Ali Mohammad*, Sameen Laeeq and Abdul Moheman
Analytical Research Laboratory, Department of Applied Chemistry, Faculty of Engineering & Technology, Aligarh Muslim University, Aligarh, India
Citation: Mohammad A, Laeeq S, Moheman A (2010) Sodium Deoxycholate Micelles Activated Separation of Coexisting Five-nucleobases by Highperformance Thin-layer Chromatography. J Bioanal Biomed 2: 055-059. doi:10.4172/1948-593X.1000022
Copyright: © 2010 Mohammad A, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
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A novel high-performance thin-layer chromatographic method has been developed for the resolution of fi ve-coexisting nucleobases (adenine, guanine, cytosine, thymine, and uracil). The nucleobases were separated on aluminum-backed cellulose 60 F 254 plates with the aid of 5.0% aqueous sodium deoxycholate (NaDC)-acetonitrile (AcN), 1:3 (v/v) as mobile phase. All the nucleobases were viewed on HPTLC plates under 254nm UV light. The order of R F value given in parentheses was guanine (0.12) < adenine (0.44) < cytosine (0.50) < uracil (0.72) < thymine (0.84). The effect of pH (acidity or basicity) of the mobile phase on the retention of individual nucleobases was examined. Furthermore, the effect of interference of mono- (Li + , Na + ), and bivalent (Mg 2+ , Ba 2+ , Co 2+ , Ni 2+ , Cu 2+ , Zn 2+ , Cd 2+ , Pb 2+ ) cations; mono- (Br - , CH 3 COO - , NO 3 - , IO 4 - ) , and bivalent (CO 3 2- , SO 4 2- MoO 4 2- ) anions, and complexing ligands (urea, and EDTA) on the retention behavior of nucleobases were also assessed. The chromatography of nucleobases was also performed on silica 60 F 254 , RP-18 F 254 , and kieselgel 60 F 254 HPTLC plates. These TLC plates failed to separate the coexisting purines and pyrimidines. The detection limit of all nucleobases on cellulose 60 F 254 layers was 5.4 × 10 -2 μ g spot -1 . The proposed method is rapid, easy, and reliable. It can be applied for routine analysis of DNA, and RNA nucleobases.
HPTLC; Nucleobases; Purines; Pyrimidines; Bile salt; Sodium deoxycholate
High-performance thin-layer chromatography (HPTLC) is well suited to the separation of nucleobases. Most of reports on this topic are limited to the use of PEI-cellulose, ODS (octadecyl silica), and silica gel as layer materials (Randerath and Struck, 1961; Bij and Lederer, 1983; Steinberg et al., 1996). Chiral plates have also been used for separation of nucleobases, and enantiomers (Hatzack and Rasmuseen, 1999). The first use of micellar mobile phase by Armstrong in 1979 for the analysis of nucleotides was 1.3 AOT in cyclohexane-water mixture. The suggested mobile phase was capable to resolve a mixture of nucleotides on silanized silica gel. Compared to normal micelle forming surfactants, bile salts are unique in forming helical aggregates in solution (Giglio et al., 1988; Campanelli et al., 1989; Williams et al., 1990). In nature, sodium deoxycholate (Figure 1) referred as “secondary bile acid” is produced in the intestine from the salts of glycocholic, and taurocholic acid by the action of bacterial enzymes. Applications of sodium deoxycholate range from cell lyses, liposome preparation, isolation of membrane proteins and lipids, a cell culture media as supplement, preventing non specific binding in affinity chromatography, micellar electrokinetic chromatography and other chromatographic techniques (Hofmann and Mysels, 1988; Williams et al., 1990; Thompson et al., 1995; Morgan et al., 2008). Versatility of bile salts led us to utilize its analytical potential as mobile phase for the separation of coexisting nucleobases from their mixtures. This is probably the first report to separate five nucleobases (two purines, and three pyrimidines) on cellulose 60 F254 HPTLC plates by utilizing sodium deoxycholate (aqueous 5.0%) plus acetonitrile (1:3, v/v) as mobile phase. Furthermore, we have successfully achieved an interesting separation of thymine from uracil. This separation is important because both pyrimidines are used to differentiate between the structures of DNA and RNA (Figure 2).
All experiments were performed at 25 ± 2°C.
Chemicals and reagents
Pre-coated cellulose 60 F254 HPTLC aluminum foils were of Merck, Darmstadt, Germany. Acetonitrile (Lichsolv, Merck, India), purines and pyrimidines (Himedia, Mumbai, India) and sodium deoxycholate
(E-Merck, India) were used.
All chemicals and reagents were of analytical reagent (AR) grade. Double distilled water (DDW) was used throughout the experiment.
Working standard solutions
Solutions of purines (adenine, and guanine), and pyrimidines (cytosine, thymine, and uracil) were prepared in a mixture of methanol and water (4: 6) to give concentration of 1.0% w/v.
HPTLC plates containing fluorescent indicator were kept under short wave 254nm UV light to locate the position of analyte.
Cellulose 60 F254, silica gel 60 F254, RP-18 F254, and Kieselgel 60 F254 aluminium foils of size 6 x 6 cm were used as stationary phases.
The buffer solutions used to prepare the solution of NaDC (5.0%) at different pH levels are listed in Table 1.
|0.04M boric acid + 0.04 M phosphoric acid||50 : 50||2.3|
|0.04M boric acid + 0.04 M phosphoric acid + 0.24 M NaOH||50 : 50 : 8||3.4|
|0.04M boric acid + 0.04 M phosphoric acid + 0.24 M NaOH||50 : 50 : 10||5.7|
|0.04M boric acid + 0.04 M phosphoric acid + 0.24 M NaOH||50 : 50 : 14||7.2|
|0.04M boric acid + 0.04 M phosphoric acid + 0.24 M NaOH||50 : 50 : 60||11.9|
Table 1: Buffer solutions used as solvents to prepare mobile phases containing 5.0 % NaDC.
A variety of mobile phases listed in Table 2 were examined to identify the most suitable solvent system for separation of nucleobases.
|Code for mobile phases||Composition|
|M1||DDW (double distilled water)|
|Aqueous micellar bile salt solution|
|M2||5.0% NaDC in DDW|
|Aqueous micellar solutions with organic additives|
|M3||M2−AcN (9 : 1, v/v)|
|M4||M2−AcN (8 : 2, v/v)|
|M5||M2−AcN (7 : 3, v/v)|
|M6||M2−AcN (6 : 4, v/v)|
|M7||M2−AcN (5 : 5, v/v)|
|M8||M2−AcN (4 : 6, v/v)|
|M9||M2−AcN (3 : 7, v/v)|
|M10||M2−AcN (2.5 : 7.5, v/v)|
|M11||M2−AcN (2 : 8, v/v)|
|M12||M2−AcN (9 : 1, v/v)|
|M13||M2−Acetone (2.5 : 7.5, v/v)|
|M14||M2−MeOH (2.5 : 7.5, v/v)|
|M15||M2−HCOOH (2.5 : 7.5, v/v)|
|M16||M2−DMSO (2.5 : 7.5, v/v)|
|M17||5.0% aqueous NaC−AcN (2.5 : 7.5, v/v)|
|M18||5.0% aqueous NaTC−AcN (2.5 : 7.5, v/v)|
|Buffer micellar NaDC solutions with AcN (2.5 : 7.5, v/v)|
|M19||5.0% NaDC (pH 2.3)−AcN|
|M20||5.0% NaDC (pH 3.4)−AcN|
|M21||5.0% NaDC (pH 5.7)−AcN|
|M22||5.0% NaDC (pH 7.2)−AcN|
|M23||5.0% NaDC (pH 11.9)−AcN|
AcN, acetonitrile; NaC, sodium cholate; NaTC, sodium taurocholate; MeOH, methanol; DMSO, dimethylsulfoxide.
Table 2: Solvent systems used as mobile phases in chromatographic studies.
Precoated HPTLC plates were activated at 60 ± 2°C in an electrically controlled oven for 20 min and stored in closed chamber until used. Spotting of 1.0 µL of sample using micropipette (Tripette 0783178, Germany) is done at 1.0 cm from the base of HPTLC plates. Spots were air dried and developed in a closed presaturated Camag TLC twin-trough chamber with desired mobile phase by ascending technique up to the ascent of 5.0 cm from the point of application. After development, the HPTLC plates were withdrawn from glass chamber, air dried and then detected under UV light to locate the position of analyte as fluorescent spots under short wavelength.
The RF values of visualized spots were calculated by using formula: RF = 0.5 (RL+ RT) where RL = RF of the leading edge, and RT = RF of trailing edge.
In order to examine the effect of volume of acetonitrile added in aqueous NaDC, RF of nucleobases, were chromatographed on cellulose layers. The affinity of the solvent systems with different concentrations of aqueous NaDC was assessed by calculating the capacity factor values.
The capacity factor (K) was determined as a function of the conventional mobility RF
It is used to indicate the relative affinity of purines or pyrimidines between the solid substrate and the solvent. Strong affinity for the substrate is indicated by high value of capacity factor and vice versa.
To study the effect of interference of mono- and bivalent cations or anions, urea, and EDTA on the RF value of nucleobases, analyte sample (1.0 µL) was spotted onto the activated TLC plates followed by the spotting of 1.0 µL of the interfering species on the same spot. The plates were developed with M10 (5% aqueous NaDC-AcN, 1:3, v/v). After development, spots were visualized under short wave 254nm UV light. The RF valued were determined and compared with those obtained in the absence of interfering species.
For the separation, equal volumes of all five nucleobases were mixed and 1.0 µL of the resultant mixture was applied on HPTLC plates. The plate was developed with M10, the spots were visualized and RF values of the separated spots of the nucleobases were calculated.
The limits of detection of nucleobases were determined by spotting a definite volume of different concentrations of nucleobases (0.01-1.0%) on the cellulose F254 HPTLC plates. The plates were then developed and the corresponding spots were detected. The method was repeated with successive lowering of the amounts of nucleobase (purine or pyrimidine) until no spot was detected. The minimum amount of nucleobase that could be visualized was taken as the limit of detection.
Selection of mobile phase
The aim of the present study was to select a useful solvent system to achieve the separation of coexisting five nucleobases (two purines, and three pyrimidines). For this, purpose chromatography of all nucleobases was performed on cellulose layers using different solvent systems. Both mono- component as well as binary mixed solvent systems was tested for the chromatography of these five nucleobases. When micellar solutions of cationic (cetyltrimethylammonium bromide, CTAB), non-ionic (Tween-20), and anionic (sodium dodecyl sulfate, SDS) surfactants were used as eluent, all nucleobases show smeared spots with high mobility. Mixed micellar solutions were also not successful for separating nucleobases; all show broadened spots. In view of these unfavorable results, linear anionic surfactant, SDS was replaced with helical sodium deoxycholate (bile salt). Aqueous 5.0% solution of sodium deoxycholate (NaDC) was selected for study because of its versatile nature of separating many isomeric compounds. It is well known that, the separation efficiency of pure micellar mobile phase system is improved by the addition of the organic additives (Sumina et al., 2003). Therefore, mixed mobile phases of 5.0% NaDC-acetonitrile in different ratio was tested, and the obtained results with these solvent systems are listed in Table 3. Addition of acetonitrile increases the hydrophobic nature of the mobile phase in order to retain the hydrophilic species. From the data of this Table, the following conclusions may be drawn:
Each value is an average of four measurements; T = Tailed spot; nd = not detected.
Table 3: RF values of five nucleobases on cellulose 60 F254 HPTLC plates developed with mobile phases (M1-M18).
• In mobile phase, M1 (DDW) both purines show lower mobility compared to pyrimidines on cellulose 60 F254 layers.
• When 5.0% aqueous bile salt, NaDC (M2) was used as the mobile phase, the RF of all nucleobases slightly reduced as compared to their mobility in M1.
• In both M1, and M2, the RF of nucleobases was in the order: adenine < guanine < cytosine < thymine ≈ uracil.
• The RF value of adenine, guanine, cytosine, thymine, and uracil was found to increase with the increase in volume ratio of acetonitrile in its mixture with 5.0%aqueous NaDC (M3-M6), followed by decline in RF value from M7 to M12. As more compact spots, and differential RF were realized in M10 (5.0% aqueous NaDC-acetonitrile, 1:3). This mobile phase was selected for further studies. To understand the effect of other organic additives on the RF values of nucleobases, acetonitrile in M10 was replaced by other organic solvents (viz: acetone, methanol, formic acid, and DMSO), and the resultant mobile phase systems (M13-M16) were used for chromatography.
• In M13, and M14, all nucleobases showed lower mobility as compared to M10. The order of RF was: M10 > M13 > M14.
• In M15 and M16 all nucleobases showed constant and high RF value (RF > 0.90). Compact spots were realized in M15, whereas in M16 spots were diffused. For investigating the effect of other bile salts, NaDC in M10 was replaced by other biological bile salts (i.e. NaC or NaTC), and the resultant mobile phase systems (M17, and M18) were used for chromatography. It was found that there was no significant change in RF values on substitution of NaDC by NaC or NaTC in M10.
The RF data of nucleobases obtained with buffered 5.0% NaDC (pH 2.3, 3.4, 5.7, 7.2 or 11.9) plus acetonitrile (M19-M23) mobile phases are compared and presented in Figure 3. It is clear from this Figure that the RF value of nucleobases is slightly influenced by pH of NaDC in all mobile phases (M19-M23), except guanine, which was not detected in M22, and M23. The RF value of guanine was increased to 0.83 from its standard RF value (0.12) in acidic media (M19-M21).
Effect of nature of sorbent layers
To establish the effectiveness of cellulose 60 F254 HPTLC layers, the retention behavior of nucleobases was also examined on different sorbent layers, and the obtained results have been plotted in Figure 4. Although more compact spots were realized on RP-18 F254 layers, but separation of coexisting five nucleobases is possible only on cellulose 60 F254 layers.
Effect of interference
Table 4 summarizes the effect of a variety of impurities on retention (RF) of nucleobases. From the data listed in Table 4, it is evident that the RF values of nucleobases were altered from its standard value in the presence of impurities in the sample. All nucleobases showed high RF value in the presence of Li+ as compared to other alkali or alkaline earth metal cations. Guanine, cytosine, thymine and uracil were not detected in the presence of Mg2+. The RF value (retention) of nucleobases was greatly influenced in the presence of transition metal cations, probably due to greater affinity of transition metal cations to bind with DNA or RNA nucleobases. In the presence of Ni2+ and Cu2+, all nucleobases showed almost similar RF values. Zn2+ converted the compact spot of nucleobases into a badly diffused spots.The detection of guanine cytosine, thymine, and uracil in the presence of Co2+ was difficult.
|Cations (mono- and bivalent)|
|Anions (mono- and bivalent)|
Table 4: RF values of nucleobases on cellulose 60 F254 HPTLC plates developed with mobile phase, M10 in the presence of organic and inorganic impurities in the working standard sample.
However, a significant increase in RF values of adenine, guanine, and cytosine was noted in the presence of anions, urea, and EDTA. Both purines (adenine, and guanine) produced double spots in the presence of MoO42- showing effective complexing tendency of molybdate ion.
Compared to adenine, the RF value of guanine was greatly modified in the presence of impurities (cations, anions, urea,or EDTA) in the sample. This might be due to the presence of two attractive centres in guanine to interact with impurities, whereas in the case of adenine there is only one attractive centre (Barda et al., 1996).
The position of spots appeared on cellulose fluorescent plate is depicted in Figure 5. From Figure 5 it is clear that, with mobile phase M10 (5% aqueous NaDC + AcN, 1: 3, v/v) purines and pyrimidines could be separated easily from their mixtures.
Limit of detection
The lowest possible detectable microgram amounts of all five nucleobases obtained on cellulose F254 HPTLC plates developed with M10 was ≈ 0.054 µg spot-1. It shows that the developed method is reasonably suitable for identifying these nucleobases at trace level.
The proposed thin layer chromatographic system comprising of cellulose 60 F254 as stationary phase , and 5.0% aqueous sodium deoxycholate (NaDC)-acetonitrile (AcN), 1:3 (v/v) as mobile phase is most favorable for the separation of coexisting five-nucleobases (adenine, guanine, cytosine, thymine, and uracil). IT is highly selective, reliable, and rapid requiring 5.0−7.0 min for resolution of DNA and RNA nucleobases.
The authors are thankful to the chairman, department of Applied Chemistry for providing necessary research facilities. One of the authors (Sameen Laeeq) is also grateful to the University Grant Commission, New Delhi for providing fi nancial assistance.
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