Separation of Dyes by Mixed Hydrotropic Thin Layer Chromatography
P. A. Salunke*, Dr. S. D. Barhate, B. R. Chavhan, S. S. Patil. R. S. Wagh, S. B. Rathod
Shree Sureshdada Jain Institute of Pharmaceutical Education and Research, Jamner, Dist: Jalgaon
*Corresponding Author E-mail: salunkepoonam@rediffmail.com
ABSTRACT:
Now a days, aqueous solubility of drugs is major problem in pharmaceutical field. Generally for solubilization of drugs organic solvents generally prefer. But these solvents are toxic in nature, high cost, flammable and acts as pollutants. Organic solvents are not environment safe. Hydrotropic method is used to enhance water solubility of poorly water soluble drug by using various hydrotropes instead of organic solvents. As a Model sample Bromocresol green and phenol red was selected. To increase water solubility of poorly water soluble dyes, single and mixed hydrotropic method was selected. Both dyes soluble in optimized single hydrotropic solution like as 20% niacinamide, 25% niacinamide, 10% sodium benzoate and mixed hydrotropic solution like 10% Sodium citrate and 10% urea. For separation of both dyes from mixture Thin layer chromatography have been used. Silica gel G used as stationary phase on glass slide and hydrotropic solution used as a mobile phase in which sample was completely soluble. When single hydrotropic solution used as a mobile phase, separation of dyes was not obtained from mixture. Mixed hydrotropic solution of 10% sodium citrate and 10% urea used as mobile phase gives better result. Rf value of Bromocresol green and phenol red in mixed hydrotropic solution was found to be 0.51 and 0.82 respectively.
KEYWORDS: Bromocresol green, Phenol red, Thin layer chromatography, Hydrotropy, solubility, Rf value.
INTRODUCTION:
Hydrotropy term originated by Scientist Neuberg describes the enhancement of solubility of solute by the addition of high concentration of alkali metal salts of various organic acids. The term has been used in literature to designate non-micelle-forming substances, either liquid or solids, organic or inorganic, capable of solubilizing insoluble compounds(1). In hydrotropic solubilization process involves intermolecular interaction with several balancing molecular forces. Hydrotropic agents have been used to enhance aqueous solubility of poorly water soluble drugs (2,3,4)
Bromocresol green is a triarylmethane dye. It is used as a pH indicator in application of growth mediums for microorganisms and titrations. In clinical practice it is used to measure serum albumin concentration in mammalian blood samples (5).
Figure 1: Structure of Bromocresol green
Phenol red is a pH indicator also known as phenolsulphonphthalein. It used in cell biology laboratories (6).
Figure 2: Structure of Phenol red
Bromocresol green and phenol red slightly water soluble and soluble in organic solvents like as ethanol, dichloromethane benzene. Organic solvent are toxic in nature, carcinogenic and causes environment pollution. To avoid this problem hydrotropic method is safe. Hydrotropy is the process by which water insoluble substances are made water soluble by hydrotropic substances. Both dyes make water soluble by using single and mixed hydrotropic solution method (7,8,9,10).
Separation of dyes by using TLC with organic solvents has been reported (11). But yet not any hydrotropic method mentioned for separation of dyes by TLC. In present research work describes simple, accurate reliable and rapid method for dyes by Thin layer chromatography for analysis and to replace the organic solvent because of their toxicity and higher volatility and introduced the hydrotropic agent and to enhance the solubility of Bromocresol green and Phenol red by employing various hydrotropic agents.
MATERIAL AND EXPERIMENTAL WORK:
Instrument:
TLC chamber, sealed capillary, were used, TLC plates coated with ̴thin layer of silica gel G (̴ 0.2 mm thickness) glass slides were used as the stationary phase.
Solvents and chemicals
Bromocresol green and Phenol red was collected from institute.
Niacinamide, Sodium citrate, Sodium benzoate, Urea was used as mobile phase in the form of hydrotropic solution. All reagents used were of analytical reagent grade procured from Loba chemie.
Thin layer chromatography was performed by following steps(12):
Silica gel G was selected as adsorbent and act as stationary phase. Slurry of silica gel G was prepared in water and methanol mixture. The stationary phase is applied onto the glass slides uniformly by pouring method.
The glass slides are allowed to dry in air (5 -10min) & it was further dried & activated by heating at about 100°C for 30 min . Glass slides made with volatile organic liquids may not require this further drying. By removing the liquids associated with layer completely, the absorbent layer was activated.
Step 3: Solubility of sample:
Solubility of dyes checked in various hydrotropes. Some single and mixed hydrotropes enhances water solubility of Bromocresol green and phenol red. By trial error basis 20% Niacinamide, 25% niacinamide, 20% sodium benzoate, mixture of 10% sodium citrate and 10% urea was selected for solubilization of dyes.
Step 4: Preparation of sample solution of dyes in selected hydrotropes:
20% Niacinamide:
Weighed accurately 10 gm of niacinamide, dissolved in sufficient distilled water and made volume up to the 50 ml. Add aliquots of this solution in 10 mg Bromocresol green and phenol red weighed separately in separate 10 ml volumetric flask. After addition of 6 ml solution dyes were completely soluble then made volume upto mark by distilled water. From this solution pipette out 1 ml and made volume up to 10 ml. Use this solution as a sample solution.
25% Niacinamide:
Weighed accurately 12.5 gm of niacinamide, dissolved in sufficient distilled water and made volume up to the 50 ml. Add aliquots of this solution in 10 mg Bromocresol green and phenol red weighed separately in separate 10 ml volumetric flask. After addition of 4 ml solution dyes were completely soluble then made volume up to mark by distilled water. From this solution pipette out 1 ml and made volume up to 10 ml. Use this solution as a sample solution.
20% Sodium benzoate:
Weighed accurately 10 gm of Sodium benzoate, dissolved in sufficient distilled water and made volume up to the 50 ml. Add aliquots of this solution in 10 mg Bromocresol green and phenol red weighed separately in separate 10 ml volumetric flask. After addition of 5 ml solution dyes were completely soluble then made volume up to mark by distilled water. From this solution pipette out 1 ml and made volume up to 10 ml. Use this solution as a sample solution.
Mixture of 10% Sodium citrate and 10% Urea: Weighed accurately 5 gm of Sodium citrate and 5 gm of urea, dissolved in sufficient distilled water and made volume up to the 50 ml. Add aliquots of this solution in 10 mg Bromocresol green and phenol red weighed separately in separate 10 ml volumetric flask. After addition of 5 ml solution dyes were completely soluble then made volume up to mark by distilled water. From this solution pipette out 1 ml and made volume up to 10 ml. Use this solution as a sample solution.
Step 5: Selection of mobile phase:
Mobile phase was selected on the basis of trial and error. Mobile phase selected in which Bromocresol green and phenol red was completely soluble. As a mobile phase single hydrotropic solution as well as mixed hydrotropic solution has been used.
Step 7: Application of sample:
Samples applied to TLC plates on origin line at the bottom of plate. Sealed capillary have used for applying the sample as a spot.
The TLC slides were placed vertically in rectangular chromatography tank or chamber. Saturate tank carefully with mobile phase. The development should be carried out at room temp.
Ascending development was preferred for development of plates. The plates after spotting of the sample were placed in chromatography chamber containing mobile phase at the bottom.
After locating the developed spots on plate & making their position & size, they are evaluated either qualitatively or quantitatively.
Step 8: Calculations:
The Rf value is defined as the ratio of the distance moved by the solute (i.e. the dye or pigment under test) and the distance moved by the solvent (known as the Solvent front) along the paper, where both distances are measured from the common Origin or Application Baseline, that is the point where the sample is initially spotted on the plate.
Distance travelled by solute
Rf value = -----------------------------------------------
Distance travelled by solvent
RESULT AND DISCUSSION:
Separation of Bromocresol green and phenol red has been achieved by using thin layer chromatography.
The solubility Bromocresol green and phenol red had been checked in different concentrations of hydrotropes. 20% Niacinamide, 25% Niacinamide, 20% Sodium benzoate, mixture of 10% sodium citrate and 10% urea selected as a hydrotropic solution and mobile phase.
20% Niacinamide:
Bromocresol green and Phenol red completely soluble in 20% niacinamide solution but when used a mobile phase separation was not observed.
Figure no.03: 20% niacinamide used as a mobile phase
25% Niacinamide:
Bromocresol green and Phenol red completely soluble in 25% niacinamide solution but when used a mobile phase separation was not observed.
Figure no.04: 25% Niacinamide used as a mobile phase
Figure no.05: 20% sodium benzoate used as mobile phase
Figure no.06: (10% Sod. Citrate & 10% urea)
CALCULATIONS:
The Rf value is defined as the ratio of the distance moved by the solute (i.e. the dye under test) and the distance moved by the solvent (known as the Solvent front) along the paper, where both distances are measured from the common Origin or Application Baseline, that is the point where the sample is initially spotted on the plate.
Distance travelled by sample
Rf value = -----------------------------------------------
Distance travelled by solvent
Table No. 01: Calculation of Rf value
|
Sr. No. |
Sample |
Distance traveled by solute (cm) |
Distance traveled by solvent (cm) |
Rf value |
|
1 |
Bromocresol green |
2.6 |
3.9 |
0.66 |
|
2 |
Phenol red |
3.1 |
3.9 |
0.79 |
|
3 |
Mixture |
|
|
|
|
1 Bromocresol green |
2 |
3.9 |
0.51 |
|
|
2 Phenol red |
3.2 |
3.9 |
0.82 |
SUMMARY AND CONCLUSION:
Summary:
In single hydrotropic solution dyes get completely soluble but chromatographic separation does not obtained properly. Mixed hydrotropic solution used for enhancement of aqueous solubility of dyes. This same solution when used as a mobile phase proper separation observed from mixture of dyes. The rate of separation was faster in mixed hydrotropic mobile phase by using Thin layer chromatography.
Table no. 02: Summary of result
|
Sr. No. |
Mobile phase |
Figure |
Result |
|
1 |
20% Niacinamide |
|
Separation was not observed |
|
2 |
25% Niacinamide |
|
Separation was not observed |
|
3 |
20% sod. Benzoate |
|
Separation was not observed |
|
4 |
10% Sodium citrate + 10% Urea |
|
Separation was observed |
CONCLUSION:
The present research work is the novel application of hydrotropic solubilization technique in the analysis of poorly water soluble samples such as Bromocresol green and Phenol red. Separation of both these dyes from mixture successfully carried out by using mixed hydrotropic solution. In single hydrotropic solution separation was not observed and it was time consuming. From results it was concluded that by using mixed hydrotropic method rate of development was faster. Developed method was easy, fast, safe, cost effective, time saving and environment safe. In future, method can be used for routine analysis of various drugs and formulations without use of organic solvents.
ACKNOWLEDGMENT:
The authors are greatful to SSJIPER, Jamner, for providing required facilities for research.
And also thanks to Dr. R.K. Maheshvari, SGSITS, Indore, for his guidance.
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Received on 11.06.2019 Accepted on 11.07.2019
© Asian Pharma Press All Right Reserved
Asian J. Pharm. Ana. 2019; 9(3):151-155.
DOI: 10.5958/2231-5675.2019.00027.9