Lab 3: Organic Fluorophores - Optical Spectroscopy | PHYS 552, Lab Reports of Optics

Download Lab 3: Organic Fluorophores - Optical Spectroscopy | PHYS 552 and more Lab Reports Optics in PDF only on Docsity! Physics 552 Optical Spectroscopy (Fall ‘08) - 1 - Lab 3: Organic Fluorophores Organic fluorophores form the heart of optical spectroscopy. Their diverse array of properties provide a seemingly endless range of available applications. Today, we will study some of the basic chemical properties of organic fluorophores, focusing on the importance of resonance delocalization and solvent interactions. Cyanines, polyenes, and resonance delocalization We have already seen that a common structure in chromophores is alternating single and double bonds (conjugated bonds), often in the form of aromatic structures. These type of structures are responsible for delocalizing the electrons over many atoms, leading to a red-shift of the electronic transitions into the optical range. This phenomena – resonance delocalization – is the topic of our first experiment. Comparing a series of cyanine and polyene molecules, we will see that resonant structures lead to complete delocalization, while unresonant structures with conjugated bonds only have partial delocalization. Conjugated bonds and partial delocalization The polyenes are hydrocarbon chains with alternating single and double bonds. Recall that a double bond consists of a σ-bond localized between two atoms and a π-bond localized above and below the atoms. In conjugated structures, the neighboring π-bonds are aligned and there is some overlap that occurs. This leads to some “leakage” (i.e. partial delocalization) between neighboring double bonds. Resonance delocalization The simplest example of a resonant structure is given by the benzene molecule C6H6. There are two equivalent structures for benzene, as shown at the top of the picture (single and double bonds inverted). In either of these structures, as drawn, the electrons would be localized in π-bonds above and below two carbon atoms (with some “leakage” between them). However, the molecule is not actually in one state or the other. Rather, it exists in a hybrid of these states (bottom figure) in which the electrons are delocalized across all six carbon atoms equally (to imagine the corresponding molecular orbital, think of doughnuts above and below the plane of the ring). Fluorescein and pH dependence Fluorescein is a bright green fluorophore commonly used in fluorescence measurements. It has two ionizable groups, –COOH and –OH. Depending on the pH, it is found in four forms: dianion, monoanion, neutral, and cation. In its dianionic form, it is a strong absorber of visible light (ε490 ~80,000 L/mol-cm) and has a high Physics 552 Optical Spectroscopy (Fall ‘08) - 2 - fluorescence efficiency (φf > 90%). This is because its structure is highly resonant, as shown in the figure, such that the electrons are delocalized across the three rings at the top. Note that the bottom ring is not part of the delocalization because the –COOH group causes steric interference and forces the ring to rotate out of the plane of the other rings. In its neutral form with the phenol and carboxylic groups protonated, fluorescein’s emission degrades considerably and the absorption shifts toward the blue and decreases. The reason is that the molecule is no longer in complete resonance. The presence of the –OH group requires a proton to be transfered from one side to the other in order to draw equivalent resonant structures. Polyenes and Cyanines: Report The Cyanine dyes are given to you in closed plastic absorption cuvettes. Please do not open/discard contents when you are done. They are to be reused by the next section. (1) Measure the absorption spectra for the cyanine dyes from 450 to 750 nm. Record the peak absorption wavelength, calculate the Stokes shift in nm, and determine the concentration or the dyes in mol/L. The relevant extinction coefficients at the peak wavelength are ε3 = 150,000 L/mol-cm, ε5 = 250,000 L/mol-cm, and ε5.5 = 250,000 L/mol-cm. If your absorption spectrum is too noisy to resolve the peak, increase the integration time (remember to take a new blank with the same integration time). Absorption Max [nm] Emission Max [nm] Stokes shift [nm] Concentration [mol/L] Cy-3 570 Cy-5 667 Cy-5.5 694 (2) From your experimental data, calculate the shift in absorption wavelength [nm] for the addition of the following chemical structures to a cyanine dye. Δλ Another double bond inserted along the chain Δλ An additional pair of phenyl rings on the ends of the dye Physics 552 Optical Spectroscopy (Fall ‘08) ® os Table 8.2 Symmetrical cyanine cations (CH3), N=CH—(CH=CH)—N(CH;)) with k = 0 to k=6(N =4 to N = 167z electrons). Calculated and experimental absorption maxima Aja, and oscillator strengths f (see Section 8.3) k N Color of solution Calculated Experiment Calculated Experiment 0 4 206 224 Colorless 0.7 1 6 332 313 Colorless 1.0 0.9 2 8 459 416 Yellow 13 1.0 3 10 587 519 Red 1.6 12 4 12 716 625 Blue 17 1.5 5 14 844 735 Green 2.2 2.0 6 16 973 848 Colorless 24 The color of the solution of the dye is complementary to the color of the absorbed light; e.g. if red light is absorbed the color of the solution is green. The absorption maxima are calculated according to equation (8.21), the oscillator strengths according to equation (8.29) with # = 1.42 (index of refraction of the solvent dichloromethane). References see Further Reading Table 8.4 Absorption maxima Ama, of polyenes with Nz electrons (N/2 Double Bonds) Polyene N AE/10-°3 AE‘/10-° J Calculated Experiment Ethene 2 10.4 13.4 150 162 Butadiene 4 6.2 9.2 220 217 Hexatriene 6 45 75 270 257 Octatetraene 8 3.5 6.5 310 290 . 10 2.8 5.8 340 317 12 24 5.4 370 344 14 2.1 S$. 390 368 16 18 48 410 386 18 16 46 430 413 20 15 45 440 420 (B-Carotene) 22 14 44 450 453 24 12 4.2 470 461 26. 1.2 42 470 471 28 Ld 41 480 500 30 1.0 40 500 504 Polyacetylene oo 0 3.0 660 650 AE is the excitation energy of a corresponding 7 electron system with equally long bonds according to equation (8.36) with do = 140pm, Aé’ is the excitation energy with correction for bond alternation according to equation (8.35). References see Further Reading. Physics 552 Optical Spectroscopy (Fall ‘08) - 6 - Prodan, environmental probes, and solvent interactions Organic fluorophores form a diverse group of probes responsive to the conditions of their local environment. This makes them useful for studying a wide-range of phenomena such as protein dynamics, calcium sensing, DNA sequencing, and membrane structures. Many fluorophores are designed (or found to have) sensitivity to parameters like pH, polarity, hydrophobicity, viscosity, temperature, and pressure. These parameters can occur on a macroscopic scale (i.e. solvent properties) or a microscopic scale (e.g. inside proteins and membranes). With the example of fluorescein discussed above, we have already seen how pH can affect the properties of a fluorophore by causing structural changes. This was also described last week with the chromophore phenol red, which undergoes a structural change altering the degree of resonance delocalizaton and leading to shifts in absorption wavelengths. In this experiment, we will study how a molecule’s dipole moment interacts with the solvent. Using the fluorophore Prodan, we will see that more polar and protic solvents lead to large redshifts in the emission relative to non-polar and aprotic solvents. Gregorio Weber No discussion of the chemistry and design of organic fluorophores is complete with first mentioning its pioneer, Gregorio Weber (UIUC, Chemistry). Weber made many fundamental contributions to the fields of fluorescence spectroscopy and protein biochemistry. His group was responsible for designing many fluorophores in common use today, including dansyl chloride, ANS, TNS, and Prodan. His design of Prodan (Weber and Farris, 1979) was based on the notion that a molecule with an electron donor and an electron acceptor located on opposite sides of an aromatic center would have good sensitivity to solvent polarity because of the large dipole moment produced upon excitation. Prodan and its derivatives The chemical structure of several Prodan related fluorophores shown in the figure. They are based on a naphthalene center with an electron donating alkylamine –NR2 group on one end and an electron withdrawing carbonyl >C=O group on the other. This leads to a sizable increase in dipole moment upon excitation and is related to its sensitivity to solvent polarity that we will measure today. The senisitivity of the emission color to the polarity of the local environment has made Prodan and several related compounds particularly useful in biological studies. The probe Acrylodan adds a –CH=CH2 group that can be directly reacted with –SH groups in proteins for labeling. Danca was designed to bind in hydrophobic pockets of proteins. Laurdan has a long aliphatic –C11H23 chain that preferentially targets to hydrophobic regions like lipid bilayer membranes. It can then survey the polarity of the local environment. Research has Physics 552 Optical Spectroscopy (Fall ‘08) - 7 - shown that two distinct phases can be found in membranes, a liquid crystalline phase that is hydrophobic and a more water-enriched, fluid phase. A Laurdan molecule localized to one of these phases has a distinct emission, bluer for the crystalline phase and redder for the fluid phase. By taking ratiometric measurements at these emission wavelengths, Laurdan has been used with fluorescence microscopy to map out the polarity microheterogeneity of natural membranes. Polarity sensitive probes and solvent reorganization One of the controlling factors in the red-shift of Prodan’s emission with solvent polarity is the reorganization of solvent molecules near the excited molecule. The probe in its ground state has a small dipole and the solvent molecules are oriented relatively randomly about it periphery. Upon excitation, the dipole moment increases as charge is transferred from the alkylamine group to the carbonyl group (intramolecular charge transfer, ICT). Rapidly, the solvent molecules reorganize around the dipole, lowering the system’s energy. The more polar the solvent, the greater the relaxation and the larger the red-shift of the emission spectrum. Prodan measurements You are provided with solutions of Prodan in 5 solvents, labeled A, B, C, D, and E. For each solvent, you are provided with two solutions – a blank solution for measuring the absorption background and cleaning and PRODAN solution for fluorescence measurements. For each solvent, carry out the following procedure. 1. Measure the absorption spectra for each sample from 250 to 500 nm using the Agilent 8453 spectrometer and Labview (from last week). Use a fresh Pasteur pipette for each solution. The vials you are provided should last for another section as well, so don’t waste/contaminate the solutions. First blank using the solvent itself, then take a Prodan spectum. Save the absorption spectrum, and record the peak wavelength in thee table below. 2. For the fluorescence spectra, you will be using the Agilent 1100 series fluorimeter and the handheld controller. The cuvette is inside the fluorimeter. You will use the syringe to inject the sample.