This article was downloaded by: [Duke University Libraries] On: 17 October 2012, At: 06:26 Publisher: Taylor & Francis Informa Ltd Registered in England and Wales Registered Number: 1072954 Registered office: Mortimer House, 37-41 Mortimer Street, London W1T 3JH, UK Journal of Liquid Chromatography & Related Technologies Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/ljlc20 A COMPARATIVE STUDY CONCERNING THE IMAGE ANALYSIS IN THIN LAYER CHROMATOGRAPHY OF FLUORESCENT COMPOUNDS Lucian Alexandru Fazakaş a , Rodica Domnica Naşcu-Briciu a & Costel Sârbu a a Faculty of Chemistry and Chemical Engineering, Babeş-Bolyai University, Cluj-Napoca, Romania Version of record first published: 18 Nov 2011. To cite this article: Lucian Alexandru Fazakaş, Rodica Domnica Naşcu-Briciu & Costel Sârbu (2011): A COMPARATIVE STUDY CONCERNING THE IMAGE ANALYSIS IN THIN LAYER CHROMATOGRAPHY OF FLUORESCENT COMPOUNDS, Journal of Liquid Chromatography & Related Technologies, 34:19, 2315-2325 To link to this article: http://dx.doi.org/10.1080/10826076.2011.587226 PLEASE SCROLL DOWN FOR ARTICLE Full terms and conditions of use: http://www.tandfonline.com/page/terms-and-conditions This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. The publisher does not give any warranty express or implied or make any representation that the contents will be complete or accurate or up to date. The accuracy of any instructions, formulae, and drug doses should be independently verified with primary sources. The publisher shall not be liable for any loss, actions, claims, proceedings, demand, or costs or damages whatsoever or howsoever caused arising directly or indirectly in connection with or arising out of the use of this material.
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This article was downloaded by: [Duke University Libraries]On: 17 October 2012, At: 06:26Publisher: Taylor & FrancisInforma Ltd Registered in England and Wales Registered Number: 1072954 Registeredoffice: Mortimer House, 37-41 Mortimer Street, London W1T 3JH, UK
Journal of Liquid Chromatography &Related TechnologiesPublication details, including instructions for authors andsubscription information:http://www.tandfonline.com/loi/ljlc20
A COMPARATIVE STUDY CONCERNINGTHE IMAGE ANALYSIS IN THIN LAYERCHROMATOGRAPHY OF FLUORESCENTCOMPOUNDSLucian Alexandru Fazakaş a , Rodica Domnica Naşcu-Briciu a & Costel
Sârbu aa Faculty of Chemistry and Chemical Engineering, Babeş-BolyaiUniversity, Cluj-Napoca, Romania
Version of record first published: 18 Nov 2011.
To cite this article: Lucian Alexandru Fazakaş, Rodica Domnica Naşcu-Briciu & Costel Sârbu (2011):A COMPARATIVE STUDY CONCERNING THE IMAGE ANALYSIS IN THIN LAYER CHROMATOGRAPHY OFFLUORESCENT COMPOUNDS, Journal of Liquid Chromatography & Related Technologies, 34:19,2315-2325
To link to this article: http://dx.doi.org/10.1080/10826076.2011.587226
PLEASE SCROLL DOWN FOR ARTICLE
Full terms and conditions of use: http://www.tandfonline.com/page/terms-and-conditions
This article may be used for research, teaching, and private study purposes. Anysubstantial or systematic reproduction, redistribution, reselling, loan, sub-licensing,systematic supply, or distribution in any form to anyone is expressly forbidden.
The publisher does not give any warranty express or implied or make any representationthat the contents will be complete or accurate or up to date. The accuracy of anyinstructions, formulae, and drug doses should be independently verified with primarysources. The publisher shall not be liable for any loss, actions, claims, proceedings,demand, or costs or damages whatsoever or howsoever caused arising directly orindirectly in connection with or arising out of the use of this material.
A COMPARATIVE STUDY CONCERNING THE IMAGE ANALYSISIN THIN LAYER CHROMATOGRAPHY OF FLUORESCENTCOMPOUNDS
Lucian Alexandru Fazakas, Rodica Domnica Nascu-Briciu,and Costel Sarbu
Faculty of Chemistry and Chemical Engineering, Babes-Bolyai University,Cluj-Napoca, Romania
& The use of image analysis software for the detection and quantitative evaluation of fluorescentcompounds in thin layer chromatography is investigated. Four different image analysis softwarepackages (Sorbfil TLC, Biodit, JustTLC, and Melanie) were applied and compared. The imageanalysis methods were developed and validated for the simultaneous determination of quercetinand kaempferol in food supplements. The chromatographic separation used Silica gel 60 TLC platesas the stationary phase and carbon tetrachloride:acetone:formic acid (35:11:3, v=v=v) as the mobilephase. The plate images were recorded by a digital camera and imported in the image analysissoftware. The final results were validated by evaluating the linearity, precision, and accuracy ofthe methods used to quantify synthetic and real samples. All the results suggest that the imageanalysis software packages are proper to investigate the fluorescent compounds in quantitative thinlayer chromatography.
Thin layer chromatography (TLC) has its origins at the beginning of thetwentieth century and it was widely used after the 1950s when the TLCprocedures were improved and standardized.[1–3] The widespread use ofthe TLC was mainly caused by some advantages offered by this chromato-graphic method. TLC was a rapid, simple, and inexpensive method providingthe possibility of processing multiple samples in parallel with a high through-put.[4,5] In spite of all these advantages offered by the TLC, the method had a
Address correspondence to Assoc. Prof. Costel Sarbu, Department of Analytical Chemistry andChemical Engineering, Babes-Bolyai University, Arany Janos No 11, RO 400028, Cluj-Napoca, Romania.E-mail: [email protected]
Journal of Liquid Chromatography & Related Technologies, 34:2315–2325, 2011Copyright # Taylor & Francis Group, LLCISSN: 1082-6076 print/1520-572X onlineDOI: 10.1080/10826076.2011.587226
Journal of Liquid Chromatography & Related Technologies, 34:2315–2325, 2011Copyright # Taylor & Francis Group, LLCISSN: 1082-6076 print/1520-572X onlineDOI: 10.1080/10826076.2011.587226
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reduced sensitivity and limited delectability and, therefore, it was only used inqualitative analysis. However, TLC became a quantitative method when it wascoupled with slit scanning densitometers and later with charged-coupleddevices (CCD) which considerably improved the accuracy of the method.Recent studies described the use of image analysis methods for quantitativelyevaluating the TLC chromatograms. In these studies, the image analysis meth-ods proved to be very accessible, simple, and able to simultaneously evaluateall the samples analyzed with the TLC.[6,7] The new image analysis methodsproved also to be faster, less expensive, and often provided a higher resolutionand sensitivity when they were compared to the scanning slit densitometryanalysis.[8,9] Some authors have recently reported the successful use of imageanalysis for both the TLC qualitative and quantitative evaluation of the UV-Visabsorbing compounds.[10–16] The successful results of using the image analy-sis methods for TLC quantitative evaluation and the practical advantages ofthese methods represented important factors that imposed them as standardprocedures in different important forensic laboratories like those of the Inter-pol.[17,18] On the other hand, currently there is no reference in the literaturedescribing the use of the of the image analysis for the TLC quantitative evalu-ation of fluorescent compounds. It is the aim of the present work to fill thisgap by presenting the latest results of fluorescent compounds detection byimage analysis methods.
Flavonoids are polyphenolic compounds with active phytoalexins(anti-microbian) properties and they are synthesized in the plants as mainpigments. Two of the most known and widely spread plant flavonoids arethe quercetin and the kaempferol with their corresponding chemical for-mula presented in Figure 1. Both quercetin and kaempferol have multiplebiological roles such as antioxidant, anti-inflammatory, antiviral, andanti-aging. They also have an inhibitor role on blood platelet aggregationpreventing in this way the cardiovascular diseases.[19,20] The important bio-logical roles of the flavonoids imposed them as one of the main ingredientsin food products and supplements with plant extracts.[21,22]
FIGURE 1 The chemical structure of the investigated flavonoids.
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Flavonoids have a weak natural fluorescence that has to be enhanced whenthey are separated on chromatographic plates. The flavonoids fluorescence isenhanced by spraying the TLC plates after elution with different complexingagents. The most common complexing agent used to increase the flavonoidsfluorescence is the diphenylboric acid 2-amino ethyl ester (DPBA).[23,24] TheDPBA reacts with the flavonoids forming chemical complexes with differentfluorescent colors as a function of the original structure of the flavonoid. Inthis way the DPBA—quercetin complex has an orange fluorescence whereasthe DPBA—has a green fluorescence under the UV light.
This paper presents a critical evaluation of the image analysis methodin the TLC quantitative evaluation of the flavonoids (quercetin and kaemp-ferol) from food supplements. Four different image analysis softwarepackages were used for processing the images of the fluorescent flavonoidsand results were compared and statistically analyzed.
EXPERIMENTAL
Materials
Samples of flavonoids (apigenin, kaempferol, luteolin, quercetin,fisetin, and galangin) were purchased from Sigma (Redox, Bucharest,Romania). The organic solvents (ethanol, ethyl acetate, n-hexane, chloro-form, methanol, carbon tetrachloride, formic acid, and acetone) were ofanalytical grade and they were purchased form S.C. Chemical CompanyS.A. (Iasi, Romania). The distillate water was produced by a MultilabGFL-2008 distillation system (Multilab, Bucharest, Romania). AntioxidantForte (Laboratoarele Remedia, Bucharest, Romania) was the food sup-plement selected to extract the natural flavonoids. It contains extract ofgreen tea (75mg), extract of grape seeds (50mg), extract of marine pinebark (20mg), and other natural ingredients.
Analytical Equipments and Software
The powdered food supplement was suspended in ethanol and soni-cated in an Elmasonic S15H ultrasonic bath (Elma Hans SchmidbauerGmbH & Co, Singen, Germany) for the extraction of the natural flavonoids.Liquid samples were applied in spots on the TLC plates by using a semiauto-matic sample applicator Camag Linomat 5 (Camag, Muttenz, Switzerland).The chromatographic separations were done on TLC Silica gel 60 plates,20� 10 cm, (Merck, Darmstadt, Germany).
The images of the developed TLC plates were recorded using a FujiFilmFinePix A920 digital camera and then they were processed with four different
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image analysis softwares: Sorbfil TLC Vidiodensitometer (Sorbpolymer,Krasnodar, Russia), JustTLC (Sweday, Sweden), Biodit TLC-1200 (BioDitTechnology Co.), and Melanie 7.0 (Geneva Bioinformatics, Switzerland).The quantification and detection limits were evaluated by regression usingthe SMAC package (statistical methods in analytical chemistry).[25] Furtherstatistical analysis of the data was done using STATISTICA 8.0 (Statsoft.Inc).
Sample Preparation
The standard solutions were prepared by dissolving in ethanol the quer-cetin (0.05mgmL�1) and the kaempferol (0.10mgmL�1). In order to buildthe calibration function, successive spots from the stock solution wereapplied onto the plates in the following order 3, 4, 5, 6, 7, and 8 mL. For eachof these calibration levels, two identical spots were applied on the plates. Inaddition to these calibration spots, three more identical parallel spots of0.55 mg of the synthetic sample were applied on the plates. These three spotswere used for evaluating the precision and the accuracy of the method.
The supplier of the Antioxidant Forte food supplement does notprovide any information regarding the chemical composition, that is, theflavonoid content of this product. Therefore, a careful analysis was donefirst in order to identify the flavonoids in Antioxidant Forte. The foodsupplement tablet was first weighed and then finely ground with a pestlein a mortar. The product powder was then suspended in a 25-mL volu-metric flask filled up with ethanol and the solution was sonicated in anultrasonic bath for 15min at 20�C. Following the ultrasonic bath procedurethe solution was filtrated through a paper filter for removing the solidcompounds of the suspension. Then, the filtrate was directly applied onthe chromatographic plate in spots of 5 and 25mL.
Five different mobile phases were tested for the separation of flavonoids(quercetin and kaempferol) standards and of the food supplement extracton the silica gel stationary phase. These five mobile phases (MP1–MP5) hadthe following compositions:
Finally the mobile phase that provided the best separation results forselected flavonoids was selected for the TLC quantitative evaluation. Thespots were applied on the chromatographic plates on a 1.5 cm distancefrom the bottom and the margin of the plates, starting from left to right
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with 1 cm space between two consecutive spots. The elution distance was8 cm. The thin layer chromatographic separation was done in an ascendantchromatographic chamber that was previously saturated with the mobilephase for 15min. The plates were developed at room temperature. Afterthe elution, the dried chromatographic plates were sprayed with the DPBAsolution prepared according to procedure published by Wagner et al.[26]
The DPBA solution increased the fluorescence of the flavonoids, improvingtheir visualization under the UV light at 365 nm.
Obtaining the Chromatographic Images and Storage
The images of the developed chromatographic plates were recordedusing the digital camera FujiFilm FinePix A920 and then they were savedas JPEG files without any compression to avoid the loss in image quality.The JPEG files were later processed by the image analysis software providingthe typical format (inverted color, gray scale, or natural color) for finalanalysis.
RESULTS AND DISCUSSION
Chromatographic Separation
The best separation of kaempferol and quercetin was obtained usingthe mobile phases MP1 and MP3 as it is shown in Figure 2. When analyzingthese two phases’ results, it was clear that although the separation with MP3provided a very good compound identification the tailing effect observedwith MP3 made the quantitative determination difficult. This tailing effectwas not present in the case of MP1 separation which also provided a higherresolution. These two advantages selected the MP1 as the best candidate forthe all other separations done in this work.
Image Analysis Software Evaluation
A digitized colored picture produced by a digital camera or a scanner isusually represented by a set of RGB-space values. This means that in everydigitized image the color intensities are evaluated as additive subtractionof the red (R), green (G), and blue (B) intensities from the color whiteby using the image analysis software.[27] Some image analysis software areable to evaluate the images for each color channel, other such softwarecan analyze only the gray scale which implies the suppression of all othercolors prior the analysis. The image analysis software used in this work eval-uated the area of the eluted spots by comparing the spot color intensity tothe color of the TLC plate background. In this way the image software
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generated a final chromatogram that allowed the quantitative evaluation ofthe TLC separation.
The DPBA-flavonoid complexes were characterized by different fluores-cence colors as a function of the chemical structure of the flavonoid. Asmentioned previously, not every image analysis software is able to processcolored images; therefore, the colored images of the chromatograms wereconverted to grey scale before they were analyzed with the Biodit, JustTLC,and Melanie software. By switching to gray scale analysis, these image analy-sis software packages were able to simultaneously analyze the two flavonoids.The Sorbfil TLC software is not sensitive to the color but for a better resol-ution, the colors of the chromatograms were also inverted (Figure 3) beforethey were analyzed with this software.
Every image of the chromatographic plates was evaluated by using allimage analysis options (RGB cannels, normal image, inverted colors image,and gray scale) and, in this way, it became possible to eliminate any inter-ference of impurities of plate imperfections in the final results.
The quantitative evaluation of the chromatographic plates using theproposed image analysis methods is based on the assumption that the colorintensity and the area size of the each individual spot on the plate are afunction of the quantity of that particular compound in the correspondingspot. Because the spot color intensity is evaluated by comparing it to the
FIGURE 2 The chromatographic separation of quercetin (Q) and kaempferol (K) from standardsolution (1) and real sample (2), with MP1 (a) and MP3 (b). (Color figure available online.)
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plate color background, the analysis should include the entire chromato-graphic plate.
The calibration solutions were applied in duplicates on the plates whilethe test solutions and the real samples were applied in triplicates for thecalibration curve generation and the evaluation of linearity. Table 1 sum-marizes the statistical parameters evaluated for the data generated by thefour image software packages. The correlation coefficients of the four soft-ware data indicate a very good linearity in the case of Sorbfil TLC, Biodit,and JustTLC, (r> 0.9952) but a poor linearity for the Melanie softwaredata. The limit of detection (LOD) and the limit of quantification(LOQ) presented in the same Table 1 were calculated using the SMACpackage based on the confidence bands generated from the calibrationresults. The confidence bands were calculated by using the ordinary leastsquares method included in the same SMAC package. The final conclusion
FIGURE 3 The chromatographic separation of quercetin (Q) and kaempferol (K): (a) real image; (b)inverted image; and (c) greyscale image. Spots: 1, calibration curve spots; 2, synthetic sample; 3–5mL ofreal sample; 4–25 mL of real sample. (Color figure available online.)
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based on the analysis of these statistical data is that all image analysissoftware are able to detect the quercetin and kaempferol at a very lowconcentration levels with the JustTLC being the most sensitive of all fourtested software.
The precision of each software was determined by analyzing three ident-ical spots each one of them containing 0.550 mg kaempferol and 0.275mgquercetin. The images of the chromatographic plate with these three spotswere scanned with all four image analysis software and the results in terms ofstandard deviation (SD) and standard error of mean (SEM) are presented inTable 2. The results in Table 2 prove that JustTLC software had the most pre-cise results followed closely in terms of precision by Biodit and Sorbfil TLC.The Melanie software had the lowest precision. In terms of accuracy, it wasBiodit software with the best performance both for kaempferol and for quer-cetine. The JustTLC and Sorbfil provided highly comparable results butwere less accurate than Biodit, and Melanie had the lowest accuracy ofall software. Summarizing all the aforementioned evaluations, it can be
TABLE 1 Regression Statistic Parameters, Limit of Detection, and Limit of Quantification
544.65 (�454.78)Correlation coefficient (r) 0.9977 0.9991F 432 1864Limit of detection (mg=spot) 0.065 0.038Limit of quantification (mg=spot) 0.113 0.074
JustTLCRegression equation y¼ 2869.77 (�73.65)xþ
34.66 (�21.03)y¼ 48.38 (�2.57)xþ
0.31(�1.33)Correlation coefficient (r) 0.9990 0.9958F 1518 355Limit of detection (mg=spot) 0.023 0.088Limit of quantification (mg=spot) 0.045 0.161
MelanieRegression equation y¼ 1065.50 (�144.90)xþ
57.84 (�40.66)y¼ 168.00 (�20.68)xþ
59.40 (�10.75)Correlation coefficient (r) 0.9820 0.9780F 54 66Limit of detection (mg=spot) 0.164 0.203Limit of quantification (mg=spot) 0.237 0.332
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concluded that Biodit software is the best proposal for an efficient quantitat-ive analysis of kaempferol and quercetin by TLC coupled with image analysisdetection.
Quantitative Determination
In the previous section it was concluded that Biodit software was thebest option in the quantitative evaluation of TLC separation, based onthe linearity, precision, and accuracy of the software results. The quantitat-ive evaluation of the quercetin and kaempferol from the Antioxidant Forte(real sample) was done using all four image analysis software but becauseBiotin had the best performance, all other results will be critically com-pared to this software. The final results of the TLC image software analysisfor the flavonoids extracted from Antioxidant Forte are presented inTable 3. The SD values included in Table 3 indicated again that Biotionhad a high precision for the real samples evaluations similar to syntheticsamples evaluations. Quantitatively, all software estimated similar valuesin the case of quercetin, and it can be concluded based on these results thatthe quercetin amount in Antioxidant Forte is approximately 1.45mg=tablet. The quantitative evaluation of kaempferol in Antioxidant Fortewas different for each of the four software. Biodit provided a lower valuefor kaempferol than JustTLC and Sorbfil TLC, which generated similarvalues but with a low accuracy. Melanie software generated quantitativevalues with a low level of significance. Analyzing these quantitativeevaluation results and taking into account the validation results of the fourinvestigated image software brought us to the conclusion that Biodit is thesoftware that indicated the real amounts of quercetin and kaempferol inAntioxidant Forte.
TABLE 2 Precision and the Accuracy Results of the Four Investigated Methods (N¼ 3)
PrecisionAccuracy
Compound Software Mean (mg) SD� SEM�� Recovery (%)
�SD, standard deviation.��SEM, standard error of mean.
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CONCLUSIONS
The thin layer chromatography coupled with image analysis detectionwas evaluated for the quantitative determination of some induced fluor-escent flavonoids. The proposed method described here was validatedand found to be precise, sensitive, and accurate for the quantitative evalu-ation of quercetin and kaempferol in food supplements. All four imageanalysis software investigated in this work were able to detect and evaluatequantitatively the two flavonoids but only one software, the Biodit providedlinear, precise, and accurate values for both synthetic and real samples.
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TABLE 3 Quantitative Determination of Quercetin and Kaempferol in Antioxidant Forte Using FourImage Analysis Software for the TLC Results Evaluation
Amount of Compound (mg=tablet)
Software Sample No� Quercetin Kaempferol
Biodit 1 1.483 1.5112 1.375 1.4603 1.443 1.421
Mean Value (�SD) 1.434 (�0.054) 1.464 (�0.045)Sorbfil TLC 1 1.493 1.888
2 1.272 1.8063 1.382 1.847
Mean Value (�SD) 1.383 (�0.110) 1.847 (�0.041)JustTLC 1 1.538 1.752
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Mean Value (�SD) 1.402 (�0.172) 1.758 (�0.092)Melanie 1 1.334 1.115
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