Abstract
Since the last few decades, the TLC has been an essential tool in the standardization and quality control of herbal medicines. Often, the traditional Thin Layer Chromatographic (t-TLC) profiling is inadequate in separating and identifying the huge numbers of phytochemicals present therein. A simple t-TLC is unable to accommodate or separate such large numbers of spots or bands on a plate. The present investigation has tried to adopt multidimensional and multiple development methods in combination to separate 21 phytochemicals and to generate a decisive chromatographic fingerprint of the sample. The most exciting point is that the qualitative profile generated here is highly reproducible for 19 phytocompounds. The limitations of traditional TLC in separating and identifying were overcome by the present investigation. The output of the present study may be an example of creating herboprint, which is generic and conclusive in the qualitative identification of complex herbal formulations. The most significant output of this work is that the presence or absence of any herb in a polyherbal formulation can easily be detected. It generates a unique herboprint, which can be referred as an authentic chromatographic fingerprint of the complex formulation. The concept adopted here is not new in the domain of TLC, but here maximized improvisation has been imposed in the field of herbs and herbal formulations. Significantly, a concept of coordinate position(s) which has been introduced is more rational than Rf value(s) for qualitative profiling in multidimensional development.
1 Introduction
There is no doubt that traditional Thin Layer Chromatography (t-TLC) has significantly taken the place as an indispensable tool in the field of natural product studies. In the traditional method, the sample is applied on the plate and developed with a suitable mobile phase in a glass chamber [1, 2]. Using one mobile phase for the entire development is called isocratic development. The major limitation of the t-TLC is that the use of a single mobile phase is not enough to separate a complex matrix; hence, the separation of phytocomponents remains partial.
The fact is that in t-TLC, a TLC plate gives a certain number of theoretical plates (N) during development [3]. Theoretical plates provide an equilibration point of solute between the mobile phase and the stationary phase [4]. If theoretical plate numbers increase, the distribution equilibrium of solute between solvent and stationary phase also increases. An Increase in the theoretical plate brings the efficient separation. These numbers of theoretical plates are the key factor in bringing separation on a chromatographic plate [5]. Therefore, any effort for enhancing the number of theoretical plates on a TLC plate can bring a better separation. This can be achieved by multiple developments or gradient development instead of isocratic development [6–8]. Multiple development is that when more than one mobile phases of varying polarity are used for the entire development distance. It suggests that the polarity of the mobile phase changed linearly with an increase in migration distance of the mobile phase and the gradient in multiple unfolding is stepwise. In multiple development the chance of achieving an equilibration point is higher because of the dynamic competition between the mobile and stationary phases to retain the solute, due to gradual changes in the polarity of the mobile phase. Multiple development is possible by changing the polarity of the mobile phase along the development direction.
Another limitation of the t-TLC method is that it usually brings the development of the plate in one direction. Unidirectional development often camouflages us by hiding two or more spots in a single Rf value. Hence, the identification of phytocomponents remains limited in numbers and gives misinformation. This can be overcome by multidimensional development, which is a sure shot modification of one-dimensional development, to reveal the hidden spots by splitting two or more merged spots residing in a single Rf value. After one-dimensional development, the plate is rotated 90° and developed again with usually different mobile phase [9]. Such development is called two-dimensional development.
Hence, it is proposed that both multiple development and multidimensional development can be used in combination to identify complex mixtures of phytocompounds and generate the most authentic and extensive chromatographic profiling [10–13].
The t-TLC methods are usually suitable for the identification of less complex mixtures. But, when the separation for highly complex mixtures comes in question, the combination of multiple development and multidimensional development is a deadly tool to come out with a comprehensive output. Previously, a group of researchers has demonstrated the two dimensional and multiple development methods for zonal separations [14]. Separation of a mixture of pesticides by strategic application of multidimensional chromatography on TLC plate has been carried out by some workers [15]. Separation of a complex mixture of the same class of compounds has also been achieved by some chromatographers [16]. Investigators of the present study have adopted a few technicalities, explained and demonstrated by a group of workers who worked with medicinal plants [17]. The present work demonstrates the combinatorial application of TLC for the analysis of complex mixture of poly herbal formulation, which is probably the first of its kind.
With this above said background, the present study is aiming in application of this combination to maximize the separation and identifications of phytocompounds present in an Indian traditional complex formulation, namely Lekhaniya Gana Kashaya [18]. The significance of the present study is that the separation and identification of phytocompounds from each single plant ingredient ensures the use of each plant in the manufacturing of the formulation. Thus, it generates a unique herboprint, which can be referred as an authentic chromatographic fingerprint of the complex formulation. Significantly, a concept of coordinate position (s) which has been introduced, was found to be more rational than Rf value (s) for qualitative profiling in multidimensional development. The present investigation very neatly and vividly explains each step of these sets of experiments to understand the need and significance of this applications. At the end, the significant outcome and the limitations associated with the work are discussed.
2 Experimental
2.1 Chemical and solvents
The solvents and High Performance Thin Layer Chromatography (HPTLC) plates used in the present investigation were purchased from E Merck, India Pvt. Ptd, Mumbai, India. Acetic acid was procured from the Finar Chemicals Ltd., Gujrat, India. The standards used in the experiments comprised of triterpenoids, sesquiterpenoids, alkaloids, curcuminoids, and flavonoids were purchased from, Fluka Chemicals, Tokyo, Japan; Natural Remedies, Bangalore, India; E Merck, Germany and Sigma-Aldrich, Germany. The standards used were listed here as, α-asarone (Sigma-Aldrich), β-amyrin (Natural Remedies), β-asarone (Sigma-Aldrich), Betulin (Sigma-Aldrich), Betulinic acid (Sigma-Aldrich), Berbamine (E Merck), Berberine (E Merck), Cucurbetaine (Sigma-Aldrich), Curcumin (Natural Remedies), Curcumol (Natural remedies), Cyperusol (Sigma-Aldrich), Lupenone (Natural Remedies), Lupeol (Natural Remedies), Malvidin (Fluka Chemicals), Picroside I (Fluka Chemicals), Picroside II (Fluka Chemicals), Plumbagin (Natural Remedies), Rotundine A (Sigma-Aldrich), Rotundine B (Sigma-Aldrich), Taraxsterol (Fluka Chemicals) and Ursolic acid (Natural Remedies). Here in parenthesis name of the manufacturers and locations are mentioned.
2.2 Test sample
The sample under investigation was a classical Indian traditional formulation Lekhaniya Gana Kashaya (LGK) prepared in the Department of Pharmacy of this Institute by following the traditional method of preparation as mentioned in the ancient text [18]. The list of plant materials used in this formulation for the preparation of LGK and the phytocompounds under investigation of corresponding plants are given in Table 1.
List of plants used in formulations and their corresponding phytocompounds under investigation
| Sl. | Plant name | Sanskrit name | Part used | Phytocompound identified |
| 1. | Aconitum heterophyllum Wall. Ex Royle. | Ativisha | Root | Betulinic acid, Cucurbetaine |
| 2. | Acoruscalamus L. | Vacha | Rhizome | α-asarone, β-asarone |
| 3. | Berberis aristata Roxb. Ex DC. | Daruharidra | Stem bark | Berberine, Berbamine |
| 4. | Curcuma longa L. | Haridra | Rhizome | Curcumin, Curcumol |
| 5. | Cyperus rotundus L. | Musta | Rhizome | Cyperusol, Rotundine A, Rotundine B |
| 6. | Holoptelea integrifolia Planch. | Chirbilwa | Stem Bark | Betulin, |
| 7. | Iris kashmiriana Thunb. | Haimavati | Rhizome | Malvidin |
| 8. | Picrorhiza kurroa Royle ex Benth. | Katukarohini | Root | Picroside I, Picroside II |
| 9. | Plumbago zeylanica L. | Chitraka | Root | Plumbagin |
| 10. | Saussurea lappa (Decne) Sch. Bip. | Kustha | Rhizome | β-amyrin, Lupeol, Lupenone, Taraxsterol, Ursolic acid |
2.3 Preparation of individual standard solution and mixture of standards (SM)
Initially, 21 standards each of 5 mg was individually dissolved in 100 mL methanol. This gave the stock solution of each standard of 50 μg mL−1. In next step, 5 mL of each stock standard solution was taken in a conical flask and mixed properly, this was working standard called as SM. As the final volume was 105 mL (5 mL x 21), the final concentration of each standard in SM became 2.38 μg mL−1. This working standard was labelled as SM and preserved at ∼4 °C for analysis.
2.4 Preparation of test solution (TS)
The test sample under investigation that is Lekhaniya Gana Kashaya (LGK) was taken 10 g in a round bottom flask and extracted with 100 mL methanol by Soxhlet for three hours. The extract was filtered through Whatman filter paper (No. 40), and the filtrate was kept in a stoppered conical by making the final volume 100 mL. This extract was labelled as TS and preserved at ∼4 °C.
2.5 Chromatographic experiment
2.5.1 Chromatographic conditions
The whole chromatographic experiment was divided into four levels. In the first level, standard mixture (SM) and LGK extract (TS) were developed in the t-TLC method. The second level of the experiment was done by two-dimensional single development of TS and SM. The third level of the experiment comprised of one-dimension multi development, and the fourth level that is the final level of the experiment was comprised of a combination of multiple development and multidimensional development. Multiple development was performed by using more than one mobile phase for the entire development throughout the plate by changing the polarity of the mobile phase along the development direction. The changes in migration distance and the polarity of the mobile phase are inversely proportional. It means with increasing migration distance, the polarity of the mobile phase is decreasing. The mobile phases and migration distances of chromatographic developments are summarized in Table 2. A schematic diagram of different developments with respect to the dimensions was outlined in Fig. 1. In the experimental setup each time the developed plate was dried for 30 min [19, 20].
Chromatographic development condition in each level of experiments
| Level of Chromatographic experiment | Dimension of development | Development steps | Mobile phase* (v/v) | Development distance |
| First level | One | Single | H: C: Et: Aa (0.5: 4.5: 6: 0.5) | 90 mm |
| Second level | Two | Single | H: C: Et: Aa (4: 3: 3: 0.5) | 90 mm |
| Third level | One | Multiple | H: C: Et: Aa (1: 1: 8: 0.5) | 30 mm |
| H: C: Et: Aa (1: 3: 6: 0.5) | 45 mm | |||
| H: C: Et: Aa (2: 3: 5: 0.5) | 60 mm | |||
| H: C: Et: Aa (4: 2: 4: 0.5) | 75 mm | |||
| H: C: Et: Aa (6: 2: 2: 0.5) | 90 mm | |||
| Fourth level | Two | Multiple | H: C: Et: Aa (1: 3: 6: 0.5) | 30 mm |
| H: C: Et: Aa (3: 2: 5: 0.5) | 45 mm | |||
| H: C: Et: Aa (4: 2: 4: 0.5) | 60 mm | |||
| H: C: Et: Aa (6: 2: 2: 0.5) | 75 mm | |||
| H: C: Et: Aa (8: 1: 1: 0.5) | 90 mm |
*H = hexane, C = chloroform, E = ethylacetate, Aa = acetic acid.
Schematic representations of (a) Unidimensional single development (first level), (b) Two dimensional single development (second level), (c) Unidimensional multiple development (third level), (d) Two dimensional multiple development (fourth level) TLC experiments
Citation: Acta Chromatographica 2025; 10.1556/1326.2025.01304
For all the chromatographic experimental steps 20 × 20 cm glass supported precoated silica gel 60F254 HPTLC plates of 0.20 mm layer thickness were used. Samples and standard solutions were applied on a plate by ATS-4 (make CAMAG, Switzerland) with the help of 25 µL contact syringe. Plates were developed in 20 × 20 cm twin trough glass chamber. Developed plates were dried for 30 min in air and then by applying hot air blow. The experimental set up was maintained at 22.7 °C with an average relative humidity of 57%. The development tank saturation was maintained for 15 min by placing a filter paper of suitable size. These conditions were maintained strictly for all experiments without any alterations.
2.5.2 Traditional Thin Layer Chromatography (Unidimensional single development): First level
Standard mixture (SM) and test sample (TS) of 2 µL each were applied by CAMAG make automatic TLC sampler ATS-4, as round spot by keeping the distance of 170 mm between the applications and by maintaining a distance of 15 mm from the bottom edge of a glass supported silica gel 60 F254 HPTLC plate of 20 x 10 cm size. The mobile phase plays a crucial role during the HPTLC analysis for the exact measurement of analytes. A solvent system which would give well resolute and compact spots with appropriate and significant resolution was highly desired. In view of this, number of mobile phases were tried, and it was found that hexane: chloroform: ethylacetate: acetic acid (0.5: 4.5: 6: 0.5, v/v) gave the best separation of the bands. Finally, the plate was developed in above mentioned mobile phase, in a twin trough chamber of 20 x 20 cm size with the migration distance of 80 mm from the base. Developed plate was dried for 30 min with hot air blow to remove traces of acetic acid and visualized at long UV (366 nm) and image was captured.
2.5.3 Traditional Thin Layer Chromatography (Two-dimensional single development): Second level
The above plate (as obtained in 2.5.2) was rotated 90° anticlockwise and developed up to 80 mm in a 20 x 20 cm twin trough chamber using the pre optimized mobile phase Hexane: Chloroform: Ethylacetate: Acetic acid (4: 3: 3: 0.5, v/v) for separation of the spots obtained from SM in first position. Plate was then removed from the developing chamber and was dried for 30 min with hot air blow to remove traces of acetic acid. Plate was rotated 90° clockwise with respect to its original development (as in first dimension) and developed up to 80 mm in a 20 x 20 cm twin trough chamber using the same mobile phase for separation of the spots obtained from TS in first dimension. Developed plate was dried for 30 min with hot air blow to remove traces of acetic acid and visualized at long UV (366 nm) and image was captured.
2.5.4 Multiple development Thin Layer Chromatography in one dimension: Third level
The third level of the chromatographic experiment was done in multiple development modes. Here, SM and TS each were applied as 2 µL by CAMAG make automatic TLC sampler ATS-4, as a round spot by keeping a distance of 170 mm between the applications and by maintaining a distance of 15 mm from the bottom edge of a glass supported silica gel 60 F254 HPTLC plate of 20 x 10 cm size. The plate was developed in five steps with increasing migration distance and decreasing the polarity of the mobile phase gradually from the first development to the fifth development as mentioned in Table 2. The changes in mobile phase combination and migration distance were graphically represented in Fig. 2a. According to these figures, the mobile phase composition was dynamic, for example, the ratio of hexane was increasing, and methanol was decreasing as the migration distance increasing gradually. These resulted in gradual fall of resultant polarity of mobile phase with increasing migration distance. After each development the plate was dried for 30 min by hot air blow to remove any traces of acetic acid and proceeded for the next development. Finally, after complete development, the plate was visualized at long UV (366 nm) and image was captured.
Gradient graphic for multi development (a) in first dimension (third level), (b) in second dimension (fourth level)
Citation: Acta Chromatographica 2025; 10.1556/1326.2025.01304
2.5.5 Multiple development and Multidimensional combination in Thin Layer Chromatography: Fourth level
The fourth level of the chromatographic experiment was performed by combining the multiple development and multi-dimensional development together. For this, the previously developed plate (as obtained in 2.5.4) was rotated 90° anticlockwise and again developed in five steps with increasing migration distance and by decreasing the polarity of the mobile phase gradually from first development to fifth development for separation of the spots obtained from SM in first dimension. After each development, the plate was dried for 30 min with a hot air blow for drying and removing any traces of acetic acid used in previous step and proceeded for subsequent development. After completing the five steps of development, the plate was rotated 90° clockwise with respect to its original development (as in first dimension) and developed in five steps with increasing migration distance and decreasing the polarity of the mobile phase gradually from first development to fifth development for separation of the spots obtained from TS in first dimension. The multiple development patterns are mentioned in Table 2. The changes in mobile phase combination and migration distance were graphically represented in Fig. 2b. According to these figures the mobile phase composition was dynamic, for example, the ratio of hexane was increasing and ethyl acetate was decreasing as the migration distance increasing gradually. These resulted in gradual fall of resultant polarity of mobile phase with increasing migration distance. After each development the plate was dried for 30 min with hot air blow for drying and removing any traces of acetic acid used in previous step. Finally, after complete development the plate was visualized at long UV (366 nm), and image was captured.
2.5.6 Checking the reproducibility of the observation in the fourth level
As per the experimental protocol, the complete separation of the standard substances was achieved in fourth level. Thus, it was decided to check the repeatability at this level. Hence, the fourth level of the experiment was repeatedly performed for eleven (11) times in 11 consecutive days without changing any chromatographic conditions.
2.5.7 Identification of Phytocompounds in Test sample (TS)
Identification of individual phytocompounds in TS on chromatographic plate was performed by application of single standard solution and extract solution of TS. Plate was developed by using the mobile phase used for the third and fourth level of the experiments. For the individual identification of each standard, 1 µL solution of standard substance and 5 µL of the test solution (TS) were applied as spot and developed as per the protocol laid down in fourth level and image of the finally developed plate was captured at 366 nm. All the compounds were identified individually. For these identifications, we used 21 plates. Each plate was dedicated for identification of one phytocompound at a time.
3 Results
The experiments produced numbers of images as a result of chromatographic separations. There separated spots were numbered on the plate images. Before going to draw any inference regarding the separation of phytocompounds, it is to be clear that numbers mentioned for the spots in Figs 3–5 were used for the counting purpose. One must not correlate these numbering with the sequence mentioned in section 2.1. In that section the names of phytocompounds were listed alphabetically. The final separation and identification of the phytocompounds were achieved in level 4 represented in Fig. 6 where the numbering of the spots corresponds to a particular phytocompounds.
Visualization at 366 nm after unidimensional single development (First level)
Citation: Acta Chromatographica 2025; 10.1556/1326.2025.01304
Visualization at 366 nm after two-dimensional single development (second level)
Citation: Acta Chromatographica 2025; 10.1556/1326.2025.01304
Visualization at 366 nm after unidimensional multiple development (third level)
Citation: Acta Chromatographica 2025; 10.1556/1326.2025.01304
Visualization at 366 nm after two dimensional multiple development (fourth level)
Citation: Acta Chromatographica 2025; 10.1556/1326.2025.01304
Now let us have a look into the images of the developed plates at different level. In the present study we tried to separate 21 phytocompounds in the LGK extract (TS). We observed 6 spots in Fig. 3 on the plate developed by the traditional TLC (Level 1) method. Hence it could be stated that 6 phytocompounds were separated. But this statement was not true, as one spot was not representing one phytocompound because of incomplete separation. Therefore, the t-TLC was modified by two-dimensional development (Level 2) of this plate to overcome this limitation in separation of t-TLC. The spots which seemed to be single in one dimensional development were split in multiple spots, which proved the incomplete separation in t-TLC method and definitely the two-dimensional development was a better option. For an example, the spot number 6 in Fig. 3 (first level) was appearing as a single spot, but in second level experiment it was split into another 5 spots in Fig. 4. Therefore, two-dimensional development is obviously a better option than t-TLC. According to the experimental set up, the standard mixture was containing 21 different phytocompounds, but this two-dimensional development in second level was able to bring down the separation of 11 spots in Fig. 4. Therefore, execution of two-dimensional development was also not enough to separate such large numbers of phytocompounds on a TLC plate. It was clear that the spots appeared in first dimension were split in second dimension and generated a greater number of spots after. Hence, the spots in first dimension were the parent spots which in later stage split to give more spots. For easy understanding of this fact, let us refer to the all images of the TLC plates. In Fig. 3, there are total 6 spots (as parent spots) which generated by single development split into 11 spots in second dimension development as in Fig. 4, whereas, in Fig. 5, there are 11 spots (parent spots) generated by multiple development in first dimension. Hence, multiple development instead of single development in first dimension was proved to be better option to maximize the separation at beginning level. Therefore, it might be wise to execute multiple development at first dimension in order to get higher number of spots in first dimension which in later stage of development gave separation of maximum phytocompounds. With this logic the multiple development in one dimension (level 3) was done which gave 11 spots in Fig. 5. Seeing the advantages of multiple development in first dimension, we introduced multiple development in second dimension (level 4) also and as a result we got 20 spots in Fig. 6. Therefore, it might be stated that, consideration of the multiple development in first dimension was one of the key points in reaching the higher separation. Similarly, by considering the multiple development also in second dimension we achieved highest separation. Hence, it might be concluded that to culminate the separation reaching up to fourth level was essential. Therefore, improvisation up to fourth level (where multi dimension and multiple development worked together) could be able to achieve the highest separation pattern. In nutshell it may also be stated that only one dimension development or single development alone was unable to separate up to the desired level, rather the combination of multiple development and multidimension was proven to be the best option to get maximum separation.
The present work was able to separate and identified 20 compounds out of 21. On repeated experiments for 21 times, each time a single phytocompound was considered for identification at level 4 by using 21 different plates, it was observed that the all the 21 compounds did not have the repeatable separation pattern. A single plate was dedicated for identification of one compound at a time. Though the present work was able to separate the 20 phytocompounds in TS, but it might be admitted that out of these 20 phytocompounds, 19 spots were highly reproducible. The individual identifications of the substances on chromatographic plate (As in Fig. 6) were marked and numbered as: Betulinic acid (1), Picroside II (2), Picroside I (3), Ursolic acid (4), Cucurbetaine (5), β-asarone (6), α-asarone (7), Rotundine A (8), Rotundine B (9), Berberine (10), Plumbagin (11), Malvidin (12), Curcumol (13), Cyperusol (14), Berbamine (14), Curcumin (16), Betulin (17), Lupeol (18), Lupenone (19), Taraxsterol (20) and β-amyrin (21). Among all these 21 substances, β-amyrin (21) could not be identified in the TS solution. The repeatability in identification of the compound Curcumin (16) was not satisfactory.
4 Discussion
4.1 Conceptualization of coordinate position of the spots on TLC plate
In t-TLC experiments we refer Rf values for the identification of the spot (s) and correlate the spots of standard and test sample. That is possible due to one dimensional development. But that was not possible and not rational, by measuring and mentioning the Rf values in case of multidimensional development. This is because, multi dimension development, eliminated all the chances of having unique movement position resulted as Rf values. After two-dimension development the relevancy of Rf values are completely vanished. So, the question arises that how one can be able to identify a spot without any Rf value in a qualitative study, and how the spot would be similarized to correlate between the chromatogram of standard and test sample.
Here, we conceptualized the coordinate values to correlate between the spots of sample and standard tracks. A detailed discussion of this concept and its application has been discussed here.
4.1.1 Traditional TLC (one dimensional) and Rf value
In t-TLC, we represent a spot or band in terms of Rf value. Rf value is a ratio of distance travelled by a spot to the distance travelled by solvent front. This distance travelled by the spot and the solvent front is in a single direction that is known as unidimensional or one-dimensional development.
4.1.2 Two-dimensional development and Rf value
Now let us consider the situation in two-dimensional development. Here the spot and solvent travel twice, initially along the Y-axis in first dimension and later along X-axis in second dimension. Therefore, a spot can acquire two different Rf values, one during first dimension and another during second dimension.
For easy understanding we have presented a few arbitrary data in Table 3 (these data table is purely imaginary and assumed for easy explanation). To understand the situations and facts let us consider that a sample containing mixture of three compounds (A, B & C) are applied on a plate and developed along Y-axis for first dimension development. After development it has been observed that A has travelled 27 mm, B has travelled 36 mm and C has travelled 45 mm, whereas the solvent front is 90 mm. Therefore, Rf values of A is 0.30, B is 0.40 and for C is 0.50 in first dimension. Now plate is rotated 90° anticlockwise and developed along X axis (which now becomes vertical in second development) with a solvent front of 90 mm. To understand, the rotation patterns please see Fig. 1. After development, it has been noted that A does not move from its original position and resides in its position where it has reached by first dimension development. B and C both move 36 mm along X-axis after second dimension development. Therefore, as per second dimension development A acquire Rf of 0 (0 mm/90 mm), B & C both acquire Rf of 0.40 (36 mm/90 mm). For easy understanding the above facts and figures Table 3 is a representative.
Calculation of Rf in unidimensional development
| Name of the spots | First Dimension Record (along Y-axis) | Second Dimension Record (along X-axis) | ||||
| Distance travelled by the spot | Distance travelled by the solvent | Calculated Rf value | Distance travelled by the spot | Distance travelled by the solvent | Calculated Rf value | |
| A | 27 mm | 90 mm | 0.30 | 0 mm | 90 mm | 0 |
| B | 36 mm | 90 mm | 0.40 | 36 mm | 90 mm | 0.40 |
| C | 45 mm | 90 mm | 0.50 | 36 mm | 90 mm | 0.40 |
Now let us answer a couple of questions. What are the Rf values for A, B and C to be represented? Can Rf value be zero? After second dimension development how can we distinguish between B and C?
Any definite answers to these questions are not logical and difficult to satisfy an inquisitive mind. Because, the concept of Rf value is not fitted rationally in this situation of multidimensional development. To overcome these limitations in defining the situation, we have postulated a method to demark and distinguish the spots by conceptualizing the idea of coordinate value instead of Rf value.
4.1.3 Proposed co-ordinate values
Considering the above situation let us find out the positions of these three spots namely A, B and C along Y-axis (after first dimension development) and along X-axis (after second dimension development). For easy understanding let us look at the Table 4.
Calculations of coordinate positions for two-dimensional development
| Name of the spots | First Dimension Record (along Y-axis) | Second Dimension Record (along Y-axis) | Coordinate value (Value of Y axis, Value of X-axis) | ||
| Distance travelled by the spot | Observed coordinate value | Distance travelled by the spot | Observed coordinate value | ||
| A | 27 mm | 27 | 0 mm | 0 | (y, x)/(27, 0) |
| B | 36 mm | 36 | 36 mm | 36 | (y, x)/(36, 36) |
| C | 45 mm | 45 | 36 mm | 36 | (y, x)/(45, 36) |
Now with this concept of coordinate values any spot residing anywhere on the plate can be distinguished and can have a unique numerical value to represent the spot as Rf does in unidimensional development. These two coordinate values can be used unambiguously for identification of spot in two-dimensional development.
4.1.4 Determining Coordinate value
The OriginPro 8.5 program was used to find the coordinates. For this purpose, the image of the 20 × 10 cm plate of the fourth level development has been divided into two 10 × 10 cm plates/images. The position for Standard Mixture (SM) is on the left side of the plate while the spot for Test Sample (TS) is on the right side of the plate, therefore the image with TS spot has been horizontally flipped for the uniform representation. Both the images were separately imported into the OriginPro 8.5 program along the X- and Y- axes of graph. As shown in Figs 7 and 8, the graph's scales were set to 0–100 mm with 10-point increments along each axis. Now, the y, x coordinates was noted as mentioned in Table 5, by pointing out each spot of both the images using the screen reader feature of OriginPro 8.5 software.
Determination of (y, x) coordinates for Standard Mixture (SM) using OriginPro 8.5 software
Citation: Acta Chromatographica 2025; 10.1556/1326.2025.01304
Determination of (y, x) coordinates for Test Sample (TS) using OriginPro 8.5 software
Citation: Acta Chromatographica 2025; 10.1556/1326.2025.01304
(y, x) coordinates for Standard Mixture (SM) and Test Sample (TS) determined using OriginPro 8.5 software
| Standard Mixture (SM) | Test Sample (TS) | ||||||
| Spot No. | SM (y, x) | Spot No. | SM (y, x) | Spot No. | TS (y, x) | Spot No. | TS (y, x) |
| 1 | (18.83, 15.33) | 11 | (81.38, 37.96) | 1 | (19.08, 15.53) | 11 | (81.63, 37.76) |
| 2 | (22.08,15.33) | 12 | (80.64, 44.03) | 2 | (22.33, 15.57) | 12 | (81.13, 43.83) |
| 3 | (24.05, 15.35) | 13 | (80.64, 51.30) | 3 | (24.76, 15.33) | 13 | (81.38, 50.90) |
| 4 | (28.28, 15.35) | 14 | (79.91, 62.01) | 4 | (28.72, 15.54) | 14 | (80.64, 61.39) |
| 5 | (31.75, 15.13) | 15 | (76.68, 69.08) | 5 | (31.99, 15.34) | 15 | (77.17, 68.28) |
| 6 | (35.98, 15.35) | 16 | (79.14, 74.73) | 6 | (35.98, 15.14) | 16 | (79.15, 74.12) |
| 7 | (38.46, 15.13) | 17 | (65.49, 44.83) | 7 | (38.71, 15.14) | 17 | (65.76, 44.04) |
| 8 | (38.46, 18.16) | 18 | (64.75, 51.60) | 8 | (38.71, 18.95) | 18 | (65.26, 50.90) |
| 9 | (38.71, 23.03) | 19 | (73.43, 59.99) | 9 | (39.20, 23.83) | 19 | (74.20, 59.59) |
| 10 | (38.71, 26.85) | 20 | (73.43, 62.61) | 10 | (39.20, 27.63) | 20 | (74.22, 61.87) |
| – | 21 | (36.21, 27.41) | – | 21 | Not identified | ||
The method of determining the coordinate values is very simple and precise. As initial development direction was along the Y-axis, so while in representing the coordinate value, Y-axis position was mentioned first and then X-axis value, because the X-axis development was later development direction. Two adjacent spots which found to be well resolute and well separated from each other are having different coordinate values. Even the well-defined values along X-axis of two adjacent spots are indicating the good separation in second dimension. The ambiguities in referring Rf values have easily been overcome by this coordinate values. In representation of coordinate values, no two different spots have same values, which is a clear indication of uniqueness of this way of representation. This method for determining the coordinate position being a very simple, precise and unique, can easily be adopted while working in two-dimensional mode.
The target of the all experimental set up was to achieve the separation of 21 phytocompounds in TS. That separation and identification was expected to achieve at level 4 experiment. Therefore, repeatability needs to be checked at fourth level. Hence, fourth level of the experiments was repeated 11 times without changing any conditions to check the reproducibility in terms of co-ordinate positions. After each experiment at level 4 the coordinate positions values were determined and found that a maximum deviation was within ± 0.21 along Y axis and ± 0.80 in X axis. It means that the co-ordinate values along the X-axis (in 11 repetitions) dwell in the range of 0.21 from the mean value, similarly, deviation along X-axis is ±0.80, and it means that co-ordinate values along the X-axis (in 11 repetitions) dwell in the range of 0.80 from the mean value. Hence, lesser the deviation value greater is the reproducibility. Here, these low values of deviation indicate that chromatogram was quite good and highly reproducible.
5 Conclusion
In regular qualitative investigation, the identification of three or four phytocompounds in complex natural product is a routine practice. Even marker based qualitative identification of plant considers two or in some cases three markers. The present work is envisaged to achieve the separation of maximum numbers of phytocompounds in a complex matrix of a traditional formulation by improvised techniques in Thin Layer Chromatography. In that sense, the present set of experiments is found to be able to separate 19 out of 21 phytocompounds with good reproducibility. This type of qualitative chromatographic profiling may be treated as a blueprint of a specific complex preparation. However, the present investigation has successfully separated 19 phytocompounds with good repeatability, but some limitations are associated with this work. First and foremost is that, the band application which is common practice in modern days TLC cannot be done, instead spot application is the only way of sample application. Secondly, the present analysis is having a validity limited to qualitative profiling. The developed plate cannot be exposed to densitometric scanning and hence the quantitative output from this work cannot be expected. The execution of the whole experimental set up was a laborious job but that would work when performed with patience and good observance. Despite having these limitations, the output of the present work, can serve the purpose of qualitative profiling of complex herbal mixtures. Another significant output of this work is that presence or absence of any herb in a polyherbal formulation can easily be detected. It is becoming a challenging issue in present era of commercialization and intentional skipping of one or more herbal ingredients in profit making malpractices.
Declaration on conflict of interest
The authors declare that there is no conflict of interest associated with this article.
Acknowledgement
Authors acknowledge the support of Sharad Daulatrao Pawar for technical help in some points of the study.
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