1. HIGH-PERFORMANCE LIQUID CHROMATOGRAPHIC EXPERIMENT
Study of condensation oscillations with L-lactic acid in pure acetonitrile and aqueous ethanol
Mieczysław Sajewicz, Dorota Kronenbach, Monika Gontarska, Teresa Kowalska
Institute of Chemistry, University of Silesia, 9 Szkolna Street, Katowice, Poland
AIM
In this study, we present high-performance liquid chromatographic and mass spectroscopic results on chemical transformation of L-lactic acid, when dissolved both in 70% aqueous ethanol and in pure acetonitrile, and then stored for certain periods of time in the stoppered glass vials at an ambient temperature. In order to gain a better insight in the nature of the investigated processes, in our experiments we employed HPLC with the three different detectors (diode array (DAD), evaporative light scattering (ELSD), and mass spectrometric (MS) detector).
INTRODUCTION
Spontaneous nonlinear chemical processes that occur in the Nature still seem not to be adequately explored. On the other hand, these very processes can play crucial role in various different metabolic and evolutionary pathways. Tracing oscillatory reactions in purely organic and colorless solutions is a challenging experimental task. High-performance liquid chromatography with the diode array detection (HPLC-DAD) is a reliable and accurate enough tool to monitor and quantify such phenomena. A bottleneck of the chromatographic analysis is, however, the time needed for a single analytical run, which makes continuous measurements of the concentration changes (needed for the kinetic assessment of the investigated reactions) virtually impossible. One way to circumvent this inconvenience is to obtain the shortest possible single analytical run.
Among chiral compounds, L-lactic acid plays a particular role due to its known biological importance, but there has been no experimental evidence prior to our own research [1,2] on its ability to undergo a spontaneous oscillatory in vitro chiral conversion. One reason is that lactic acid is poorly retained in the HPLC systems [3] and in that way it causes considerable analytical problems.
HIGH-PERFORMANCE LIQUID CHROMATOGRAPHIC EXPERIMENT
For the spontaneous condensation experiment, we used solutions of L-lactic acid in acetonitrile (ACN) and in 70% ethanol (EtOH) in the concentration of 2 g L−1.
Samples were stored in the tightly stoppered colourless glass vials and a spontaneous ageing thereof was carried out for 900 min (ACN), or 400 min (70% EtOH) at the temperature of 22oC.
HPLC analysis was carried out using the Varian model 920 liquid chromatograph equipped with the Varian 900-LC model autosampler, the gradient pump, the Varian 330 PDA model DAD detector, the Varian 380-LC model ELSD detector, and the Galaxie software for data acquisition and processing. The analyses were carried out for the 20-μL aliquots of the L-lactic acid solutions in the isocratic mode, using the Pursuit 5 C18 (5 m particle size) column (250 mm  4.6 mm i.d.) and methanol – water (5:5, v/v) mobile phase at a flow rate of 0.5 mL min-1. The chromatographic column was thermostated at 30oC with use of the Varian Pro Star 510 model column oven.
Liquid chromatographic analysis with mass spectrometric detection (LC-MS) was carried out using an LC-MS System Varian equipped with a Varian ProStar model pump, Varian 100-MS mass spectrometer, and Varian MS Workstation v software for data acquisition and processing. The LC analyses were carried out for the 20-μL aliquots of the L-lactic acid solutions in the isocratic mode, using a Pursuit X RS 3-C18 column (50 mm x 2.0 mm i.d.) and methanol–water (5:5, v/v) mobile phase at the flow rate of 0.20 mL min-1. Mass spectrometric detection was carried out in the ESI mode (full ESI-MS scan, positive ionization, spray chamber temperature 45oC, drying gas temperature 200oC, drying gas pressure 25 psi, capillary voltage 70 V, needle voltage 5 kV). A B a c
Fig. 1. Sequence of the chromatographic concentration profiles registered with use of the DAD detector at (a) 220 nm, (b) 250 nm, and (c) with use of the ELSD detector for the L-lactic acid solution in (A) ACN and (B) 70% EtOH after 0, 170, and 750 min storage time at 22ºC. d b
Fig. 2. Spectrochromatograms of the L-lactic acid solution registered with use of the DAD detector after (a) 0 min and (b) 900 min storage in ACN, and after (c) 0 min and (d) 400 min storage in 70% EtOH; samples storage temperature, 22oC.
Fig. 3. Time changes of the chromatographic peak heights for the L-lactic acid solution in ACN stored at 22ºC for 900 minutes. Registration of the chromatograms by means of (a) the DAD detector at the wavelength λ = 220 nm; (b) the DAD detector at the wavelength λ = 250 nm; and (c) the ELSD detector. Plots shown in (a) and (b) are valid for the peak retention times, tR, equal to 4.61 and 5.26 min, respectively, and plots shown in (c) are valid for the peak retention times, tR, equal to 5.07 and 5.57 min, respectively. a b c
Fig. 4. Time changes of the chromatographic peak heights for the L-lactic acid solution in 70% EtOH stored at 22ºC for 400 minutes. Registration of the chromatograms by means of (a) the DAD detector at the wavelength λ = 220 nm; (b) the DAD detector at the wavelength λ = 250 nm; and (c) the ELSD detector. Plots shown in (a) and (b) are valid for the peak retention times, tR, equal to 4.85 , 5.01, 5.36, and 5.53 min, respectively, and plots shown in (c) are valid for the peak retention times, tR, equal to 5.07, 5.20, 5.38, and 5.42 min, respectively. a b c
258,34
330,67
403,05
475,38
547,51 a
60,24
102,52
160,71
262,23
672,03
60,28
160,76
262,18
627,79
305,57
627,89 b
We used two different UV wavelengths (the DAD detector) and additionally the ELSD detector, to stronger emphasize an evident evolution of the concentration profiles (fingerprints) of the analyzed samples. This evolution clearly witnesses to the processes running spontaneously in the investigated reaction systems. Fingerprints valid for the ACN solution considerably differ from those valid for the 70% EtOH solution. The reason is a different ability of ACN on the one hand, and that of EtOH and H2O on the other, to participate in the intermolecular interactions through hydrogen bonds with lactic acid. Apparently, the tendency to form mixed H-bonded associates is far less pronounced with ACN than with 70% EtOH. Consequently, the concentration profiles registered for lactic acid in the former solvent are more compact and showing a lesser number of peaks which can be attributed to the condensation products than in the latter solvent. Finally, it has to be stated that the results recorded with use of the ELSD detector (Figs 3(c) and 4(c)) better reflect the concentration changes than the results recorded with use of the DAD detector.
An additional liquid chromatographic proof of ageing of the lactic acid samples, when dissolved in ACN and 70% EtOH, was obtained with use of the LC-MS system. With the fresh lactic acid sample in ACN, there was only one mass spectrum of the column effluent, as shown in Fig. 5(a). The predominant signal in this spectrum is at m/z , which might correspond with the following structure: [trimeric LA condensate + Na + He]+. After the storage period of 900 minutes, from the different parts of the chromatogram the different mass spectra were recorded and in none of them the m/z signal predominated (Fig. 5(b)). In that case, however, in each mass spectrum a characteristic group of signals can be seen with the distribution of their respective percentage yields resembling normal distribution and with central predominant peak characterizing with m/z 627. This signal might correspond with the following structure: [octameric LA condensate + Na + He]+.
With the fresh lactic acid sample in 70% EtOH, the structure of the concentration profile is richer than with the analogous sample prepared in ACN (Fig. 5(c)). However, mass spectra recorded for the different parts of this chromatogram are very simple and besides, very similar to one another. The predominant signals in the three recorded mass spectra are either at m/z 306, or 319. The signal at m/z 306 very well corresponds with the tetrameric LA condensate. Mass spectra recorded for the lactic acid solution in 70% EtOH and stored for 400 min at ambient temperature give evidence of a considerably greater abundance of the condensation products than in the fresh sample (Fig. 5(d)). Again, in each mass spectrum a characteristic group of signals can be seen with the distribution of their respective percentage yields resembling normal distribution and with the central predominant peaks characterizing with the m/z values between 700 and 800. In these mass spectra, one also encounters an abundance of signals with the m/z values considerably higher than 1000.
319,77
69,28
305,67
69,33 c
305,57
231,24
637,14
420,84
669,18
89,27
415,10
319,72
337,76
422,79 d
Fig. 5. The chromatogram of the L-lactic acid solution registered with use of the LC-MS system after (a) 0 min and (b) 900 min storage in ACN, and after (c) 0 min, and (d) 400 min storage in 70% EtOH; samples storage temperature, 22oC. Insets show the mass spectra of the separated species recorded at the maxima of the respective peaks.
ADFSF
REFERENCES
[1] M. Sajewicz, M. Gontarska, D. Kronenbach, T. Kowalska.
Acta Chromatogr. 20: (2008)
[2] M. Sajewicz, E. John, D. Kronenbach, M. Gontarska, T. Kowalska. Acta Chromatogr. 20: (2008)
[3] F. Duprat and V. Coyard. Chromatographia. 34: (1992)[
The work of two of the authors (M.G. and D.K.) was partially supported by the PhD scholarship granted to her in 2010 within the framework of the ‘University as a Partner of the Economy Based on Science’ (UPGOW) project, subsidized by the European Social Fund (EFS) of the European Union.
CONCLUSION
With each employed high-performance liquid chromatographic technique (i.e., HPLC-DAD, HPLC-ELSD, and LC-MS), we managed to amass an important, diversified, and convincing empirical evidence pointing out to the oscillatory condensation of L-lactic acid, running spontaneously and irrespectable of the nature (aqueous or non-aqueous) of a solvent employed.