Figure 1. Behaviour of pyrene molecules in micellar systems during dilution process.

Recently I have done some experiments about critical micellar concentration (CMC). The CMC values of polymeric nano-micelles were determined by fluorescence spectroscopy, using pyrene as a polarity-sensitive fluorescent probe, of which the operations, data recording as well as the curve fitting are totally done according to the relevant literature reported previously.¹

Principle

Pyrene, a hydrophobic fluorescent dye, could be used as a microenvironmental polarity-sensitive probe for the determination of CMC values. Pyrene molecules could be excited by light at a wavelength of 339 nm and emitted fluoresces with maxima at wavelengths of 373 nm and 384 nm.²

• To use the ratio of wave length at 373/384 instead of single wavelength is to normalise the fluorescence intensity, which may change as the amount of pyrene varies.
• More importantly, the fluorescent ratio will change dramatically according to the polarity of microenvironment in which the probes stay. Sensing a more hydrophobic environment, the fluorescent ratio of 373/384 will be much smaller than one. However, if in a more hydrophilic microenvironment (such as water and alcohol), the ratio would increase, basically much bigger than that in a more hydrophobic environment.
• The plot of pyrene ratio 373/384 as a function of the logarithm of polymer concentration was fitted to a Boltzmann sigmoidal curve, and the CMC values can be obtained through two approaches: (i) from the interception of the rapidly varying part and the nearly horizontal part where pyrene apparently appears in a more hydrophobic environment; (ii) from the inflection point of the pyrene fluorescence ratio plots.

Methods

• In a typical procedure, a stock solution of pyrene in acetone was firstly prepared by dissolving 0.61 mg of the fluorescent dye into 50 ml of acetone followed by ten minutes’ ultra-sonication to make a clear pyrene solution with a concentration of $6.03 \times 10^{-5} mol/L$.
• Into each Eppendorf thin-walled polypropylene (EP) tube with a capacity of two millilitres, 10 μL of the formed pyrene solution was added and the acetone was left to evaporate completely in a fume hood.
• Then the micellar solutions of known concentrations ranging from 1149.47 μg/mL to 0.035 μg/mL were prepared by serial dilutions of the previously prepared stock micellar solution with Milli Q water.
• At last one millilitre of the diluted micellar solution was transferred into each pyrene-containing EP tube and the final concentration of pyrene in each tube was $6.03 \times 10^{-7} mol/L$.
• The prepared tubes containing micelle and pyrene were ultra-solicated for twenty minutes and left to equilibrate in darkness for 24 hours before test.
• Then the emission spectra were scanned from 350 nm to 450 nm at an excitation wavelength of 339 nm on a lumina fluorescence spectrometer (Thermo Scientific) at 25 °C. Both the excitation and emission splits were set at 5 nm and the fluorescent intensity ratio of wavelength at 373 nm to that at 384 nm from the emission spectra was analysed as a function of the logarithm of the copolymer concentration ³, which was then used to determine the CMC values.

Results

The trends of fitted curve (fluorescent wave length ratio at 373/384 versus polymer concentration, Figure 5) are totally opposite compared with that of the reported trend curves (Figure 2) After many experiments, all the operations and data processing are as same as the reported methods, however, the trends of the curves are totally opposite! What strange curve trends!

Figure 2. The curve trends of reported curves ⁴

Analysis

Pyrene

Pyrene is a hydrophobic dye, it’s every difficult to be dissolved in polar environment, such as in water. With the help of amphiphilic material, the emission fluorescent intensity of pyrene increased greatly as the polymer concentration went up, indicating the increase of total content of pyrene in the tested water solutions, as shown in Figure 3.

Figure 3. Fluorescence emission spectra of pyrene.

The previous reports explained this phenomenon: After certain preparative process, the amphiphilic polymers (concentration higher than CMC) in polar conditions can form some micellar systems which consists of a hydrophilic surface and a hydrophobic inner core. With help of the micellar systems, the pyrene molecules can crowd into the inner hydrophobic core of micelles so that the solubility of pyrene can be relatively improved and leads to fairly strong fluorescent peak.⁵

Polarity-sensitivity

Pyrene could be used as a microenvironmental polarity-sensitive probe for the determination of CMC values.

Figure 4. Fluorescent intensity ratio of pyrene in solvents with different polarities, tested at 25°C.

As shown in Figure 4, when the polarity of solvents decrease, the ratio values also decrease. In Figure 5, when the polymer concentration (high concentration) is above CMC, the fluorescent intensity ratio goes up to a roughly constant value as the polymer concentration rises, which is bigger than that in DCM while smaller than that in acetone (Figure 4), indicating that pyrene in this micellar system faced a microenvironment of which the polarity is between DCM and acetone. What’s more, the polarity of this microenvironment is much smaller than that of water. All these indicate that the solubility of pyrene molecules indeed have been improved with help of the amphiphilic polymers in solution and pyrene molecules stay in a relatively more hydrophobic environment.

Figure 5. The Boltzmann sigmoidal curve of pyrene fluorescence ratio, as polymer concentration decrease, the ratio goes down.

While polymer concentration is below CMC, only few polymer aggregates form. And inside the formed polymer aggregates, only few pyrene molecules has been crowded. Besides, the solubility of pyrene molecules are too small in pure water. That’s why the pyrene fluorescent intensity is so weak below CMC (Figure 3) and the pyrene fluorescence ratio is much smaller than one (Figure 5).

Increase of water-solubility

Figure 6. Fluorescent intensities of pyrene in pure water or Polymer Con. above CMC.

Figure 7. Fluorescent intensities of pyrene in pure water or Polymer Con. below CMC.

Figure 6 illustrates the solubility of pyrene in different solvents, when polymer concentration is above CMC, the pyrene fluorescence increases dramatically. And in pure water, little fluorescence can be observed. In Figure 7, when polymer concentration is below CMC, the emission fluorescent intensity of pyrene in polymer solution is as same as that in pure water, indicating polymer concentration below CMC has no effect on improvement of pyrene solubility in water. However, the pyrene fluorescent ratios in pure water (Figure 4) and water with polymers (concentration below CMC, Figure 5) are totally different. The pyrene fluorescent ratio in pure water is much higher than one, indicating a more hydrophilic environment. While in polymer solutions (concentration below CMC), the pyrene fluorescent ratio is smaller than one, demonstrating a more hydrophobic environment. Though very little polymers in water, they can significantly change pyrene fluorescent ratio. Polymers can form some aggregates in water, which pyrene molecules can stay with. Therefore the fluorescent ratio indicating that they are in a more hydrophobic environment.

The curve trends

• [Figure 1A]: Above CMC, with help of micellar systems, the fluorescent intensity increased dramatically. And the emission fluorescence ratio indicates pyrene molecules were in a microenvironment whose polarity was between DCM and acetone, a little lower than water. These results indicate that pyrene molecules should be embedded in the shell of micelles. (If in the inner cores, this demonstrates that the inner core of this prepared micelles should be a more hydrophilic environment, at least the polarity value should be higher than that of DCM.)
• [Figure 1B]: As polymer concentration decreased (like micellar solution was diluted), the fluorescent intensity dropped and the fluorescent ratio also decreased, indicating the amount of pyrene molecules went down and they were in a relatively more hydrophobic environment.
• [Figure 1C]: Below CMC, the fluorescent intensity and fluorescent ratio dropped further. Polymers in water can assemble some aggregates to form a hydrophobic microenvironment.

That’s why the trend of the fitted curves was different from the ones reported previously.

A little more

Someone told me that if you could find the inflection from your fitting curve and get the accurate results of your experiments, that would be ok. Just forget others. But I still want to know why the strange phenomenon occurred to my fitting curves and wanna know the truth, at least one step closer to the truth. Just a little curious about that :) .