Pyrene Concentration distribution variation with migration distance via Thin-Layer Chromatography

Adam Bielski, Sean McCrea, Ryne SternbergThe Pennsylvania State University, University Park, PA

In nature, pyrene (Py) exists in the lowest possible energy state known as the ground state, but may be excited by photons to its excited state (Py*). For pyrene, the excited state may react with the ground state to create an excimer (Ex*), which then decays back to the ground state as shown in Eq. (1a-b): 

(1a) (1b)

Exciting pyrene with a laser creates an absorption spectrum that can be measured with a spectrometer. This spectrum gives a ratio of the amount of excimer and monomer present in the sample. From this data, it is possible to determine the amount of pyrene present in a given sample. However, the Py* lifetime is relatively short and will only have the opportunity to react with ground state in concentrated solutions1. To take it one step further, Ex* competes with *Py luminescence decay and will create a concentration gradient.

As a spot of pyrene moves down a thin-layer chromatography (TLC) plate, it will gradually widen. The spectrum of pyrene at different points along the plate will yield the concentration distribution before and after the sample elutes down the plate.

Pyrene, shown above consists of four aromatics rings and it is because of this aromaticity, that pyrene is easily excited by UV lasers. The transition from ground to excited state also has an energy penalty associated with it that can be characterized by the Frank-Condon Principal2. It’s aromatic structure makes it an ideal subject to observe the relative concentration distribution of the monomer and excimer during elution along a TLC plate.

Introduction

Materials and MethodsDilutions of concentrated pyrene (2x, 4x, 8x, 16x) were spotted and excited with an SRS NL100 337 nm laser to generate a calibration curve relating pyrene concentration to the monomer/excimer ratio. Concentrated pyrene was spotted in a line across a TLC plate and excited with the laser at different distances. The volume of the Pyrene spotted on the sample was approximately 24µL, and will allow us to calculate total number of moles present in our concentration gradient. Pyrene eluted (Mobile: 70/30 toluene-ethyl acetate, Stationary: silica) and the sample was excited to find concentration at various migration distances.

Figure 1 – Schematic setup with the SRS NL100 337 nm laser, theOcean Optics spectrometer, and the TLC

Figure 2 - Emission Spectra of Pyrene, excited with 337 laser along a TLC plate. The monomer occurs at 392 nm and the Excimer is active at 492 nm.

Figure 3- By diluting the concentrated Pyrene solution and observing the Excimer to Monomer ratio, we created a plot to relate the ratios to the concentration of the ground state Pyrene.

A = 344.17 mM/(nm)^2

Figure 4- Using the E/M ratios constructed from figure 2, and sixteen other spectrums, and the calibration plot, the concentration distribution across the TLC plate is plotted as a function of migration distance. The areas under each curve show the ground state Pyrene distributed across a given area.

DiscussionThe main objectives of this experiment include, studying the migration distance effects of pyrene concentration, understanding the photophysical properties of pyrene and how they are applied to specroscopy. Thin layer chromatography with silica gel as a stationary phase is used as a separation method for pyrene and its components. After this and applying a laser enables one to calculate the intensity ratio between the excimer and monomer of pyrene. When these ratios are converted into concentrations, seen in Figure 4, and plotted against migration distance, a Gaussian distribution is revealed. This distribution shows how the ground state Pyrene distributes itself in the spot, dilute around the edges and concentrated in the center3 demonstrated by an E/M ratio greater than 1. This Gaussian distribution is what we expected to observe, because the excimer and monomer migrate at different rates due to differences in photo-physical properties.

The areas on under each curve, in Figure 4, are representative of the total pyrene present on the TLC plate. We must also account for the dimensionally analysis in our experiment. At the beginning, a volume of pyrene is spotted onto the plate, however, what we measure with the laser is an area and then the concentration at a given distance from the baseline, Rf. The sum of each point in the concentration profile is equal to the total amount of pyrene spotted on the plate at the beginning of the experiment, only distributed across a larger area. Theoretically, the area under each cureve should be equal because the total amount of pyrene remains constant. Error in the areas due can be attributed to the calibration plot and the E/M ratios.

ConclusionUpon running the experiment and processing our results, the data behaved in a Guassian distribution as expected. Each spectrum was processed to find the E/M ratio, and diluting the concentration solution allowed us to create a calibration curve. This calibration curve allowed us to determine the concentration profile of ground state pyrene as it eluted along the TLC plate. The areas under each curve demonstrate that although the pyrene migrates, the total amount does not change, only creating a larger distribution. We’ve learned that by exciting pyrene, its excimer and monomer migrate at different rates creating a concentration profile and thus different photo-physical properties.

Future Work Had more time been allotted, there are several steps we would have done to take this experiment one step farther. We would have run the experiment again with more Rf values to compare the concentration distributions and added them to Figure 4. We would have also used different aromatic structures and compared their photo-physical properties to that of Pyrene.

References and Acknowledgements We would like to thank Dr. Bratoljub Milosavlevic and Tony Zidell for assisting in performing this experiment and also conceptual topics.

1. Milosavljevic, B. H.. Lab Packet for CHEM 457 Experimental Physical Chemistry. 2014, 8-1-42. Lochmuller, C.H, Colborn A.S, Hunnicutt, M.L, Harris, J.M. Bound Pyrene Excimer Photophysics and the Organization and Distribution of Reaction sites on Silica. J. Am. Chem. Soc.1984, 106, 4077-4082 3. McCoy, E.F, Ross, I.G. Electronic states of aromatic Hydrocarbons the Frank-Codon Principle and Geometries in Excited States. Australian Journal of Chemistry. 1962