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Ultra Violet-Visible (UV-Vis) Spectroscopy is absorption spectroscopy in the UV and visible regions of light. This technique works on the principle that, across the wavelength region concerned, electrons from bonding and non-bonding orbitals in the molecules of interest absorb light and are promoted to excited states. Small energy gaps between the highest occupied and lowest unoccupied molecular orbitals correspond to absorption at longer wavelengths. Measuring the absorption across a range of wavelengths results in spectral peaks which can be correlated to the bonds present in certain molecules and thus the technique can be used to quantitatively determine the presence of certain analytes in solution. These include biological molecules, metal ions and conjugated organic molecules. The concentration of a given analyte in solution can be determined from the Beer- Lambert Law (equation 2.3), where A is the absorbance,  is the molar absorptivity, l is the path length and c is the concentration. The molar absorptivity is an intrinsic property of chemical species which is a measure of how strongly they attenuate light at a given wavelength.

A = εcl (2.3)

In this work UV-Vis is used exclusively to determine the concentration of peptoids in solution. This is possible given that every peptoid studied in this work contains a certain fraction of N spe residues, with side chains containing an aromatic group like that in the amino acid phenylalanine (illustrated in Figure 2.6). This has an absorption band centred around 257 nm. The concentration measured by this method is the concentration of N spe residues which can then be divided by the number of N spe residues in each peptoid sequence to give the peptoid concentration of the solution.

Fig. 2.6: Structure of (a) phenylalanine residue in peptides and (b) N spe residue in peptoids. Both contain the aromatic group responsible for a peak at 257 nm in the UV-Vis spectrum.

This method was used to measure the concentration of stock solutions of peptoids 23

2. Methodology and Technical Details

which were then further diluted for the collection of the CD spectra presented in Chapters 3 and 5. Additionally, UV-Vis was used to calculate peptoid concentrations in partitioning experiments designed to measure Log D, the PBS-octanol distribution coefficients, which are presented in Chapter 5.

Experimental Details

In order to make peptoid solutions of the desired concentration for CD measurements, a high concentration stock solution was made up for each peptoid. The concentration of each stock solution was determined by UV-Vis spectroscopy. UV-Vis measurements were carried out using a ThermoFisher Scientific Nanodrop 1 microvolume UV-Vis spec- trophotometer. Measurements were made over the wavelength range 200-350 nm using 3 µL samples. Though measurements are performed on microvolume samples the nanodrop normalizes the path length to 1 cm. The spectrum for each peptoid solution was averaged over 5 measurements and a baseline was fitted and subtracted to account for scattering. The concentration of peptoid was then calculated according to the Beer-Lambert Law (given in equation 2.3), with l = 1 cm,  = 195 cm−1 M−1 and the measured absorbance at 257 nm.

PBS-octanol Partitioning Protocol

The nanodrop was used to determine the relative concentrations of peptoid in both phases in the PBS-octanol partitioning experiments reported in Chapter 5. These experiments were carried out with a protocol adapted from that presented by Bolt et al. in their 2017 study of peptoid partitioning [23]. The key change made to the protocol was that instead of preparing large volumes of sample and measuring the peptoid concentration via bulk UV-Vis measurements, microvolume samples were prepared and measured according to the following protocol.

Powdered peptoid was dissolved in PBS at concentration in the region of 1 mM. The concentration was measured as detailed above, using the nanodrop. 50 µL of peptoid solution was deposited in an eppendorf tube and 50 µL of octanol was then deposited in the same tube. The sample was then gently agitated for a period of 24 hours following which it was allowed to rest for 1 week to allow the peptoid to partition between the two solvents and the system to equilibrate. The concentration of peptoid in each phase was then measured (again using the nanodrop) by taking a 3 µL aliquot from each solvent phase. This protocol was carried out in triplicate for each peptoid.

Great care was necessary in extracting the PBS aliquot from the mixed phase sample. As the pipette tip has to pass through the octanol to reach the PBS phase and small amounts of octanol tend to cling to the plastic tip this can result in the PBS samples

2. Methodology and Technical Details

being contaminated by octanol, which distorts the spectrum. To circumvent this, the pipette plunger was not depressed until the end of the tip was submerged in the PBS phase, expelling the air and any octanol. The PBS aliquot was then drawn up into the tip and the tip removed from the sample. The tip was rested briefly on the clean inner side of the sample tube to let any external octanol run off before the sample itself was deposited on the nanodrop stage. It was clear from the signal to noise in the collected spectra whether a clean PBS sample had been collected successfully and in any cases where contamination was evident the data was discarded and the process repeated.

Discussion of Experimental Errors Associated with Concentration Measurement by UV-Vis

Inconsistency in sample concentration is one of the main sources of experimental error in CD spectra, therefore accurately measuring the concentration of each peptoid sample is important. Each peptoid stock solution was prepared at least 12 hours prior to measuring the concentration to ensure all powder was fully dissolved. For each sample 5 UV-Vis spectra were collected and averaged before the calculation of the peptoid concentration. The concentration of the peptoids in solution is particularly important in the calculation of the Log D values reported in Chapter 5. Errors in Log D were calculated from the standard deviation in results from repeat measurements. The errors associated with particular peptoids that partitioned strongly into one phase (PBS or octanol) are much larger than those where the peptoid was more evenly distributed between the two phases. This is due to the difficulty of obtaining accurate concentration measurements of low concentration solutions using micro-volume samples on the nanodrop.