxy 818 M

30 No name

31 No name

32 Zebra Rubber 101

33 SANFORD SAGA

34 Bensia

35 Girls

36 EUROPEN

37 PARS swiss Refill 606

38 STAEDTLER stick 430 M TBRITAIN

39 Lus HF 500

40 No name

41 STABILO bill 508

TLC analysis was carried out on Merck (Darmstadt, Germany) 20 cm x 20 cm silica gel 60 TLC plates without fluorescent indicator. The plates were activated at 60 °C for 20 min and immediately spotted after cooling in a desiccator. The plates were developed in a developing chamber. The mobile phase used was: ethyl acetate/ absolute ethanol/ distilled water (70:35:30, v/v/v). Chromatographic development of the plates was carried out at room temperature for 40 min. All mobile phases were prepared daily with analytical grade chemicals. Enough was prepared to supply the tank for each run. The plates were air-dried after development. The separated compounds were visible on the plate by their natural colour and the plates were scanned into a computer using an office scanner.

An IBM compatible PC (Pentium IV) with a 2.6 GHz microprocessor, 256 MB random access memory (RAM) and a hard disk with 40 GB capacity for external storage was used for processing the colour images. The computer was equipped with an on-board graphic card (NviDiA Geforce 7300LE) and a scanner (CanoScan 4200F) was connected to the computer for scanning (300 dpi) TLC plates as digital images. The images were saved as bmp files. Matlab (Version 6.5, The Math Works, Inc.) was used to write a new program to process the previously saved images.

Previous studies indicated that Pyridine is the solvent used with ballpoint pen inks. Djozan et al preformed extraction with different solvents using various extraction modes. These modes were immersion of paper into solvent and simple agitation for 1 min, ultra-sound assisted extraction and micro-wave assistance extraction. The results showed that the immersion of paper into methanol or pyridine and simple agitation resulted in complete extraction of the inks from paper (Table 2).

Table 2. List of solvents used for extraction of ink components from paper

Solvent

Solubility of ink colours

Ethyl acetate

Ethanol

Acetic acid

Acetone

Butanol

1,2-Dichloroethane

Butyl acetate

Tetrachloroethane

Acetyl acetate

Cyclohexane

Methanol

Pyridine

Slightly

Soluble

No improvement was found using ultra-sound or micro-wave assisted extraction. Methanol was chosen as the extraction solvent due to the safety of the solvent. The selection of the plate was down to the fact that silica gel plates provided the best resolution of dye spots. They selected five mobile phases (Table 3) and found that ethyl acetate/ absolute ethanol/ distilled water (70:35:30, v/v/v) was effective in separating nearly all the dye mixtures. The spot capacity obtained was more than 15.

Table 3. Different solvent systems used to develop plate

Solvent System

Ratio

Spot capacity

Butanol:ethanol:H2O

Ethyl acetate:cyclohexane:methanol:NH3

Ethyl acetate:Butanol:NH3

Ethyl acetate:ethanol:H2O

Toluene:acetone:ethanol:NH3

50:15:10

70:15:10:5

60:35:5

70:35:30

30:60:7:2

Fig. 1. Typical TLC results of 10 different ink samples (Djozan et al, 2008)

Fig. 1. shows a typical chromatogram that they achieved in their experiment. To confirm complete separation of all components in the studied sample, two-dimensional (2D) TLC was carried out using various solvent systems. The results proved that the one-dimensional (1D) TLC is able to provide sufficient separation.

The first stage carried out was colour image normalization. A function of the input images was computed that is invariant to confounding scene properties but was discriminative with respect to desired scene information. The calculation is as follows:

Stage 2 is to compute a colour image profile. The intensity profile of an image is the set intensity values taken from regularly spaced points along a line segment in an image the intensity values are interpolated for points that don’t fall on the centre pixel they computed an intensity profile for r, g and b images along the line passing through the centre of the image on the chromatographic development straight of each ink spot.

Fig. 2. RGB characteristic of an ink after TLC (Djozan et al, 2008)

In stage 3 the colour image profiles were correlated. The intensity profiles were considered as sequences and the normalized cross-correlation of sequences were computed. Cross-correlation is a measure of similarity of two signals. It is used to find features in an unknown signal and compared to a known signal. It is calculated as follows for discrete functions:

Eq. (1)

For image-processing applications in which the brightness of the images can be due to lighting and exposure conditions, the images can be first normalized. It is calculated as follows:

Eq. (2)

Stage 4 involved computing image similarity. The weighted mean of and were computed as follows:

Eq. (3)

The ability of the method to differentiate between various blue ballpoint pens was evaluated by comparing the similarity of different inks according to Eq. (3).

Fig. 3. Screen shot of Matlab software running (Djozan et al, 2008)

Fig. 4. All possible combination of comparing inks with TLC-IA (Djozan et al, 2008)

In 2006 Liu et al published a paper on the classification of black gel pen inks by ion-pairing high-performance liquid chromatography. They stated that black gel inks usually contain several dye components. These components all have different colours and are mixed together proportionally to give the black colour.

They used reverse-phase ion-pairing high performance chromatography (RP-IP-HPLC). It was done in such a manner as the dyes couldn’t be reversed on the C18 column due to their high polarities. The maximum UV absorption bands of the black gel pen inks obtained were between 500 and 700 nm. The wavelength of the detector was set to 580 nm as most of the dyes had a maximum UV adsorption near 580 nm.

They investigated the influence of both volatile and non-volatile ion-pairing reagents on the HPLC analysis of black gel pen ink dyes. All the reagents had different alkyl chain, ammonium acetate, triethylamine (TEA), tributylamine (TBA), dihexylamine (DHA) and tetrabutylammonium bromide (TBABr). The results revealed that the dyes were nearly not retained using ammonium acetate or TEA as the ion-pairing reagent. Using TBABr, TBA and DHA as the ion-pairing reagent, individually, the dyes were separated. TBABr was selected as the ion-pairing reagent as the retention times were shorter than the others and sharper peaks were obtained.

They also investigated the buffer solution concentrations and the effect of pH on the separation. The optimum result was: 40 mmol/L TBABr buffer solution (pH 7) with acetonitrile as the organic modifier for IP-HPLC analysis and an identical proportion of the buffer and acetonitrile (v/v = 40:60) at a flow rate of 1.0 mL/min. these optimum conditions were used to separate 50 dye-based black gel pen inks by IP-HPLC.

Liu et al carried out another study on ion-pairing HPLC in 2006. This time, however, they studied the degradation of blue gel pen dyes and also used electrospray tandem mass spectrometry.

They used ion-pairing reversed phase liquid chromatography as the inorganic compounds they were analysing have weak retention on the ordinary reversed stationary phases when separating on HPLC. This is due to their high polarities. The UV detector was set at 580 nm for the analysis as most dyes have a normal maximum absorption near 580 nm. The UV absorptions of the fluorescence whitening reagents in paper are usually below 500 nm and they had no interference for the detection of the gel pen dyes at 580 nm.

Fig. 5. Chromatograms of blue gel pen inks using different ion pairing reagents (Liu et al, 2006)

The tested various mobile phases: eluent A: eluent B (acetonitrile) = 50:50 (v/v); eluent A was the buffer of ion pairing reagent with concentration of 40 mmol Lâˆ’1 (pH 7.0), and the ion pairing reagent was (a) ammonium acetate, (b) TEA acetate, (d)

TBA acetate, (e) DHA acetate and (f) TBABr, respectively. (c) Ammonium carbonate as eluent A (40 mmol Lâˆ’1, pH 9.5) and eluent A:eluent B (acetonitrile) = 50:50

(v/v). they found that 10 mmol-1 TBA acetate (pH 7.0) was suitable ion-pairing agent for the purpose and ink samples stored in different conditions were analyzed by IP-HPLC. Significant changes of ink composition were observed. The noticed that the natural aged inks had the similar but weaker degradation trend than the light aged inks. They used HPLC-MS/MS with ammonium carbonate as ion-pairing reagent to obtain the information of the light aged inks and their photodegradation mechanism.

In 1994, Varshney et al analysed ink from typed script of electronic typewriters by HPTLC. They used script from seven electronic typewriters. They used the resultant Rf values and in-situ visible spectra of each resolved band of all the chromatograms indicated that the same chemical composition is being used in six typewriter ribbon inks. However, the seventh one is completely different.

Fig. 6. Wavelength maxima values of in-situ visible spectrum bands of electronic typewriter scripts (Varshney et al, 1994)

Fig. 6. shows the densitograms obtained after scanning and integration of the chromatograms of tracks of individual typewriters and blank paper. The seven electronic typewriter inks could be categorised into two groups after analysis. The first group resolved the sample to four bands including the base. The second group did not resolve the samples at all with the solvent systems used.

Several varieties of blue ballpoint pen inks were analysed by HPLC and IR spectroscopy by Kher et al in 2006. The chromatographic data extracted at four wavelengths (254, 279, 370 and 400 nm) was analyzed individually and at a combination of these wavelengths by the soft independent modeling of class analogies (SIMCA) technique. They used principal components analysis (PCA) to estimate the separation between the pen samples. Linear discriminant analysis (LDA) measured the probability with which an observation could be assigned to a pen class. The best resolution was obtained by HPLC using data from all four wavelengths together, differentiating 96.4% pen pairs successfully using PCA and 97.9% pen samples by LDA. PCA separated 60.7% of the pen pairs and LDA provided a correct classification of 62.5% of the pens analyzed by IR. They stated that HPLC coupled with chemometrics provided a better discrimination of ballpoint pen inks compared to IR.

Kher et al effectively combined LDA and PCA to classify the HPLC and IR data. PCA gives a general idea of how different a given pair of pens is, whereas LDA can quantify the predictive ability of a generated classification model. The two techniques of PCA and LDA were shown to be complimentary to each other. The PCA and LDA results indicated that although IR cannot differentiate between all classes of pen inks, it can still provide a reasonable discrimination, which can be enhanced further by improving the quality of the spectra. The analysis of such an enhanced IR data with chemometric analyses would provide a valuable non-destructive tool for forensic analyses.

Raman Spectroscopy Studies

Mazzella and Buzini used Raman spectroscopy to analyse blue gel pen inks in 2004. They sampled 55 blue gel pens. They first separated them into two groups using a preliminary solubility test in methanol. They discovered that 36 were pigmented inks, which aren’t soluble in methanol, and 19 were dye-based inks, which are soluble in methanol.

They applied Raman spectroscopy to the 36 pigmented blue gel inks. Raman spectroscopy is a non-destructive technique. Spectra were first obtained using the 514.5 nm argon ion laser which proved the observation of 4 different groups. They then used the 830 nm NIR diode laser and divided the inks into three groups. They then combined the two lasers and a separation into 5 groups was obtained.

They then attempted to identify the pigments contained in the gel by comparison to standard pigments. Two main pigments were detected in the analysed samples: PB15 and PV23. PB 15 is pigment blue 15 and belongs to the class of phthalocyanines. PV23 is the pigment violet 23 and belongs to the class oxazines. The argon laser allowed the detection of a mixture of PB 15 and PV 23. This was a better result than using the NIR diode laser.

The results showed that the same gel pen ink (same model and brand) from different geographical locations showed the same Raman spectra. However, it was stated that the Raman technique obtained low discriminating values.

2. Materials and Method

2.1 Materials:

Blue ballpoint pens

Merck silica gel 60 TLC plates (20 cm x 20 cm)

Methanol

Ethyl acetate

Ethanol (absolute)

Paper

Dessicator

Developing chamber

Puncher

Glass tubes (0.1 ml)

Capillary tubes

2.2 Experimental

13 blue ball-point pens (Table 1) were bought from a number of different shops in the town. A circle with a diameter of 5mm was written by the pen on paper. One fourth of it was punched out for extraction. The samples are placed in 1 ml glass tubes. 0.1ml of methanol was added and vigorously shaken for 1 min. The ink component was fully dissolved in methanol. The supernatant methanoic solutions were used for spot application on TLC plate. A blank of paper only is also treated as was a control which was a permanent marker. TLC analyses were preformed using Alugram 20 cm x 10 cm silica gel/UV plates (Macherey-Nagel). The plates were activated at 60°C for 20 min and immediately after, cooled in a desiccator, and spotted. The plates were developed in a horizontal developing chamber. The solvent system included: ethyl acetate/absolute ethanol/ distilled water (70:35:30, v/v/v). Development was preformed at room temperature for 40 min. All mobile phases were prepared daily. After development the plates were air-dried. All 13 different pens were tested in triplicate. Retention factors were calculated using the results from the plates and photographs taken using a digital camera were loaded onto the computer and analysed using image analysis software.

Table 1:

List of pens analysed

Number

Description

No Brand (blue)

Pilot G-207

BIC ReAction

BIC Medium (Bought in Tesco)

BIC Medium (Bought in Dunnes)

No Brand (Purple)

Staedtler Stick 430M

Roller Pen

Papermate 1.2M

Scripto Stick Pen

Papermate Write Bros.

Comfort Touch

No Brand (Tesco Click Pen)

3. Results and Discussion

Before carrying out the experiment it needed to be researched. This research pointed out the importance of the correct solvent to remove the ink from the document. Djozan et al used methanol as their choice of solvent after considering other solvents (Table 2). They stated that Pyridine was the reported solvent used with ball-point pen inks. However, they carried out extractions with different solvents using various extraction modes. They realised that immersion of the paper into methanol with agitation resulted in complete extraction of the inks from the paper. Methanol was also chosen because of its safety.

Table 2:

List of solvents used for the extraction of ink components from paper

Solvent

Solubility of ink colours

Ethyl acetate

Slightly

Ethanol

Slightly

Acetic Acid

Slightly

Acetone

Slightly

Butanol

Slightly

1,2-Dichloroethane

Slightly

Butyl acetate

Slightly

Tetrachloroethane

Slightly

Acethyl acetate

Slightly

Cyclohexan

Slightly

Methanol

Soluble

Oyridine

Soluble

Different concentrations of the solvent system (Table 3) were analysed to see which gave the greater separation. It was found that the concentration given by Djozan et al, (ethyl acetate, ethanol, and water (70:35:30, v/v/v)) gave the best results. The Alugram silica Gell/UV plates were also found to work better than the suggested, Merck silica gel 60, plates without fluorescent indicator.

Table 3:

Concentrations of solvent system investigated

Number

Solvent system

ethyl acetate, ethanol, water (70:35:30, v/v/v)

ethyl acetate, ethanol, water (70:30:35, v/v/v)

ethyl acetate, ethanol, water (70:25:40, v/v/v)

ethyl acetate, ethanol, water (70:40:25, v/v/v)

Table 4:

Retention factors for all separated components

pen

spot 1

spot 2

spot 3

spot 4

spot 5

Solvent

RF1

RF2

RF3

RF4

RF5

1.1

0.958904

1.2

0.958904

1.2

0.958904

2.1

0.849315

81.24194

2.2

0.849315

81.24194

2.3

0.849315

81.24194

3.1

0.814286

73.68421

0.841429

3.2

0.814286

73.68421

0.841429

3.3

0.814286

73.68421

0.841429

4.1

0.547945

76.65

0.74364

80.68421

4.2

0.547945

76.65

0.74364

80.68421

4.3

0.547945

76.65

0.74364

80.68421

5.1

0.557143

102.3077

0.576692

5.2

0.557143

102.3077

0.576692

5.3

0.557143

102.3077

0.576692

6.1

0.685714

86.04167

0.732203

6.2

0.685714

86.04167

0.732203

6.3

0.685714

86.04167

0.732203

7.1

0.8

72.5

0.841379

74.87705

7.2

0.8

72.5

0.841379

74.87705

7.3

0.8

72.5

0.841379

74.87705

8.1

0.547945

76.65

8.2

0.547945

76.65

8.3

0.547945

76.65

9.1

0.8

72.5

0.827586

9.2

0.8

72.5

0.827586

9.3

0.8

72.5

0.827586

10.1

0.657534

85.16667

0.681018

89.57184

0.71451

10.2

0.657534

85.16667

0.681018

89.57184

0.71451

10.3

0.657534

85.16667

0.681018

89.57184

0.71451

11.1

0.753425

76.98182

0.792395

83.2918

11.2

0.753425

76.98182

0.792395

83.2918

11.3

0.753425

76.98182

0.792395

83.2918

12.1

0.814286

72.45614

0.841889

12.2

0.814286

72.45614

0.841889

12.3

0.814286

72.45614

0.841889

13.1

0.753425

76.98182

0.792395

13.2

0.753425

76.98182

0.792395

13.3

0.753425

76.98182

0.792395

No Retention factor was calculated for the blank. The control all had a retention factor of around 0.971.

The blue ballpoint pen inks were discriminated against using image processing software.

Fig. 1:

Pen 3

Fig. 2:

Pen 10

Shown above are the results for the image processing software of pen 3 and pen 10. As clearly shown the spectra of both pens are considerably different.

Conclusion

The aim of this study is to find methods to analyze and classify different pen ink samples by TLC. This is particularly useful in the field of forensic science so as to collect evidence from crime scene and to analyze forged documents.

This experiment shows a fast and straightforward method for evaluating thin layer chromatograms after photographing them with a digital camera. The software used was cell phone spectrometer software available to download for free. It is based on the intensity profile of RGB characteristic. Using this software and the retention factors calculated allow the discrimination between different pen inks with high reliability. We successfully analyzed and discriminated 13 blue ballpoint pen inks of different trade marks available at local shops at the time of the study.

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