THIN-LAYER CHROMATOGRAPHY AND IMAGE ANALYSIS FOR QUANTITATION

Authors

  • PANADDA PHATTANAWASIN Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Silpakorn University, Sanamchandra Palace Campus, Nakhon Pathom
  • UTHAI SOTANAPHUN Department of Pharmacognosy, Faculty of Pharmacy, Silpakorn University, Sanamchandra Palace Campus, Nakhon Pathom
  • LAWAN SRIPHONG Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Silpakorn University, Sanamchandra Palace Campus, Nakhon Pathom
  • JANKANA BURANA-OSOT Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Silpakorn University, Sanamchandra Palace Campus, Nakhon Pathom
  • JARIYA AKKARAWARANTHORN Regional Medical Sciences Center 3, Chonburi
  • ARUNYA NANTANAKORN Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Silpakorn University, Sanamchandra Palace Campus, Nakhon Pathom

DOI:

https://doi.org/10.69598/tbps.13.1.79-92

Keywords:

โครมาโทกราฟีแบบชั้นบาง, การวิเคราะห์เชิงภาพ, การวิเคราะห์เชิงปริมาณ, thin-layer chromatography, image analysis, quantitation

Abstract

การใช้วิธีโครมาโทกราฟีแบบชั้นบาง (thin-layer chromatography, TLC) ร่วมกับเทคนิคการวิเคราะห์เชิงภาพได้นำมาประยุกต์ใช้สำหรับการหาปริมาณสารสำคัญโดยเฉพาะในตัวอย่างประเภทสมุนไพรและผลิตภัณฑ์ธรรมชาติ ขั้นตอนโดยรวมของวิธีประกอบด้วย การแยกสารที่ต้องการวิเคราะห์หาปริมาณออกจากสารอื่นและตรวจพบได้อย่างชัดเจนบนแผ่น TLC   การบันทึกและเก็บข้อมูลภาพของแผ่น TLC ด้วยเครื่องสแกนเนอร์หรือกล้องดิจิทัล การวิเคราะห์ภาพ TLC เพื่อหาความสัมพันธ์ระหว่างความเข้มสีกับปริมาณสารแต่ละจุดด้วยโปรแกรมคอมพิวเตอร์ที่เหมาะสม และการตรวจสอบความใช้ได้ของวิธี  ข้อดีหลักของวิธีนี้คือ ความสะดวก ความรวดเร็ว การใช้อุปกรณ์และเครื่องมือที่จัดหาได้ง่ายในราคาไม่สูงมาก  และหากมีการพัฒนาวิธีการอย่างเหมาะสมยังพบว่าผลการวิเคราะห์ที่ได้มีความถูกต้อง แม่นยำ และไม่แตกต่างจากผลการวิเคราะห์ด้วยเครื่องเดนสิโตมิเตอร์หรือเครื่องมือขั้นสูงประเภทอื่น ๆ

The use of thin-layer chromatography (TLC) combined with image analysis is applied for the quantitation of active ingredient especially in herbal samples and natural products. The overall process of the technique includes the separation of the compound to be quantified and clearly detected on a TLC plate, an image record of the TLC plate with a scanner or a digital camera, TLC-image analysis for relative correlations between color intensity and quantity of a compound in each spot using suitable computer programs, and method validation. The advantages of this method are convenience, rapidness and the use of simple and low cost equipment. Also, if properly developed, the analytical results obtained from this method are shown to be accurate, precise and no different from the results determined using a densitometer or other advanced instrument.

References

1. History of planar chromatography. In: Spangenberg B, Poole CF, Weins Ch, editors. Quantitative thin-layer chromatography: A practical survey. Heidelberg: Springer, 2010; p.1-12.

2. Popovic N, Sherma J. Comparative study of the quantification of thin-layer chromatograms of a model dye using three types of commercial densitometers and image analysis with ImageJ. Trends in Chromatog. 2014;9: 21-8.

3. Mustoe SP, McCrossen SD. A comparison between slit densitometry and video densitometry for quantitation in thin layer chromatography. Chromato Suppl. 2001;53: 474-7.

4. Fazaka LA, Nacu-Briciu RD, Sârbu C. A comparative study concerning the image analysis in thin layer chromatography of fluorescent compounds. J Liq Chromatog Rel Tech. 2011;34:2315-25.

5. Ford-Holevinski TS, Agranoff BW, Radin NS. An inexpensive, microcomputer-based, video densitometer for quantitating thin-layer chromatographic spots. Anal Biochem. 1983;132(1):132-6.

6. Prosek M, Medja M, Korsic J, Kaiser RE. Quantitative evaluation with image processing scanner. In: Piemonte G, Tagliaro F, Marigo M, Frigerio A, editors. Developments in analytical methods in pharmaceutical, biomedical, and forensic sciences. New York: Springer Science & Business Media, 1987;p.37-43.

7. Sherma J. Planar chromatography. Anal Chem. 2008;80: 4253-67.

8. CCD and CMOS image sensor [homepage on the Internet]. c2009 [updated 2009 Jun 12; cited 2016 Dec 26]. Available from http://dtv.mcot.net/mcot_one.php? dateone=1244772701

9. Systems of digital camera [homepage on the Internet]. No date [cited 2016 Dec 26]. Available from http://www.the-than.com/Gallery/ZphotoZ/3.html

10. Download [homepage on the Internet]. No date [cited 2016 Dec 26]. Available from https://imagej.nih.gov/ ij/download.html

11. Ferreira T, Rasband W. ImageJ user guide [homepage on the Internet]. c2012 [updated 2012 Oct 2; cited 2016 Dec 26]. Available from https://imagej.nih.gov/ij/docs/ guide/ user-guide.pdf

12. Schneider CA, Rasband SW, Eliceiri KW. NIH Image to ImageJ: 25 years of image analysis. Nat Methods. 2012;9(7):671-5.

13. Troscianko J, Stevens M. Image calibration and analysis toolbox – a free software suite for objectively measuring reflectance, colour and pattern. Methods Ecol Evol. 2015;6:1320-31.

14. ImageJ [homepage on the Internet]. c2016 [updated 2016 Sep 20; cited 2016 Dec 26]. Available from http://imagej.net

15. Schindelin J, Rueden CT, Hiner MC, Eliceire KW. The ImageJ ecosystem: an open platform for biomedical image analysis. Mol Reprod Dev. 2015;82:518–29.

16. Sotanaphun U, Phattanawasin P, Sriphong L. Application of Scion image software to the simultaneous determination of curcuminoids in turmeric (Curcuma longa). Phytochem Anal. 2009;20(1):19-23.

17. Phattanawasin P, Burana-Osot J, Sotanaphun U, Kumsum A. Stability-indicating TLC–image analysis method for determination of andrographolide in bulk drug and Andrographis paniculata formulations. Acta Chromatogr. 2016;28(4):525-40.

18. Peng-ngummuang K, Palanuvej C, Ruangrungsi N. Pharmacognostic specification and coumarin content of Alyxia reinwardtii inner bark. Eng J. 2015;19(3):15-20.

19. Hoeltz M, Welke JE, Noll IB, Dottori HA. Photometric procedure for quantitative analysis of aflatoxin B1 in peanuts by thin-layer chromatography using charge coupled device detector. Quim Nova. 2010;33(1):43-7.

20. Samten, Wetwitayaklung P, Kitcharoen N, Sotanaphun U. TLC image analysis for determination of the piperine content of the traditional medicinal preparations of Bhutan. Acta Chromatogr. 2010;22(2):227-36.

21. Sakunpak A, Suksaeree J, Monton C, Pathompak P. Development and quantitative determination of barakol in Senna Siamea leaf extract by TLC-image analysis method. Int J Pharm Sci. 2014;6(3):267-70.

22. Yukongphan P, Thitikornpong W, Palanuvej C, Ruangrungsi N. The pharmacognostic specification of Ardisia elliptica fruits and their embelin contents by TLC image analysis compared to TLC densitometry. Bull Health Sci Technol. 2013;11(2):21-8.

23. Mongkolrat S, Palanuvej C, Ruangrungsi N. Thin layer chromatography and image analysis of selected liriodenine bearing plants in Thailand. J Health Res. 2013; 27(2): 67-72.

24. Chaowuttikul C, Thitikornpong W, Palanuvej C, Ruangrungsia N. Quantitative determination of usnic acid content in Usnea siamensis by TLC-densitometry and TLC image analysis. Res J Pharm Bio Chem Sci. 2014;5(1):118-25.

25. Sasiwatpaisit N, Thitikornpong T, Palanuvej C, Ruangrungsi N. Dioscorine content in Dioscorea hispida dried tubers in Thailand by TLC-densitometry and TLC image analysis. J Chem Pharm Res. 2014;6(4):803-6.

26. TLC Analyzer [homepage on the Internet]. No date [cited 2016 Dec 26]. Available from http://www. sciencebuddies.org/science-research-papers/tlc_analyzer. shtml

27. Hess AVI. Digitally enhanced thin-layer chromatography: an inexpensive, new technique for qualitative and quantitative analysis. J Chem Educ. 2007;84(5):842-7.

28. Soponar F, Mot¸ AC, Sârbu C. Quantitative evaluation of paracetamol and caffeine from pharmaceutical preparations using image analysis and RP-TLC. Chromatographia 2009;69(1/2):151-5.

29. Sima IA, Casoni D, Sârbu C. High sensitive and selective HPTLC method assisted by digital image processing for simultaneous determination of catecholamines and related drugs. Talanta. 2013;114:117-23.

30. Sorbflil TLC Videodensitometer [homepage on the Internet]. c2016 [updated 2016 Oct 05; cited 2016 Dec 26]. Available from http://sorbflil-tlc-videodensitometer. software.informer.com

31. Sweday. JustTLC [homepage on the Internet]. No date [cited 2016 Dec 26]. Available from http:// www.sweday.com/JustTLCTry.aspx

32. Tozar T, Stoicu A, Radu E, Pascu ML. Evaluation of thin layer chromatography image analysis method for irradiated chropromazine quantification. Rom Rep Phys. 2015;67(4):1608-15.

33. Sima IA, Casoni D, Sârbu C. Simultaneous determination of carbidopa and levodopa using a new TLC method and a free radical as detection agent. J Liquid Chromatogr Rel Tech. 2013;36:2395-404.

34. Phattanawasin P, Sotanaphun U, Sriphong L. Validated TLC-image analysis method for simultaneous quantification of curcuminoids in Curcuma longa. Chromatographia 2009;69(3):397-400.

35. Phattanawasin P, Sotanaphun U, Sriphong L, Kanchanaphibool I, Nussara P. A comparison of image analysis software for quantitative TLC of ceftriaxone sodium. Silpakorn U Sci Technol J. 2011;5(1):7-13.

36. UN-SCAN-IT graph digitalizing demo [homepage on the Internet]. No date [cited 2016 Dec 26]. Available from https://www.silkscientific.com/demo/demograph.htm# Demo-b3

37. Phattanawasin P, Sotanaphun U, Sukwattanasinit T, Akkarawaranthorn J, Kitchaiya S. Quantitative determination of sibutramine in adulterated herbal slimming formulations by TLC-image analysis method. Forensic Sci Int. 2012;219:96-100.

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Published

2018-03-15

How to Cite

PHATTANAWASIN, P., SOTANAPHUN, U., SRIPHONG, L., BURANA-OSOT, J., AKKARAWARANTHORN, J., & NANTANAKORN, A. (2018). THIN-LAYER CHROMATOGRAPHY AND IMAGE ANALYSIS FOR QUANTITATION. Thai Bulletin of Pharmaceutical Sciences, 13(1), 79–92. https://doi.org/10.69598/tbps.13.1.79-92

Issue

Vol. 13 No. 1 (2018): JANUARY - JUNE 2018

Section

Review Articles

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