I have previously made a lot of nucleotide analyses with tetrabutylammonium as ion pairing agent but decided out of curiosity to try primary amines instead and then made a strange observation. One of the nucleotide peaks was split into two peaks (all the others were okay). Can anyone explain this phenomenon?
The ion pairing agent is octylamine which is pH-adjusted to pH 6 with H3PO4. I use isocratic conditions where three solutions are mixed: A (250 mM phosphate buffer pH 6 in low organic) + B (only low organic) and C (11 mM ion pairing agent + low organic) and use isocratic mode with 20% solution C (the other solutions I have varied). I have tried to made changes in phosphate concentration (by changing the ratio of solution A and B) and organic content (5-10% MeOH or MeCN) and then I can move around the splitting point so other nucleotides are affected instead but the splitting is always there. The same phenomenon appears if I use heptylamine instead. I also tried hexylamine but then the retetion time starts to be too low.
The only solution I can think of is if the ion pairing agents form micelles and the level of micelles is different in the loading solution compared to the mobile phase (the splitting point could then be the migration time of the micelles). Howver, it is very difficult to find what the critical micelle concentration of octylamine and heptylamine is and if it occurs at all at this low pH (they should be fully ionized at this pH which decreases the propensity to form micelles).
My loading solution is usually that I mix my sample 1:1 with a solution that contains 40%C, variable solution B (depending on what I have in the mobile phase and water.
Anders Hofer
I was very interested in your question. It is not clear what peaks you have in mind. Two peaks already speak of duality in the process of micelle formation, which I am investigating.
I load a nucleotide mixture and dATP comes out as two peaks at around 25 min. If I decrease the potassium phosphate concentration (changing from 60 to 40% A), dATP comes out as one peak and instead dGTP becomes double. In this case the two dGTP peaks are close to 25 min. So, it feels like there is a UV-invisible compound (possibly a micelle) with a retention time close to 25 min and that the migration time of this compound is insensitive to potassium phosphate concentration. The splitting is very strong and there is baseline separation between the two peaks.
Apparently you load the mixture (what?) Into the chromatographic column and watch for the separation of substances according to the dependence of the absorption intensity (UV spectrum) on the time of separation of the mixture.
Yes that is correct and the sample is a mixture of dNTPs (dATP, dGTP etc) and NTPs.
In an acidic environment, octylamine forms octylammonium phosphate and micelles form at your concentration. CMC octylammonium chloride 0.175 M at 25 degrees. In the case of tetrabutyl ammonium bromide with nucleotides, ion pairs are formed due to electrostatic and hydrophobic interactions. Micella has dual properties and is a supplier of a hydrophobic cation for binding to a nucleotide. Moreover, the hydrophobic interaction is cooperative. It depends on the hydrophobic groups of the ion pair. Depending on the gain (gain in Gibbs energy), a micelle can supply one or another cation. In this complex cooperative formation 1.one ion pair (as with tetrabutylammonium) can be separated by water and 2.a contact ion pair with nucleotides . They regulate the appearance of your peaks. For a better understanding of this phenomenon, read the articles.
https://www.researchgate.net/publication/274011380_Liquid_Polyamorphous_Transition_and_Self-Organization_in_Aqueous_Solutions_of_Ionic_Surfactants
https://www.researchgate.net/publication/325169774_Quantum_entanglement_in_micellar_solutions_of_ionic_surfactants
https://www.researchgate.net/publication/333694580_Structure_of_Micelles_of_Sodium_Dodecyl_Sulphate_in_Water_an_X-ray_and_Dynamic_Light_Scattering_Study?_sg=6s9bfO5sWpTG4NMi7O5Obvku0l2VeMoZjXg9vbJmq0epRcIEiUh7Z7Nr0oJ0VtUhHYbcqn_h83lu5Blju04tWW3QGZQuiLXqF98QSnLN._h6wd_CgXebSi2Lf69UtV3dXFtJlxmKRkLOzs-hUUj1KVCeJQewQxFrz1_uHaw7B8wx1uWomWhL3GK3Dq0Cxtw
Interesting, but if the CMC is 175 mM, micelles should not form in my case where the concentration is 10 mM in the stock solution (in solution C) and 2 mM in the final solution. Do you know the kinetics of micelle formation and dissociation? Is it within seconds, minutes or hours?
The presence of nucleotides and inorganic electrolyte reduces CMC surfactant. Therefore, micelle formation is possible.
Kinetics of micelles see link
Article Structure of Micelles of Sodium Dodecyl Sulphate in Water: a...
See also a similar system
Yu.A. Mirgorod. Oscillations of Fluorescence Intensity in Sodium Dodecylsulfate Aqueous-Solutions with Protonoporhine (IX) Dimethyl Ether-Zinc Complexes. Russian Journal of General Chemistry. 1995. V.65. N1. P.155-156. (Rus.)
Investigated the fluorescence intensity in Sodium Dodecylsulfate Aqueous-Solutions with Protonoporhine (IX) Dimethyl Ether-Zinc complexes at concentrations where it forms spherical micelles. Results the fluorescence intensity of emission spectrum at 580 nm undergoes fluctuations over time 7 min. The period and the waveform is not always reproduced. In the methanol solution fluctuations are absent.
https://www.researchgate.net/publication/295699653_Oscillations_of_Fluorescence_Intensity_in_Sodium_Dodecylsulfate_Aqueous-Solutions_with_Protonoporhine_IX_Dimethyl_Ether-Zinc_Complexes
See also a similar system
Yu.A. Mirgorod. Oscillations of Fluorescence Intensity in Sodium Dodecylsulfate Aqueous-Solutions with Protonoporhine (IX) Dimethyl Ether-Zinc Complexes. Russian Journal of General Chemistry. 1995. V.65. N1. P.155-156. (Rus.)
Investigated the fluorescence intensity in Sodium Dodecylsulfate Aqueous-Solutions with Protonoporhine (IX) Dimethyl Ether-Zinc complexes at concentrations where it forms spherical micelles. Results the fluorescence intensity of emission spectrum at 580 nm undergoes fluctuations over time 7 min. The period and the waveform is not always reproduced. In the methanol solution fluctuations are absent.
See also a similar system with a 7-minute fluorescence intensity fluctuation period.
Yu.A. Mirgorod. Oscillations of Fluorescence Intensity in Sodium Dodecylsulfate Aqueous-Solutions with Protonoporhine (IX) Dimethyl Ether-Zinc Complexes. Russian Journal of General Chemistry. 1995. V.65. N1. P.155-156. (Rus.)
Investigated the fluorescence intensity in Sodium Dodecylsulfate Aqueous-Solutions with Protonoporhine (IX) Dimethyl Ether-Zinc complexes at concentrations where it forms spherical micelles. Results the fluorescence intensity of emission spectrum at 580 nm undergoes fluctuations over time 7 min. The period and the waveform is not always reproduced. In the methanol solution fluctuations are absent.
See also a similar system with a 7-minute fluorescence intensity fluctuation period.
Yu.A. Mirgorod. Oscillations of Fluorescence Intensity in Sodium Dodecylsulfate Aqueous-Solutions with Protonoporhine (IX) Dimethyl Ether-Zinc Complexes. Russian Journal of General Chemistry. 1995. V.65. N1. P.155-156. (Rus.)
Investigated the fluorescence intensity in Sodium Dodecylsulfate Aqueous-Solutions with Protonoporhine (IX) Dimethyl Ether-Zinc complexes at concentrations where it forms spherical micelles. Results the fluorescence intensity of emission spectrum at 580 nm undergoes fluctuations over time 7 min. The period and the waveform is not always reproduced. In the methanol solution fluctuations are absent.
Thanks a lot for all your help. I have investigated everything a bit more and I came to the following conclusions:
1: If I premix the A, B and C solutions to make one solution (with the same A-B-C ratio as before) to be sure that the final solution has time to equiibrate, the dATP peak is still double. The concentration of octylamine is then 2 mM, which means that the effect occurs at almost 100 times lower concntration than the CMC in a salt-free solution. So if it is caused by micells, the salt has an extremely high effect on the CMC. I rather think it is the salt that has this effect than the nucleotides whichf are used at only 5 uM concentration.
2. If I prepare the sample in a solution that is the same as the mobile phase, the dATP peak is not split any more. It is important to have exactly the same final concentration of octylamine and acetonitrile in the sample as in the mobile phase. The phosphate concentration could be lower (it could be half and it still worked), indicating that the salt concentration is not supercritical. The question is then if it still could be that the salt in point 1 is able to decrease the CMC nearly hundred times. An alternative could be that the octylamine forms a yet unknown smaller aggregate than a typical micelle that is non-detectable by currently used methods and formed more easily at much lower concentrations. Also with tetrabutylammonium ions, which are not supposed to form micelles, I also see tendencies of the same phenomenon (one of the peaks is slightly split) but the effect is much weaker.
Anders Hofer
Your answers do not come to me. My name is required. The robot does not understand what needs to be reported to me. I accidentally found your answer. To determine the mechanism, you need to simplify the system. From my experience in the interaction of two hydrophobic ions of dATP- and octylammonium + I can say the following:
1.interaction depends on the hydrophobicity of two ions (cooperativity)
2. dATP has three places for directional interaction
3. This interaction is affected by the concentration of both ions and extraneous electrolyte.
4. interaction with the addition of octylammonium to the dATP begins with the formation of an ion pair, first one ion —O—, then the second, then the third. Moreover, one or three may have a contact pair. Then a precipitate will fall out. If we add octylammonium further, a common micelle can form and the precipitate will dissolve as a result of solubilization.
5. When you add a stationary phase, the system gets complicated.
6. I repeat, the system is cooperative, due to water. Water, changing its structure, adapts to each molecule, both in DNA and the human body. Your system is very interesting as a model system with further complication and with the transition to proton tunneling. I work in this direction. Therefore, I have devoted so much time to you.
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