Cheap and relatively easy tool for quantitative determination by TLC.
Digitally Enhanced Thin-Layer Chromatography: An Inexpensive, New Technique for Qualitative and Quantitative Analysis
Amber Victoria Irish Hess
Massachusetts Institute of Technology, Cambridge, MA 02139-4310
J. Chem. Educ., 2007, 84 (5), p 842
DOI: 10.1021/ed084p842
Publication Date (Web): May 1, 2007
The objective: Thin-layer chromatography (TLC) is a widely used method for qualitative analysis to determine the number of components in a mixture, to
determine the identity of two substances, or to monitor the progress of a reaction.
The more accurate high-performance TLC (HPTLC) is better suited for quantitative analysis. Unfortunately, HPTLC requires expensive equipment which
most high schools and colleges cannot afford.
I demonstrate that if digital photography is combined with regular TLC, it can perform highly improved qualitative analysis as well as make accurate
quantitative analysis possible.
Methods/Materials
This novel, "digitally-enhanced" TLC (DE TLC) is very easy to use. A fluorescent TLC plate is illuminated with UV light and a picture of the plate is
taken with a digital camera. Then, on a computer, using either TLC Analyzer, the public domain software I wrote, or common photo-editing software, one
can quickly produce multi-spectral scans, densitograms, and calibration curves--output previously available only from more expensive equipment or
complex procedures
Results
With high linearity (R^2 ~ 0.97 - 0.99), good repeatability RSD < 5%), and detection limits approaching those of HPTLC, DE TLC produces
surprisingly good results for such inexpensive equipment..
Conclusions/Discussion
Digitally-Enhanced TLC is a valuable tool that can be added to every chemist's TLC toolbox. Since this technique is much less expensive than other
quantitative chromatographic methods, DE TLC is ideal for high school and college labs.
This project developed an inexpensive technique using digital photography that is an alternative to a $30,000 piece of equipment for chemical
analysis.
Digitally enhanced thin layer chromatography: further development and some applications in isotopic chemistry.
Improvements to thin layer chromatography (TLC) analysis can be made easily and cheaply by the application of digital colour photography and image
analysis. The combined technique, digitally enhanced TLC (DE-TLC), is applicable to the accurate quantification of analytes in mixtures, to reaction
monitoring and to other typical uses of TLC. Examples are given of the application of digitally enhanced TLC to: the deuteromethylations of
theophylline to [methyl-(2)H3]caffeine and of umbelliferone to [(2)H3]7-methoxycoumarin; the selection of tertiary amine bases in
deuterodechlorination reactions; stoichiometry optimisation in the borodeuteride reduction of quinizarin (1,4-dihydroxyanthraquinone) and to the
assessment of xanthophyll yields in Lepidium sativum seedlings grown in deuterated media.
[Edited on 4-12-2016 by Mush]Mush - 11-9-2019 at 15:06
12-2018
Studies in hydrogel microfluidics and developmentof low cost imaging for quantitative TLC in the undergraduate teaching laboratory
Alexandra Marie
Anderson University of Tennessee
....For this undergraduate lab, we chose the approach of digitally-enhanced TLC (DE-TLC), which combines a digital camera with regular TLC equipment
to enable quantitative analysis (10). DE-TLC was introduced in 2007 with an accompanying public domain analysis software, but the softwareis now
obsolete and inoperable on many modern computer systems. In this work, the DE-TLC method was revisited using free public domain software(i.e.NIH
ImageJ) for quantitative TLC analysis using indirect fluorescence detection. To interest students, counterfeiting of the anti-malarial drug
chloroquine was chosen as the proposed application.
An Image Analysis System for Thin-Layer Chromatography Quantification and Its Validation
Tang Tie-xin and Wu Hong*
Abstract
Quantitation of thin-layer chromatography (TLC) using image analysis is attractive for its low cost and convenience. The image analysis is
investigated by designing a digital imaging system with simple equipment, developing an image analysis software based on our algorithm, and validated
the system in the TLC quantitative assay of cichoric acid present in Echinacea purpurea (L.) Moench. TLC used a polyamide thin-layer plate with
chloroform-methanol formic acid-water (3:6:1:1) as the mobile phase and 3% (m/v) aqueous aluminum chloride solution as the visualization reagent.
Images are acquired with a standard digital camera under a UV viewing lamp (365 nm) in a dark room. The three-dimensional grayscale digital image
dataset (x, y, gray) is reduced to twodimensional dataset (distance, accumulative gray) and then plotted as a curve. The area under the peak
corresponding to the cichoric acid spot is integrated and used for quantitation. The whole method was validated by the assay tests of detection limit,
calibration curve, repeatability, reproducibility, and recovery. The results showed that our digital imaging method and image analysis algorithm were
applicable for the quantification of TLC. The whole method is convenient, efficient, and moderately accurate for the quantitative assay of cichoric
acid present in Echinacea purpurea (L.) Moench.
Attachment: tie-xin2008.pdf (341kB) This file has been downloaded 594 times
Software for Image Analysis
Profiles of five programs for quantifying data from Westerns, dot blots, gels, and colony cultures
Abstract
Thin-layer chromatography (TLC) is one of the basic analytical procedures in chemistry and allows the demonstration of various chemical principles in
an educational setting. An often-overlooked aspect of TLC is the capability to quantify isolated target compounds in an unknown sample. Here, we
present a suitable route to implement quantitative analysis in a lesson plan. We provide both a stand-alone software and an online webapp that allow
students to obtain quantitative information from a developed TLC plate and present two suitable experiments, namely, the absorbance-based
quantification of the colorant Sudan IV and the fluorescence-based quantification of rhodamine 6G, a fluorophore widely used in biotechnology.
Students conduct TLC experiments following established protocols, take pictures of their TLC plates with mobile phones, and subsequently quantify the
different compounds in the separate bands they observe.
The Supporting Information is available on the ACS Publications website at DOI: 10.1021/acs.jchemed.8b00144.
Supporting Information with qTLC software and webapp tutorials; details on MinPeakProm, Divisor, the signal-integration process, and background
subtraction; image-acquisition guidelines; choice of concentration range; and overview of experimental errors (PDF)
Student handout with instructions on experimental TLC (PDF)
Student handout with instructions on image analysis and data acquisition with the qTLC software (PDF)
Student handout with instructions on image analysis and data acquisition with the qTLC webapp (PDF)
All MATLAB components for customization and further development of the qTLC software (ZIP)
Links for installation of the qTLC software on MacOS with separate MATLAB Runtime installation, standalone installation of the qTLC software on MacOS,
installation of the qTLC software on Windows with separate MATLAB Runtime installation, and standalone installation of the qTLC software on Windows
(PDF)
Video
Code:
https://www.youtube.com/watch?v=rSNVhVJRvws
qTLC A webapp to quantify chemical compounds via thin layer chromatography.
Application of smartphone in detection of thin-layer chromatography: Case of salvia miltiorrhiza
Journal of Chromatography A 1637 (2021) 461826
Abstract
In this work, a smartphone-based device was constructed for thin-layer chromatography (TLC) detection and semi-quantitative analysis of the components
of Salvia miltiorrhiza . The key construction and shoot- ing parameters were investigated by the relative peak area and signal-to-noise ratio. The
best conditions were as follows: shooting height, 17 cm; angle between the UV lamp and TLC plate, 58 °; exposure com- pensation, 0~0.2 EV; and
shutter speed under daylight and UV 365 nm, 1/50 s and 1/5 s, respectively. These ideal conditions could be replicated by smartphones from different
brands with different versions of software. With good precision, repeatability and stability, the developed device was used for the semi- quantitative
analysis of salvianolic acid B, rosmarinic acid, cryptotanshinone, tanshinone I, tanshinone IIA, and miltirone in the TLC analysis of 10 batches of S.
miltiorrhiza . The results were compared with those obtained by a TLC densitometric scanner and two common types of image processing software, i.e.,
Gel- analyzer and ImageJ . Except for salvianolic acid B in the TLC densitometric scanner, all results were not significantly different among these
methods, which suggested that smartphones might be a useful tool for the quality control of traditional Chinese medicines.
[Edited on 2-8-2021 by Mush]Mush - 30-7-2022 at 09:59
Digitally enhanced thin layer chromatography for simultaneous determination of norfloxacin and tinidazole with
the aid of Taguchi orthogonal array and desirability function approach: Greenness assessment by analytical Eco-Scale
In this study, an eco-friendly fast simple method was developed for simultaneous determination of norfloxacin and tinidazole based on
thin-layer chromatography and image-processing analysis. The binary mixture was separated using reversed phase - thin layer
chromatography plates and 30% trifluoroacetic acid only as mobile phase. Mobile phase composition was optimized using Taguchi orthogonal array and
Derringer's desirability function. The plates were viewed under UV lamp and photographed by iPhone camera followed by image processing with
Fiji software using integrated density as the measured response. As decreasing illumination increases the sensitivity of the method, this
method was applied on two different ranges for each drug. The first one was 0.6–6.0 and 0.9–9.0 µg/spot for norfloxacin and tinidazole,
respectively measured on the original image with normal illumination. The second one was measured after decreasing the illumination of the captured
images at 0.06–0.60 and 0.09–0.90 µg/spot for norfloxacin and tinidazole, respectively. The proposed method was successfully applied for the
determination of both drugs in tablets dosage form without interference from the commonly encountered excipients. Analytical Eco-Scale was used to
evaluate the greenness profile of the proposed method and it was found to be excellent green analytical method.
Quantitative thin layer chromatography for the determination of medroxyprogesterone acetate using a smartphone
and open-source image analysis
Journal of Chromatography A
Volume 1669, 26 April 2022, 462942
10.1016/j.chroma.2022.462942
Abstract
Intramuscular medroxyprogesterone acetate (MPA) products are commonly used to treat endometriosis and are the most widely used injectable
contraceptives worldwide. Therefore, dependable quality screening of MPA injectables is a crucial measure necessary for ensuring that consumers are
provided with safe and effective medications. Here, a thin-layer chromatography (TLC) method for MPA identification is combined with image
analysis using a smartphone, 3D-printed light box, and open-source ImageJ software. The method's validation included two brands of MPA
injectables, both at 150 mg mL−1 dosage. The TLC procedure used was based on the identity test found in The International Pharmacopoeia's
Medroxyprogesterone injection monograph. Spots produced on the TLC plates were then photographed using a smartphone camera and quantified using
ImageJ's image analysis software. The pixel data collected from each plate's standard spots were compared to the data generated from its sample spots.
Data sets collected across multiple TLC plates and numerous days of method performance were evaluated to assess linearity, accuracy, precision,
specificity, and robustness. Across the range of 75–125% of the target concentration, the method was found to have linearity of standard spots (with
R2 generally greater than 0.99), overall accuracy of 101.0% (4.1% RSD), repeatability pooled standard deviation of 2.44%, intermediate precision
pooled standard deviation of 3.68%, and observed demonstration of specificity and robustness. In low and middle-income countries (LMICs), quality
screening of pharmaceutical products like MPA injectables can be challenging when testing resources are expensive, difficult to procure, or complex to
utilize. The results of the TLC/ImageJ method validation suggest that this simple procedure that requires minimal resources may serve as a viable
option for reliable quality screening of MPA levels in injectable suspensions.
Attachment: j.chroma.2022.462942.pdf (1.3MB) This file has been downloaded 301 timesMush - 30-7-2022 at 10:18
Next-Generation TLC: A Quantitative Platform for Parallel Spotting and Imaging
J Org Chem. 2020 Aug 7;85(15):9447-9453.
doi: 10.1021/acs.joc.0c00349
Alexander A Boulgakov 1 , Sarah R Moor 2 , Hyun Hwa Jo 2 , Pedro Metola 2 , Leo A Joyce 3 , Edward M Marcotte 1 , Christopher J Welch 3 , Eric
V Anslyn 1
Abstract
A high-throughput screening approach for simultaneous analysis and quantification of the percent conversion of up to 48 reactions has been developed
using a thin-layer chromatography (TLC) imaging method. As a test-bed reaction, we monitored 48 thiol conjugate additions to a Meldrum's acid
derivative (1) in parallel using TLC. The TLC elutions were imaged using a cell phone and a LEGO brick-constructed UV/vis light box. Further, a
spotting device was constructed from LEGO bricks that allows simple transfer of the samples from a well-plate to the TLC plate. Using software that
was developed to detect "blobs" and report their intensity, we were able to quantitatively determine the extent of completion of the 48 reactions with
one analysis.
Smartphone-based thin layer chromatography for the discrimination of falsified medicines
10.1109/ICSENS.2016.7808847
Abstract:
Identification of counterfeit and substandard drugs, which pose severe risks to patient safety is increasingly important, as inauthentic drugs become
more commonplace in developing parts of the world. Though thin layer chromatography (TLC) performed with laboratory-based instruments enables accurate
analysis of suspect medicines, there is tremendous interest in development of an inexpensive mobile platform that would broaden the applicability of
TLC to remote pharmacies and clinics that presently do not have access to laboratory analysis. In this work, we demonstrate identification and
characterization of pharmaceutical products via TLC using a custom cradle that interfaces with a smartphone. A UV lamp integrated within the cradle
illuminates a TLC plate loaded with calibration standards and an aliquot of a drug of unknown concentration. Phosphorescence from the plate surface
excited by UV light reveals principal spots. Two independent image processing approaches were developed to enable image processing to be performed
locally with the smartphone processor, or remotely by a server running MatLab routines on uploaded images. Both approaches report the intensity and
travel distance of spots within a TLC plate. The system is able to discern 5% medicine concentration differences and to deliver analytical results
that are identical to those obtained by a laboratory TLC densitometer.
TLC-smartphone in antibiotics determination and low-quality pharmaceuticals detection
10.1039/D1RA01346G
Free article on RSC
Abstract
Thin layer chromatography (TLC) is a powerful and simple technique for screening and quantifying low quality and counterfeit pharmaceutical products.
The detection methods used to detect and quantify separate analytes in TLC ranges from the densitometric method to mass spectrometric or Raman
spectroscopic methods. This work describes the development and optimization of a simple and sensitive TLC method utilizing a smartphone CCD camera for
verification of both identity and quantity of antibiotics in dosage form, namely ofloxacin and ornidazole. Mixtures of ofloxacin and ornidazole were
chromatographed on a silica gel 60 F254 plate as a stationary phase. The optimized mobile phase is n-butanol : methanol : ammonia
(8 : 1 : 1.5 by volume). Iodine vapor has been used as a “universal stain” to visualize the spots on the TLC plates in order to obtain a
visual image using the smartphone camera and a desk lamp as an illumination source, thus eliminating the need for a UV illumination source. The
recorded images were processed to calculate the Rf values (Rf values for ofloxacin and ornidazole were 0.12 and 0.76, respectively) which provide
identity of the drugs while spot intensity was calculated using a commercially available smartphone app and employed for quantitative analysis of the
antibiotics and “acetaminophen” as an example of a counterfeit substance. The smartphone TLC method yielded a linearity of ofloxacin and
ornidazole in the range of 12.5–62.5 μg/band and 500–1000 μg/band, respectively. The limit of detection was found to be 1.6 μg/spot for
ofloxacin and 97.8 μg/spot for ornidazole. The proposed method was compared with the bench top densitometric method for verification using a Camag
TLC Scanner 3, the spot areas were scanned at 320 nm. The Rf value of ofloxacin and ornidazole was calculated to be 0.12 and 0.76, respectively. The
densitometric method yielded a linearity of ofloxacin and ornidazole in the range of 5–40 μg/band and 5–50 μg/band, respectively. The limit of
detection was found to be 0.8 μg/spot for ofloxacin and 1.1 μg/spot for ornidazole. The proposed method has been successfully applied for the
determination of ofloxacin and ornidazole present in more than one pharmaceutical dosage form and was comparable to the densitometric method.
Smartphone as a Portable Detector for Thin-Layer Chromatographic Determination of Some Gastrointestinal Tract
Drugs
10.1021/acsomega.2c02482
Abstract
Thin-layer chromatography (TLC) is an effective and simple technique for screening, evaluating, and quantifying low-quality and counterfeit
pharmaceutical products. Smartphones have recently been used as accessible, cheap, and portable detectors that can replace more complicated analytical
detectors. In this work, we have developed a simple and sensitive TLC method utilizing a smartphone charged-coupled device (CCD) camera not only to
verify and quantify some gastrointestinal tract drugs, namely, loperamide hydrochloride (LOP) and bisacodyl (BIS), but also to detect acetaminophen
(ACT) as a counterfeit drug. Both drugs (LOP and BIS) were chromatographed separately on a silica gel 60 F254 plate as a stationary phase under
previously reported chromatographic conditions, using ethyl acetate:methanol:ammonium hydroxide (24:3:1, by volume) and ethyl acetate:methanol:glacial
acetic acid (85:10:5, by volume) as developing systems to determine LOP and BIS, respectively. Universal stains, namely, iodine vapors and vanillin,
were used to visualize the spots on the TLC plates to get a visual image using the smartphone camera and a spotlight as an illumination source with no
need for a UV illumination source. The spot intensity was calculated using a commercially available smartphone application for quantitative analysis
of the studied drugs utilizing ″acetaminophen″ as an example of a counterfeit substance. Rf values were calculated using the recorded images and
found to be 0.77, 0.79, and 0.74 for LOP, BIS, and ACT, respectively, providing drug identity. Linear calibration curves using the smartphone–TLC
method were obtained between the luminance and the corresponding concentrations over the ranges of 2.00–10.00 μg/mL and 1.00–10.00 μg/mL with
limits of detection of 0.57 and 0.10 μg/mL for LOP and BIS, respectively. The suggested method was validated according to the International
Conference of Harmonization (ICH) guidelines. The method was then successfully applied for the qualitative and quantitative determination of LOP or
BIS as an example for gastrointestinal tract drugs in pure form and in their pharmaceutical dosage formulations. The proposed method is considered as
a perfect alternative to traditional reported densitometric methods due to its simplicity, easy application, and inexpensiveness. No previously
reported methods utilizing smartphones have been published for the determination of the studied drugs. The developed approach is considered the first
TLC method using smartphones for the determination of some gastrointestinal tract drugs in their pure form and in pharmaceutical formulations.
An open-source smartphone app for the quantitative evaluation of thin-layer chromatographic analyses in
medicine quality screening
10.1038/s41598-022-17527-y
Abstract
Substandard and falsified medicines present a serious threat to public health. Simple, low-cost screening tools are important in the identification of
such products in low- and middle-income countries. In the present study, a smartphone-based imaging software was developed for the quantification of
thin-layer chromatographic (TLC) analyses. A performance evaluation of this tool in the TLC analysis of 14 active pharmaceutical ingredients according
to the procedures of the Global Pharma Health Fund (GPHF) Minilab was carried out, following international guidelines and assessing accuracy,
repeatability, intermediate precision, specificity, linearity, range and robustness of the method. Relative standard deviations of 2.79% and 4.46%
between individual measurements were observed in the assessments of repeatability and intermediate precision, respectively. Small deliberate
variations of the conditions hardly affected the results. A locally producible wooden box was designed which ensures TLC photography under
standardized conditions and shielding from ambient light. Photography and image analysis were carried out with a low-cost Android-based smartphone.
The app allows to share TLC photos and quantification results using messaging apps, e-mail, cable or Bluetooth connections, or to upload them to a
cloud. The app is available free of charge as General Public License (GPL) open-source software, and interested individuals or organizations are
welcome to use and/or to further improve this software.
Development and validation of a simple thin-layer chromatography–smartphone method for plasma paracetamol
quantification
10.1007/s00764-023-00247-y
Abstract
Paracetamol is the most widely used analgesic drug in the world. However, the risk and the severity of poisoning with this medication are often
underestimated. A simple and cost-effective method for screening paracetamol poisoning by using thin-layer chromatography (TLC) and a smartphone has
been developed. Successful liquid‒liquid extraction of the drug from plasma was carried out. Then, samples were spotted on a TLC plate and eluted by
a mobile phase of acetone‒hexane‒ammonia (4:5:0.1, V/V). JustTLC software was used to analyze and quantify paracetamol spots. The results of the
analytical validation showed that our method is linear in the range of 80–180 µg/mL with a correlation coefficient of
R2 > 0.99. The precision, the accuracy, and the robustness of the method were also verified. The limits of detection and
quantification were 8.79 and 26.65 µg/mL, respectively. The comparison with an enzyme immunoassay and high-performance liquid chromatography
(HPLC) method was satisfactory. This methodology opens up new promising perspectives in toxicology laboratories.
Application of TLC-smartphone method for the analysis of carbamazepine in plasma
10.1080/10826076.2023.2284722
Abstract
A new, simple, rapid, low-cost, and robust thin layer chromatography-smartphone method has been developed for the qualitative and quantitative
determination of carbamazepine in human plasma. Liquid–liquid extraction (LLE) with dichloromethane was used for sample purification. The drug of
interest was separated on TLC aluminum coated with silica gel; and a mixture of cyclohexane, chloroform, and acetic acid (4:4:2 v/v/v) was used as the
mobile phase. The sample spot was visualized under a UV chamber at 254 nm then photographed by a smartphone. The intensity of each spot was
quantified by videodensitometry. The method was validated according to the guideline of the ICH Q2 (R1). The results showed good linearity
(R > 0.99), high sensitivity, and reliable robustness. For accuracy and precision, acceptable relative standard deviations (RSD) were reached
(<15%). The limits of detection and quantification were 0.94 µg/mL and 2.8 µg/mL, respectively. The comparison to HPLC method didn’t show a
significative difference (p = .314). The analysis of carbamazepine in samples charged with several interferences showed good recovery (>90%),
except for phenobarbital (recovery = 72%). It was concluded that the proposed TLC-smartphone is a viable method that can be used for the analysis of
carbamazepine in human plasma.
