Separation and identification of multicomponent mixture by thin-layer chromatography coupled with Fourier transform–infrared microscopy

Spectrochimica Acta Part A 61 (2005) 1965–1970

Separation and identification of multicomponent mixture by thin-layer chromatography coupled with Fourier transform–infrared microscopy Wenxuan Hea,∗ , Guangqiang Chenga , Fei Zaoa , Yingchun Lina , Jianming Huanga , Robert Shanksb a

Department of Organic Analysis, Fujian Institute of Testing Technology, Beihuan Middle Road 61, Fuzhou 350003, Fujian, PR China b School of Applied Science, RMIT University, Melbourne, Vic. 3001, Australia Received 15 May 2004; accepted 27 July 2004

Abstract A fast and convenient method based on coupled thin-layer chromatography (TLC) with Fourier transform infrared (FTIR) microscopy has been established for separation and identification of multicomponent mixtures. In this study, the method was developed and consummated with more perfect TLC spots transferral process and consistent FTIR testing conditions. A newly developed technique, solid-phase extract (SPE) was introduced for sample pre-treatment instead of using traditional column chromatography. It is a new field for SPE that has already been widely applied in many other fields. It not only overcomes the backwards (low separation efficiency, time consuming and solvent consumption) of column chromatography but also makes it much easier to choose an optimum TLC sheet and to set suitable TLC loading. With all the above-mentioned modifications and supplements, the analytical method of coupled TLC with FTIR microscopy for separation and identification of multicomponent mixtures becomes more convenient and more efficient. In addition, a very complex sample (a die-cast release agent) was used as an example to demonstrate the technique. © 2004 Elsevier B.V. All rights reserved. Keywords: Thin-layer chromatography; Infrared spectroscopy; Microscopy; Solid phase extraction; Deformulation

1. Introduction In many cases, deformulation of multicomponent mixtures will be confronted in situations such as analysis of the causes of products not meeting specifications, forensic investigation, and new product research and development [1]. In general, deformulation includes separation and identification. Liquid chromatography–mass spectrometry (LC–MS), thin-layer chromatography–FTIR (TLC–FTIR), TLC–MS and gas chromatography–MS (GC–MS) are all effective methods for this purpose because all of them take advantage of the benefits (separation and identification). TLC–FTIR is the most economical of these coupled techniques described above [2]. TLC coupled with Fourier transform infrared microscopy was first proposed by the authors [3] and not only avoids the interference of background from in situ ∗

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diffuse reflectance infrared Fourier transform spectroscopy (DRIFTS)–TLC but also overcomes the disadvantage of time consuming and low efficiency transfer of components from the spots on the TLC slides for FTIR analysis. In this study, the method of TLC coupled with FTIR microscopy was developed and consummated, using more efficient transfer of components and optimisation of conditions of FTIR microscopy. Usually, the separation of multicomponent mixtures by TLC is complicated by large differences in polarity and content of the various components [4]. Traditionally, a common column chromatograph is used to provide a preliminary separation then TLC is used to further separate components. However, column chromatography is time consuming, of low separation efficiency and environmentally unfriendly (using much solvent). Solid-phase extraction (SPE) is a newly developed experimental technique that has diverse application prospects for sample preparation in many fields such as forensic analysis, pharmaceutical quality, environmental investigation and agrochemical/food analysis

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W. He et al. / Spectrochimica Acta Part A 61 (2005) 1965–1970

[5]. SPE has not yet been widely applied to deformulation field due to lack of available methodology. The number of theoretical plates for SPE is just 10–50, so, it separates analytes in digital on–off mode, which is most suitable for approximate grouping of components of multicomponent mixtures according to component polarity and solubility. The subject of this study is the application of SPE for sample pre-treatment instead of using traditional column chromatography, and then FTIR microscopy coupled with TLC was used to further separate and identify various components. In this paper, a very complex sample (a release agent for diecast aluminium) was defomulated as an example to demonstrate how these combined techniques were applied. 2. Experimental 2.1. Materials SPE was performed on Phenomenex C18, 3 mL, 500 mg and Phenomenex Sil, 3 mL, 500 mg. TLC analysis was performed on TLC plates (2.5 cm × 7.5 cm) pre-coated with a 250 ␮m layer of silica gel 60 F254 and TLC plates (2.5 cm × 7.5 cm) pre-coated with a 250 ␮m layer of RP-1860 F254 on a glass support (Merck, Germany). The solvents (methanol, acetone, ethyl acetate, and toluene) were of GR grade also from Merck. A water-based release agent for die-casting was from one company. 2.2. Analytical procedures 2.2.1. Preparation of sample and sample pre-treatment by using SPE The water-based releaser is usually composed of water, surfactants, oil, and a small amount of other additives such as

preservatives. The water was first evaporated under vacuum, 760 mmHg at 50 ◦ C. SA (I) was obtained after the water was removed, and then 50% SA (I) in methanol solution and 50% SA (I) in toluene solution were prepared for SPE. For the C18 SPE column, about 8 mL methanol and 8 mL water were used to solvate the column. About 0.05 mL of 50% SA (I) methanol solution was loaded onto the column at a rate of 1 drop min−1 . Water (8 mL), methanol (8 mL) and toluene (8 mL) were used as eluants, respectively at a flowrate of about 1 drop min−1 , after which three eluates were obtained, which then were evaporated in a nitrogen stream to remove the solvents. Then groups 1, 2, and 3 were obtained, respectively. For the Sil SPE column, about 8 mL of toluene was used to solvate the column. About 0.05 mL of 50% SA(I) toluene solution was loaded onto the column at a flow-rate of 1 drop min−1 . Toluene (8 mL), acetone (8 mL), and methanol (8 mL) were used as eluants, respectively at a flow-rate of about 1 drop min−1 . As same, groups 4, 5, and 6 were obtained, respectively. All the above flow-rate control processes were performed with pressure applied to the column inlet. 2.2.2. TLC chromatographic procedures A 2.0-␮L aliquot of 10% group 1 methanol solution was applied onto the RP-18 TLC plate with a 10-␮L syringe. The sheet was then developed with a mixture of methanol–water (7:3) as the mobile phase to a distance of 60 mm. The developed spots were marked under ultraviolet light. For group 1, RF = 0.9 (spot 1) and RF = 0.3 (spot 2) were obtained (Table 1). A 2.0 ␮L of 10% group 2 methanol solution was applied onto the RP-18 TLC plate and then developed with methanol as the mobile phase. The developed spots were marked under UV light. For group 2, RF = 0.5 (spot 3) and RF = 0 (spot 4) were got (Table 1). A 2.0 ␮L of 10% group 2 methanol so-

Table 1 TLC results of groups 1–6 under different conditions Development solvents

Groupsa Group 1

RP-18 TLC sheets Methanol–water (7:3)

Group 2

Group 3

0.9 (spot 1) 0.3 (spot 2)

Methanol

0.5 (spot 3) 0.0 (spot 4)

Methanol–toluene (3:7)

0.8 (spot 5) 0.3 (spot 6)

0.3 (spot 7)

Group 4

Group 5

Group 6

0.9 (spot 8)

0.9 (spot 9) 0.3 (spot 10) 0.0 (spot 11)

Silica gel TLC sheets Toluene

Toluene–acetone (1:1) Acetate–acetone–water (5.5:3.5:1) a

RF values for different groups.

0.9 (spot 12) 0.0 (spot 13)

0.5 with long tail (spot 14) 0.3; 0.4; 0.5; 0.6; 0.7; 0.8; 0.9

W. He et al. / Spectrochimica Acta Part A 61 (2005) 1965–1970

lution and a 2.0 ␮L of 10% group 3 methanol solution were applied onto the RP-18 plate. The sheet was then developed with a mixture of methanol–toluene (3:7) as the mobile phase. The developed spots were marked under ultraviolet light. For group 2, RF = 0.8 (spot 5) and RF = 0.3 (spot 6) were obtained for group 3, RF = 0.3 (spot 7) was obtained (Table 1). Before analysis, silica gel 60 F254 TLC plates were cleaned by developing in methanol. The plates were dried in air, heated for 20 min at 110 ◦ C then stored in desiccators until use. A 2.0 ␮L of 10% group 4 and 10% group 5 toluene solution were applied onto the plate with 10-␮L syringes. The sheet was then developed with toluene as the mobile phase to a distance of 60 mm. The developed spots were marked after being visualised by iodine vapour. For group 4 RF = 0.9 (spot 8) was obtained, and for group 5 RF = 0.9 (spot 9), RF = 0.3 (spot 10) and RF = 0 (spot 11) were obtained (Table 1). A 2.0 ␮L of 10% group 5 and 10% group 6 methanol solution were applied onto the plate. The sheet was then developed with a mixture of toluene–acetone (1:1) as the mobile phase. The developed spot was marked after being visualised by iodine vapour. For group 5, RF = 0.9 (spot 12) and RF = 0(spot 13) were obtained, and for group 6, a spot (spot 14) with long tail was received (Table 1). According to [6], a 2.0 ␮L of 10% group 6 methanol solution then was developed using a mixture of acetate–acetone–water (5.5:3.5:1) as mobile phase. The result was shown in Table 1 using iodine vapour as visualized agent. 2.2.3. Transfer of analytes According to [3], with the exception that the capillary was not packed with facial tissue, and a white porcelain plate with

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12 pits was used to instead 5-mL beaker. Then methanol was used as an eluent for spots 1, 2, 10 and 12; toluene for spots 3, 5, 6, 7, 8, and 9. A 3-␮L glass capillary micropipette (d = 0.5 mm), made by Drummond Scientific Co., U.S.A., was used to draw about 0.1 ␮L of solution from the capillary. Then, the 0.1 ␮L solution was dropped onto EZ-Spot Micro Mount Sample SlidesEZ-SpotTM purchased from Thermo Nicolet. Following this method, all of the other separated components were transferred onto the slide. The slide was placed inside a fume cupboard for evaporation of the solvent. A stream of nitrogen was used to remove any remaining trace of solvent. 2.2.4. FTIR microscopy measurement A Nicolet Nexus 470 FTIR spectrometer, equipped with Spectra-Tech CentaurusTM Microscope, was used to identify the separated components. Ominic Spectrum Search Plus software was used for spectrum processing. All FTIR spectra were obtained under following conditions: mode reflection, aperture 100 ␮m × 100 ␮m, scan times 64, resolution 8 cm−1 .

3. Results and discussion Fig. 1 indicates that spots 1 and 2 were polyoxyethylene and polyoxyethylene-fatty acid ester, respectively, which means group 1 mainly consisted of polyoxyethylene and polyoxyethylene-fatty acid ester. Fig. 2 showed that spots 3 and 5 were sulfonate plant oil, and spots 6 and 7 were plant oil. So, group 2 mainly consisted of plant oil and sul-

Fig. 1. FTIR spectra of spots 1 and 2.

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W. He et al. / Spectrochimica Acta Part A 61 (2005) 1965–1970

Fig. 2. FTIR spectra of spots 3–7.

fonate plant oil and group 3 of plant oil. Fig. 3 indicated that spots 8 and 9 were modified silicone, and spot 10 was plant oil. Fig. 4 showed that spot 12 was plant oil plus modified silicone. So, group 4 was made of modified silicone, and group 5 mainly consisted of modified silicone, plant oil and an unknown compound (spots 11 and 13), which should

be sulfonate plant oil according the above analytical result for group 2. Table 1 and Fig. 1 deduced that group 6 mainly consisted of polyoxyethylene and polyoxyethylene-fatty acid ester. All these analytical results showed that the release agent mainly consisted of modified silicone, plant oil, polyoxyethylene, polyoxyethylene-fatty acid ester and sulfonate

Fig. 3. FTIR spectra of spots 8–10.

W. He et al. / Spectrochimica Acta Part A 61 (2005) 1965–1970

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Fig. 4. FTIR spectrum of spot 12.

plant oil besides water. When only TLC was used to separate the components in SA (I), the minor components such as sulfonate plant oil and polyoxyethylene-fatty acid ester would not be able to be detected, because it is impossible to choose a suitable amount of sample both for major and minor components. For example, when the amount of sample is suitable for the main components, such as modified silicone, the amount of minor components will be insufficient to visualize. However, when the amount of sample is suitable for minor components, some minor components will also be obscured by the main component tailing. In addition, it is difficult to choose right TLC for all components. In this case, it was difficult to separate sulfonate plant oil or elute polyoxyethylene when using only Sil–TLC sheet, similarly, it was difficult to separate and elute modified silicone when using only RP-18 TLC sheet. In this analysis, with the application of SPE, different category components were grouped in advance according to their polarity and solubility, which resulted in a relative concentration of minor components and at same time made it easy to choose of the most suitable TLC sheets. In [3], a small piece of facial tissue was used as filter, but sometimes the facial tissue acted like a cellulose column effecting elution. This time the tissue was eliminated. By careful transfer, a discriminating image could be obtained, as shown in Fig. 5. In Fig. 5, a liquid-like analyte (marks 3 and 4) was easy to distinguish from solid-like silica (marks 1 and 2). Sometimes, the analyte was a powder-like solid and potassium bromide could be used as filter instead of tissue. Transmittance mode was chosen in FTIR microscopy by other authors [3]. Although with transmittance mode, a

more intense spectrum could be obtained, the spectral region with barium fluoride was limited to 4000–800 cm−1 . With the current method, reflectance mode was chosen, and a wider spectrum region (4000–650 cm−1 ) could be used. What is more with the EZ-Spot Micro Mount Sample Slides EZ-SpotTM (Fig. 6), FTIR test was more con-

Fig. 5. A microscopic image of spot 1 on an EZ-Spot micro mount sample Slides EZ-SpotTM .

Fig. 6. An illustration of EZ-Spot micro mount sample Slides EZ-SpotTM .

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W. He et al. / Spectrochimica Acta Part A 61 (2005) 1965–1970

venient especially in the case of samples with multiple components.

Acknowledgments We wish to thank Fujian Financial Department for the financial support.

4. Conclusions The results demonstrate that using SPE instead of traditional column chromatography to perform a preliminary grouping of the components of multicomponent samples before the TLC process makes it much easier to choose of the optimum TLC sheet and to determine a suitable loading. With more perfect TLC spots transferring, more consistent FTIR microscopy testing conditions, the analytical method of TLC coupled with FTIR microscopy for separation and identification of multicomponent mixtures has proven rapid and discriminant.

References [1] J.W. Gooch, Analysis and Deformulation of Polymeric Materials, Plenum Press, New York, 1997, chapter 1, p. 1. [2] T. Tajima, K. Wada, K. Ichimura, Vib. Spectro. 3 (1992) 211. [3] W. He, R. Shanks, G. Amarasinghe, Vib. Spectro. 30 (2002) 148. [4] J. Sherma, B. Fried, Handbook of Thin-Layer Chromatography, Marcel Dekker, New York, 1991, chapter 6, p. 135. [5] H. Zhang, P. Zhu, Chin. J. Anal. Chem. 28 (2000) 1172–1180. [6] Y. Gao, The Application of Chromatography in the Field of Refine Chemical Industry, China Petrochemistry Press, Beijing, 1997, chapter 3, p. 177.