Research Article ReductionofBackgroundFluorescencefromImpuritiesinProtein Samples for Raman Spectroscopy Marco Pinto Corujo , 1 Pavel Michal , 2 Rod Wesson, 1 Don Praveen Amarasinghe, 1 Alison Rodger , 3 and Nikola P. Chmel 1 1 MAS, Centre for Doctoral Training, University of Warwick, CV4 7ALUK, Coventry, UK 2 Department of Optics, Palack´ y University Olomouc, 17. listopadu 12, Olomouc 77146, Czech Republic 3 School of Natural Sciences, Macquarie University, Sydney, NSW 2109, Australia CorrespondenceshouldbeaddressedtoMarcoPintoCorujo;marco.pinto@agilent.com Received 7 April 2022; Revised 10 October 2022; Accepted 13 October 2022; Published 24 November 2022 AcademicEditor:MohdSajidAli Copyright©2022MarcoPintoCorujoetal.TisisanopenaccessarticledistributedundertheCreativeCommonsAttribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background fuorescence remains the biggest challenge in Raman spectroscopy because of the consequent curvature of the baseline and the degradation of the signal•to•noise ratio of the Raman signal. While the concentrations of the fuorophore impurities are usually too low to be detected by other analytical methods, they are often sufcient to prevent Raman data collection.Amongthediferentexistingmethodstoremovethe fuorescencesignal,photobleachingremainsthemostpopulardue to its simplicity. However, using the spectrometer laser to photobleach is far from optimal. Most commercially available in• struments have little or no choice of wavelength, and their output powers are in many cases not suitable for highly fuorescent samplessuchasthosefrombiologicalsystems(e.g.,proteins).Inthisarticle,weassesspracticalaspectsofphotobleachingsuchas theapparentreversibilityoftheprocessandtheefectofconvectioncurrentsduetowhatwespeculatetobetemperaturegradients across the bulk of the solution. We also introduce an afordable custom made external photobleaching unit with a choice of excitationwavelengthanddemonstrateitsviabilitywithahighlyfuorescentbovineserumalbuminproteinsolution,whichhad proved most challenging for Raman spectroscopy as it contained ∼10% w/w impurities. 1. Introduction Manystudiesinthefeldofmolecularanalyticalscienceare carriedoutwithbiologicalsamples,whichhavebeentreated to extract the desired components (e.g., DNA, RNA, lipids, and proteins) [1, 2]. Although these molecules are put throughdiferentpurifcationprocedures,e.g.,ionexchange chromatography,afnitychromatography,gel fltration,and gelelectrophoreses,toseparatethemfromoneanotherand other cellular components, trace concentrations of impu• ritiesoftenremaininthesampleinadditiontothemolecules of interest, proteins in our case [2, 3]. Because Raman spectrometersmeasurealltheStokesshiftedfrequenciesthat reach the detector, whether it is from Raman or other phenomena, and because fuorescence has a yield several orders of magnitude larger than that of inelastic scattering, even a very low concentration of fuorescent impurities in the sample can cause signifcant background fuorescence [1, 4]. Among the main efects caused by fuorescence in a Raman measurement are the degradation of the signal•to• noiseratio,theriseofabroadGaussianshapedbaseline,and the saturation of the detector that in extreme cases can completely mask the signals of interest [5–7]. Fluorescence can be quenched by adding components withdiferentmolecularinteractionssuchasenergytransfer, ground•state complex formation, and collisional quenching [8, 9]. Tey are based on diferent physical•chemical inter• actions between a quenching agent and the fuorophore that results in a lower fuorescence quantum yield and therefore fuorescence intensity [8]. Quenching efects can be reversed byremovingthequenchingagent.Moreover,simplefltration to remove solid particles suspended in a solution removes Hindawi Journal of Spectroscopy Volume 2022, Article ID 1928091, 8 pages https://doi.org/10.1155/2022/1928091