This work is supported by grants from the National Institutes of Health (NIH) R00HD103954 and R21EB035823. Research reported in this publication was supported by the Washington University Institute of Clinical and Translational Sciences grant UL1TR002345 from the National Center for Advancing Translational Sciences (NCATS) of the National Institutes of Health (NIH). The content is solely the responsibility of the authors and does not necessarily represent the official view of the NIH. The authors would like to acknowledge assistance from O&#39;Brien Lab members, Michael Vahey and his lab members, and Huanzhu Jiang.

1. Preparation of samples
<ol>
	<li>Preparation of water sample
	<ol>
		<li>To prepare the sample, fill a clean container with ultrapure water and filter it using a 0.22 &#181;m syringe filter. Make sure the output of the syringe is directed to a clean glass or quartz cuvette to take absorption spectroscopy measurements of the sample.</li>
		<li>To prepare the reference, fill a clean container with deuterated water and filter it using a 0.22 &#181;m syringe filter. Make sure the output of the syringe is directed to an identical clean glass or quartz cuvette as the sample cuvette to serve as the reference. 
		NOTE: The filtering step will minimize optical scattering from the sample by filtering out small particulates in the water, which have strong contributions in the visible region.</li>
	</ol>
	</li>
	<li>Preparation of melanin sample
	<ol>
		<li>Prepare a 1.7 mg/mL solution of melanin by adding powdered melanin to a centrifuge tube filled with 3.5 mL of dimethyl sulfoxide (DMSO). To begin, measurements should start at the wavelengths with the lowest expected absorption, where the highest concentration of melanin is required. Begin melanin measurements with approximately 1.7 mg/mL for the SWIR and dilute it with DMSO later to a 10x dilution (approximately 0.17 mg/mL) for the VIS/NIR regions.</li>
		<li>Sonicate the tube for 10 min to ensure the powdered melanin dissolves in the DMSO and transfer the solution to a clean glass or quartz cuvette for spectroscopy measurements.</li>
		<li>Stay within the linear absorbance range of the spectrometer and ensure high SNR measurements by changing the concentration and/or pathlength (e.g., by switching from a short pathlength (1 or 2 mm) to a 10 mm pathlength cuvette). Melanin absorption has a wide range spanning from VIS to SWIR. For the VIS, use the lowest concentration and/or pathlength cuvette of melanin due to its high molar extinction coefficient; for the SWIR, use the highest concentration of melanin and/or pathlength cuvette to obtain a qualitatively clean spectroscopy measurement.</li>
	</ol>
	</li>
	<li>Preparation of corn oil sample
	<ol>
		<li>Add corn oil to a clean glass or quartz cuvette for spectroscopy measurements.</li>
		<li>Use open air as the reference. No cuvette or solution will be used as the reference.</li>
	</ol>
	</li>
	<li>Preparation of oxygenated hemoglobin sample with whole heparinized human blood 
	NOTE: Whole heparinized human blood was purchased and was collected in FDA-licensed collection centers located in the United States.
	<ol>
		<li>To isolate hemoglobin from the red blood cells (RBCs) and remove as much water from the blood as possible, first pipette 1,800 &#181;L of whole heparinized human blood into microcentrifuge tubes and centrifuge at 9.6 &#215; g for 10 min. 
		NOTE: Approximately 4-6 microcentrifuge tubes are recommended</li>
		<li>After centrifugation, remove the tubes from the microcentrifuge, then remove and discard the supernatant (will be approximately 850 &#181;L) from the centrifuged blood sample without disturbing the pellet (Figure 2). The supernatant comprises the liquid (mainly plasma and platelets) above the red blood cell pellet. Look for the red blood cell pellet at the bottom of the microcentrifuge tube.</li>
		<li>Reconstitute each pellet with the same volume of deuterated water as the volume of supernatant that was discarded. Centrifuge again at 9.6 &#215; g for 10 min.</li>
		<li>Remove the tubes from the centrifuge, then discard the supernatant without disturbing the pellet.</li>
		<li>Reconstitute the pellet with the same volume of deuterated water as the volume of supernatant that was discarded. Re-centrifuge at 9.6 &#215; g for 10 min.</li>
		<li>Remove the tubes from the centrifuge, then remove the supernatant using a pipette and discard it.</li>
		<li>Lysing red blood cells (RBCs): Reconstitute the RBC pellets with a large volume (4.5 mL) of deuterated water, which will cause a hypotonic burst. If the pellet is sticking to the tube, rinse it with some of the measured 4.5 mL of deuterated water to loosen it. 
		NOTE: The volume of supernatant will increase after each centrifugation step. This volume of supernatant can range from 800 &#181;L to 1,600 &#181;L. The volume of deuterated water for lysing could change depending on the desired sample concentration but needs to be high enough to cause hypotonic burst.</li>
		<li>Remove the lysed blood solution from the tube with a needle and syringe; then, carefully remove the blunt-tip syringe needle and discard it in a biohazard sharps container. Connect the syringe to a 0.22 &#181;m syringe filter and push the syringe contents through the filter into a 10 mm pathlength, 3.5 mL glass or quartz cuvette. 
		NOTE: The filtering step will minimize optical scattering from the sample by filtering out the cellular organelles and membranes in the solution. If the sample is highly concentrated and the syringe filter becomes difficult to push, use multiple syringe filters.</li>
		<li>Secure the cuvette with an air-tight stopper over the cuvette to prevent water vapor from the air contaminating the sample. Parafilm the outside of the stopper to further prevent contamination and ensure an air-tight seal. 
		NOTE: For measurements from 600 nm to 1,600 nm, approximately 1,200-1,600 &#181;L of pellet dissolved in 4.5 mL of deuterated water will give qualitatively clean measurements. 
		NOTE: For spectral regions with high expected absorbance (400 nm to 600 nm), samples will need to be diluted using deuterated water to obtain non-saturated absorbance values and ensure absorbance values are within the linear range of the detector. In addition, the samples and the reference should be placed in short pathlength cuvettes (1 or 2 mm). For the spectra shown herein, samples were diluted 10x for 400-500 nm and 5x for 500-600 nm and measured in 2 mm pathlength cuvettes. 
		&#8203;The shorter pathlength (1 or 2 mm) will allow for less light absorption in highly absorbing wavelengths, helping ensure some photons transmit through to the detector and remain within the linear detection range.</li>
		<li>Obtain a clean cuvette with the same pathlength and volume as the sample cuvette containing the filtered blood solution and fill it with deuterated water for baseline and reference measurements. If changing cuvette pathlengths for different spectral ranges, record a new reference spectrum for each cuvette pathlength.</li>
	</ol>
	</li>
	<li>Preparation of deoxygenated hemoglobin sample with whole heparinized human blood
	<ol>
		<li>Follow steps 1.4.1-1.4.8.</li>
		<li>Add sodium dithionite to the blood solution in the cuvette and gently stir. Secure the cuvette with an air-tight stopper over the cuvette to prevent water vapor from the air contaminating the sample. Parafilm the outside of the stopper to further prevent contamination and ensure an air-tight seal. Carefully tilt the cuvette side to side to ensure that the sodium dithionite dissolves in the solution; the blood solution will change from a red color to a darker purple-red color. 
		NOTE: To fully deoxygenate hemoglobin, the mass of sodium dithionite that is required is approximately 0.007 g per mL of blood solution. According to Briley-S&#1237;b&#248; and Bj&#248;rnerud, at least 2.5 mg of sodium dithionite/g of blood is needed to fully deoxygenate whole blood18. However, this concentration was found to be insufficient in the studies here. A concentration of approximately 3.4 mg/g of whole blood was found to completely deoxygenate the blood solution without the introduction of optical scattering. For measurements from 400 to 600 nm, the blood solution will need to be diluted with deuterated water. Calculate the required sodium dithionite mass based on the volume of bood solution used and not the volume after dilution. For measurements from 600 nm to 1,600 nm, approximately 1,200-1,600 &#181;L of pellet dissolved in 4.5 mL of deuterated water will give high SNR measurements. 
		A shorter pathlength cuvette (1 or 2 mm) will allow for less light absorption in highly absorbing wavelengths, helping ensure some photons transmit through to the detector and remain within the linear detection range.</li>
		<li>Fill another cuvette with matching pathlength as the cuvette with filtered blood solution with deuterated water for baseline and reference measurements. If changing cuvette pathlengths for different spectral ranges, record a new reference spectrum for each cuvette pathlength.</li>
	</ol>
	</li>
</ol>
2. Obtaining high-quality absorption measurements
<ol>
	<li>Prepare samples and reference as described in section 1 and place them in glass or quartz cuvettes for VIS NIR-SWIR measurements. Turn on the spectrometer lamp and detectors and allow the system to warm up for at least 5-20 min, depending on the spectrometer system and light source. 
	NOTE: Five minutes provided sufficient detector cooling and lamp output stability for the spectra collected herein, but this should be characterized for each system before the capture of absorbance measurements. 
	Keep the sample and reference handy during this period of collection. Make sure the lids on cuvettes are secure. Keep measurement space as dark as possible to prevent distortion of the absorption measurements by ambient light.</li>
	<li>Repeat the following steps for different regions of the spectrum due to wide variance in absorption across the VIS-SWIR.
	<ol>
		<li>If using a dual-beam configuration (two separate detectors for sample and reference), make sure the system is baselined. Acquire the baseline by placing identical reference samples containing the solvent in front of each detector and collect an absorbance measurement using the exact acquisition parameters to be used for the sample absorbance spectrum collection. While the detectors should theoretically show zero absorption, it is usually not perfect. Subtract the resulting absorbance spectrum from all sample absorbance spectra that are acquired using the same settings. 
		NOTE: The baseline should be repeated any time the acquisition settings for data collection are changed.</li>
		<li>Place the sample in the spectrometer and choose the detector that is relevant to the region to be measured (VIS-NIR = silicon or UV-VIS PMT, SWIR = InGaAs) and view absorbance in Live Mode, in which the measurement continuously updates as measurement parameters are tuned.</li>
		<li>Starting with default spectrometer parameters, adjust incident light power and exposure time until a qualitatively clean absorption spectrum without saturation is obtained.
		<ol>
			<li>If tuning the parameters does not result in qualitatively clean spectra, modify parameters such as incident light power, exposure time, pathlength, and sample concentration. First, try adjusting power and exposure time. If tuning these parameters does not result in a high SNR spectrum, change the sample concentration or the pathlength of the sample and the reference. 
			NOTE: Default parameters used in the collection of the spectra shown herein were incident light power of 15 nW at 400 nm and 20 nW at 1,000 nm, exposure time of 30 ms, cuvette pathlength of 10 mm, and sample specific concentrations: 100% water, melanin 1.7 mg/mL, 100% corn oil, oxygenated hemoglobin (15.9 g/dL), and deoxygenated hemoglobin (15.9 g/dL).</li>
			<li>To increase the SNR, increase the number of data reads averaged per data point. The higher the number of data reads averaged, the longer the time per measurement.</li>
			<li>To increase spectral resolution, reduce the step size (nm) of the measurement down to the spectrometer&#39;s resolution limit. This will increase the time per measurement. Additionally, reduce the incident light power bandwidth to 1/10th of the narrowest peak&#39;s full width at half maximum19.</li>
			<li>Once the measurement settings are optimized for the sample, document the settings for each spectrum acquired.</li>
			<li>To obtain a spectrum, use the optimized settings to take a measurement of the reference only. Next, replace the reference cuvette with the sample cuvette and take another measurement. Make sure to save both the reference and sample measurements separately. 
			NOTE: It is imperative that the reference measurement does not saturate. If it does, reduce the incident light power and/or exposure time until there is no saturation. Keep all system parameters the same between the reference and sample measurements; if one needs to be changed, the other will also need to be repeated to match it.</li>
			<li>For each paired set of reference and sample measurements, calculate the absorbance A (Eq 1) by treating the reference measurement as Io and the sample measurement as I.</li>
			<li>To stitch together different regions that required differences in sample concentration or pathlength, apply a multiplication factor to one of the spectral regions. For example, for a first region that had 1/10 of the concentration and identical pathlength as a second region, multiply the first spectral region by 10 to appropriately scale the two regions together. 
			NOTE: The multiplication factor should be the difference in concentration and/or pathlength between the regions.</li>
			<li>Once the spectra have been corrected for differences in pathlength or concentration, choose an overlapping wavelength in the two spectral regions for stitching the two regions together.</li>
			<li>Truncate the two spectra to end at the overlap wavelength; choose one spectrum to include its transition spectral value. Concatenate the two truncated spectra to create a single resulting spectrum of a wider wavelength range.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>

Optimizing VIS-SWIR Spectroscopy for Biological Molecules

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L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+properties+of+biological+tissues:+a+review.">Optical properties of biological tissues: a review.</a> Phys Med Biol. 58 (11), R37 (2013).</li><li>. Optical absorption of hemoglobin Available from: <a target="_blank" href="https://omlc.org/spectra/hemoglobin/">https://omlc.org/spectra/hemoglobin/</a> (1999)</li><li>Zhang, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Penetration+depth+of+photons+in+biological+tissues+from+hyperspectral+imaging+in+shortwave+infrared+in+transmission+and+reflection+geometries.">Penetration depth of photons in biological tissues from hyperspectral imaging in shortwave infrared in transmission and reflection geometries.</a> J Biomed Opt. 21 (12), 126006 (2016).</li><li>Du, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hyperspectral+imaging+and+characterization+of+allergic+contact+dermatitis+in+the+short-wave+infrared.">Hyperspectral imaging and characterization of allergic contact dermatitis in the short-wave infrared.</a> J Biophotonics. 13 (9), e202000040 (2020).</li><li>Roggan, A., Friebel, M., D&#246;rschel, K., Hahn, A., Mueller, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+properties+of+circulating+human+blood+in+the+wavelength+range+400-2500+nm.">Optical properties of circulating human blood in the wavelength range 400-2500 nm.</a> J Biomed Opt. 4 (1), 36-46 (1999).</li><li>Kuenstner, J. T., Norris, K. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spectrophotometry+of+human+hemoglobin+in+the+near+infrared+region+from+1000+to+2500+nm.">Spectrophotometry of human hemoglobin in the near infrared region from 1000 to 2500 nm.</a> J Near Infrared Spectrosc. 2 (2), 59-65 (1994).</li><li>Spectral subtraction. Available from: <a target="_blank" href="https://www.spectroscopyonline.com/view/spectral-subtraction">https://www.spectroscopyonline.com/view/spectral-subtraction</a> (2021)</li><li>Sjoding, M. W., Dickson, R. P., Iwashyna, T. J., Gay, S. E., Valley, T. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Racial+bias+in+pulse+oximetry+measurement.">Racial bias in pulse oximetry measurement.</a> N Engl J Med. 383 (25), 2477-2478 (2020).</li><li>Fawzy, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Racial+and+ethnic+discrepancy+in+pulse+oximetry+and+delayed+identification+of+treatment+eligibility+among+patients+with+COVID-19.">Racial and ethnic discrepancy in pulse oximetry and delayed identification of treatment eligibility among patients with COVID-19.</a> JAMA Intern Med. 182 (7), 730-738 (2022).</li><li>Sudat, S. E. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Racial+disparities+in+pulse+oximeter+device+inaccuracy+and+estimated+clinical+impact+on+COVID-19+treatment+course.">Racial disparities in pulse oximeter device inaccuracy and estimated clinical impact on COVID-19 treatment course.</a> Am J Epidemiol. 192 (5), 703-713 (2023).</li><li>Keller, M. D., Harrison-Smith, B., Patil, C., Arefin, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Skin+colour+affects+the+accuracy+of+medical+oxygen+sensors.">Skin colour affects the accuracy of medical oxygen sensors.</a> Nature. 610 (7932), 449-451 (2022).</li><li>Butler, W. 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Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Shortwave-infrared+(SWIR)+emitters+for+biological+imaging:+a+review+of+challenges+and+opportunities.">Shortwave-infrared (SWIR) emitters for biological imaging: a review of challenges and opportunities.</a> Nanophotonics. 6 (5), 1043-1054 (2017).</li><li>Briely-Sabo, K., Bjornerud, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Accurate+de-oxygenation+of+ex-vivo+whole+blood+using+sodium+dithionite.">Accurate de-oxygenation of ex-vivo whole blood using sodium dithionite.</a> Proc Intl Sot Mag Reson Med. 8, 2025 (2020).</li><li>Skoog, D. A., Holler, F. J., Crouch, S. 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Action spectra and quantum yields for eumelanin and synthetic melanin.</a> Photochem Photobiol. 39 (1), 69-74 (1984).</li><li>Sarna, T., Swartz, H. A., Nordlund, J. 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Christine O'Brien and Leonid Shmuylovich have a financial ownership interest in Armor Medical Inc. and may benefit financially if the company is successful in marketing its products that are related to this research.

The protocol(s) outlined in this article can be used to obtain VIS-SWIR absorption spectra of biological absorbers and contrast agents. Herein, we show specific steps to obtain spectra of water, melanin, corn oil, oxygenated hemoglobin, and deoxygenated hemoglobin spanning from 400 nm to 1,600 nm. The following discussion covers critical steps, tips for measurement optimization, limitations of the method, and applications of this protocol within biomedicine.
While all the protocol steps are important, a few critical steps must be followed to obtain accurate spectra with this protocol. First, the sample must be non-scattering; for example, when obtaining oxygenated and deoxygenated hemoglobin from blood, the scatterers in the sample must be filtered out (here, a 0.22 &#181;m syringe filter was used). When preparing deoxygenated hemoglobin, the amount of sodium dithionite added should not exceed the weight provided, as it will turn the sample turbid at higher weight percentages and invalidate the Beer-Lambert law requirements.
Second, deuterated water must be used as the sample solvent and reference for water-soluble solutes such as hemoglobin. In the SWIR, water absorption is strong and can mask other absorbers. Third, measurements must be acquired within the linear range of the detector, defined as the region in which a change in photons reaching the detector results in a linear change in detector signal. If minimal absorption occurs, the detector may register an absorbance of zero, even if there is absorbance occurring. This occurs when the sample is too dilute, the exposure time is too low, and/or the cuvette pathlength too short, but can be remedied by an increase in sample concentration, cuvette pathlength, and/or exposure time. Similarly, if high absorption occurs, the detector may not register any photons and yield a flat line termed &#34;saturation&#34; that is not sensitive to changes in absorbance. This occurs when the sample is too concentrated, the exposure time is too low, and/or the pathlength is too long, but can be fixed by increasing sample concentration, increasing exposure time, and/or decreasing cuvette pathlength.
Fourth, the detectors used must be linear. If uncertain whether the detector shows a linear response to the number of photons reaching the detector, various dilutions of a sample can be measured with the same acquisition settings to check whether their absorbance values show the expected linear trend between absorbance and concentration. Alternatively, a single sample can be measured and the exposure time increased to test whether there is a linear increase in detector intensity. Both tests should yield a linear relationship if the user is operating within its linear limits (see the third critical point above).
Fifth, to stay within the linear range of the detector for the entire VIS-SWIR spectral range, the sample concentration, detector exposure time, and/or cuvette pathlength will need to be changed for different spectral regions with significantly higher or lower absorbance from the starting spectral region.
Sixth, spectra acquired using different sample concentrations or pathlengths for different spectral regions must be stitched together by linear scaling of one region based on known changes in concentration or pathlength. After scaling, the spectra should be stitched together at an overlapping wavelength between the two regions. If a measurement will span measurements from different detectors, there should be overlapping absorbance spectra that contain the desired detector transition wavelength (800 nm is commonly used when stitching UV-VIS and SWIR regions). Each spectrum is truncated at the detector transition wavelength, and one spectrum is chosen to include its original detector transition absorbance value. Then, the two truncated spectra are concatenated to create a single elongated spectrum.
Additional tips for acquiring high-quality absorption spectra include using a previously published spectrum of the sample of interest, even if it does not cover the full spectral range to be measured, to guide the measurements. Knowing a priori the wavelength range with the lowest absorbance, measurements can be started in this region with the highest concentration sample and then adjusted and diluted from there. It can help inform how many separate regions will need to be measured based on the magnitude of absorbance changes expected. It can also provide confidence that the expected spectrum has been obtained if it matches published spectra, or if it does not match published spectra, the sample preparation method can be re-examined along with issues that could be distorting the spectrum.
Absorption spectra may require postprocessing. For example, a digital moving average filter can be used to smooth absorption measurements and points of known error can be removed with a data set repair in which the erroneous points are deleted, and interpolation is performed between non-erroneous points.
Limitations of this method include inaccuracy for samples with optical scattering such as whole blood. To accurately measure hemoglobin, the blood sample is lysed and then syringe-filtered to remove most other residual cellular components that cause optical scattering. Similarly, this method is not used in vivo due to tissue scattering. While Beer-Lambert Law-based absorbance measurements may be tedious and sometimes difficult to perform, it is the gold standard method for pure optical absorption measurements. In addition, some solvents make it particularly challenging to acquire a pure absorbance spectrum of certain solutes. For example, oxygenated and deoxygenated hemoglobin&#39;s native solvent is water, but water&#39;s absorption is strong in the SWIR, dominating the oxygenated hemoglobin absorption. Therefore, we attempt to remove water from the blood via repeated centrifugation, removal of the supernatant, and re-constitution with deuterated water which has weaker SWIR absorption. The resulting oxygenated and deoxygenated hemoglobin spectra from whole heparinized human blood is suspected to contain small residual absorption due to water absorption from approximately 1,300 nm to 1,600 nm as seen in Figure 3. While likely not a perfect disentanglement of water from oxygenated and deoxygenated hemoglobin, we observe close alignment with highly cited published oxygenated and deoxygenated hemoglobin spectra from 400 nm to 1,000 nm and we extend the spectrum by several hundred nanometers2,3.
The protocols described herein will help researchers expand absorption spectra libraries to include VIS-SWIR spectra of a host of biological components and contrast agents. This knowledge can be used to improve understanding of basic biology and physiology through non-invasive biomedical imaging and sensing at deeper wavelengths than most systems currently operate. The addition of SWIR wavelengths broadens the type of tissue components that can be monitored non-invasively and could inform the development of novel monitoring and diagnostics. Furthermore, devices operating in the NIR and SWIR should help minimize skin pigmentation bias that has been reported for optical devices operating in the VIS and NIR ranges and is a promising strategy for creating equitable medical devices.

Most biological absorbers have been optically characterized in the visible (VIS, ~400-700 nm) and near-infrared (NIR, ~700-1,000 nm) spectral ranges but not the short-wave infrared (SWIR, ~1,000-3,000 nm)1,2,3,4 (Figure 1). This is despite the SWIR offering deeper light penetration due to lower tissue scattering and reduced melanin absorption as well as additional label-free biological contrast for water, lipid, and protein1,5,6. The lack of absorption spectra spanning the VIS-SWIR has created a gap in knowledge that prevents the development of biomedical optical devices that take advantage of these SWIR benefits. While several attempts to characterize the spectra have been presented in the past, high-quality VIS-SWIR absorption spectra of pure components without significant solvent artifacts are lacking1,7,8. For example, water-soluble substances such as hemoglobin have been characterized in the SWIR using water as a solvent and subtracting pure water absorption from the resultant spectra9. However, the accuracy of this approach is unclear because water is a dominant absorber in this region.
Working in the SWIR has the additional advantage of reducing or possibly removing bias caused by strong melanin absorption in the VIS when using in vivo optical diagnostics. Numerous studies have found that pulse oximeters overestimate oxygenation in Black patients, and as such Black patients are more likely to have dangerously low oxygen levels that pulse oximeters did not detect10. This skin pigmentation bias has resulted in delayed and inadequate medical treatment of many Black hypoxic patients11,12 and prompted a call to action in the optics and engineering communities to improve the accuracy of optical devices for all skin tones13. Moving from the high melanin-absorbing VIS wavelengths to the NIR and SWIR is one exciting technical innovation that could reduce racial disparities in critically important biomedical optics technologies.
Recording ground-truth absorption spectra of biomolecules is essential for understanding the optical properties of tissue samples that are used in a variety of engineering and biomedical applications14. Absorption spectroscopy measures molecule-specific light absorption as a function of wavelength. The gold standard method of acquiring absorption measurements in solution is to direct collimated, monochromatic light through a dissolved sample and record the transmitted light with a detector placed on the opposite side of the sample from the light source. The absorption (A) of the sample is then defined by Eq. (1).
A = log10(I0/I)&#160; &#160; &#160;(1)
Where Io is the incident light intensity on the sample, and I is the intensity of light transmitted through the sample that hits the detector. Io is acquired by measuring a reference, which is typically the solvent of the solute of interest, using the same acquisition settings as I. Absorption spectroscopy provides quantitative absorption properties and is governed by the Beer-Lambert Law (Eq. 2), which does not apply to samples that have optical scattering. This equation states that optical absorption is determined by a sample&#39;s molar absorptivity coefficient (&#949;, units L&#183;mol&#8722;1&#183;cm&#8722;1) at wavelength &#955; multiplied by its molar concentration (C, units mol&#8729;L-1) and the pathlength (L, units cm), which is defined as the distance traveled through the sample15:
A(&#955;) = &#949;(&#955;)*L*C&#160; &#160; &#160;(2)
Importantly, the molar absorptivity coefficient is unique to each biological absorber and indicates how strongly a sample absorbs light at a particular wavelength16.
When taking absorption spectroscopy measurements spanning from the VIS to the SWIR, adjustments to hardware, sample preparation, and spectral postprocessing are required beyond standard VIS-NIR spectra:
Hardware 
VIS-NIR measurements typically use a silicon detector or photomultiplier tubes (PMT) with photocathodes sensitive to UV-VIS photons, while SWIR absorbance requires a photodetector that is sensitive to SWIR photons, such as an Indium Gallium Arsenide (InGaAs) detector17. VIS, NIR, and SWIR systems also typically require wavelength-specific dispersion elements, such as optical gratings, for light dispersion in these optical ranges. In addition, SWIR absorption requires glass or quartz cuvettes, which have higher transmission than plastic cuvettes in the SWIR, making them more suitable for VIS-SWIR spectra. A light source or sources with a broad and relatively uniform emission covering the VIS-SWIR, like a tungsten-halogen lamp (used in this protocol), should be selected. Adjusting the incident light power, the exposure time, or the duration for which the photodetector is exposed to light can help optimize absorbance measurements.
Sample preparation 
The sample&#39;s concentration or cuvette&#39;s pathlength will linearly alter the amount of absorption that occurs as described above in Eq (2), and thus, either can be changed to achieve a high signal-to-noise ratio (SNR, herein calculated as the ratio of the peak amplitude divided by the standard deviation of a detrended region of the spectrum, typically SNR &#62; 30 is considered high quality) measurements in different sections of the spectrum with relatively high or low absorbance. To optimize SWIR measurements in an aqueous solution, deuterium oxide (D2O), also referred to as heavy water or deuterated water, can be used instead of water (H2O), because it has significantly lower absorption peaks in the SWIR1. This allows for the characterization of the solute without interference of the dominant water absorption in the SWIR.
Spectral postprocessing 
Postprocessing is required because VIS-SWIR spectra require two detectors, one for the visible-NIR (typically silicon or UV-VIS-sensitive PMT) and one for the SWIR (typically InGaAs), and the spectra must be stitched together to achieve a full VIS-SWIR spectrum. When spectra span the VIS-SWIR, the absorption can vary significantly depending on the wavelength region, and the sample may need to be diluted or the pathlength decreased in some areas to prevent saturation. Spectra must be linearly scaled to account for changes in sample concentration or pathlength and then stitched together to create a single spectrum that includes two wavelength regions.
The following protocols will detail the preparation and spectroscopic measurement from the VIS to the SWIR of five example biological absorbers: water (comprises ~60% of body weight), melanin (primary absorber for skin pigmentation), corn oil (used as an analog for human lipids because it has low variability between batches, close chemical composition to human and animal lipids, and minimal scattering), and oxygenated and deoxygenated hemoglobin (the primary absorbers in blood).

<table><tbody><tr><td>0.22 &amp;micro;m diameter syringe Filters</td><td>Sigma-Aldrich</td><td>SLGPM33RS</td><td></td></tr><tr><td>1000 &amp;micro;L Pipette</td><td>Millipore Sigma</td><td>EP3124000121</td><td></td></tr><tr><td>200 &amp;micro;L Pipette</td><td>Millipore Sigma</td><td>EP3124000083</td><td></td></tr><tr><td>Conical Tubes</td><td>Avantor VWR</td><td>21008-089</td><td></td></tr><tr><td>Corn Oil</td><td>Happy Belly</td><td>n/a</td><td>48 Fl Oz</td></tr><tr><td>Deuterium Oxide</td><td>Cambridge Isotope Laboratories</td><td>DLM-4-100</td><td></td></tr><tr><td>Deuterium Oxide</td><td>Sigma-Aldrich</td><td>7789-20-0</td><td></td></tr><tr><td>Dimethyl Sulfoxide (DMSO)</td><td>Sigma-Aldrich</td><td>D5879-1L</td><td></td></tr><tr><td>Disposable 1 mL Syringe</td><td>BH Supplies</td><td>BH1LL</td><td></td></tr><tr><td>Disposable Needle</td><td>Atshuhut/Amazon</td><td>66941</td><td></td></tr><tr><td>Freezer (-80 &amp;deg;C)</td><td>Fisher Scientific</td><td>IUE386FARK</td><td></td></tr><tr><td>Glass Beakers</td><td>Millipore Sigma</td><td>CLS1000PACK</td><td></td></tr><tr><td>Glass or Quartz Cuvettes</td><td>Sigma-Aldrich</td><td>Z802875-1EA</td><td>700 &amp;micro;L volume</td></tr><tr><td>Glass or Quartz Cuvettes</td><td>Thorlabs</td><td>CV10Q35EP</td><td>3500 mL volume</td></tr><tr><td>Heparinized Human Blood</td><td>Lampire</td><td>7203710</td><td></td></tr><tr><td>Microcentrifuge Tubes</td><td>Costar</td><td>3213</td><td></td></tr><tr><td>Microcentrifuge--accuSpin Micro 17</td><td>Fisher Scientific</td><td>13-100-675</td><td></td></tr><tr><td>MilliQ Water</td><td>Millipore Sigma</td><td>ZMQSP0D01</td><td></td></tr><tr><td>OSRAM FCS 64640 150 W 24 V HLX Halogen Light Bulb</td><td>Amazon</td><td>B0001221DG</td><td></td></tr><tr><td>Parafilm</td><td>Millipore Sigma</td><td>HS234526B</td><td></td></tr><tr><td>Pipette Tips</td><td>Eppendorf</td><td>22492055</td><td></td></tr><tr><td>Powdered Synthetic Melanin</td><td>Sigma-Aldrich</td><td>8049-97-6</td><td></td></tr><tr><td>Scale</td><td>Sartorius</td><td>UX-11976-09</td><td></td></tr><tr><td>Sodium Dithionite</td><td>Sigma-Aldrich</td><td>1065070500</td><td></td></tr><tr><td>Sonicator</td><td>Fisher Scientific</td><td>CPX1800</td><td></td></tr><tr><td>Spatula&amp;nbsp;</td><td>Aozita</td><td>000 00 0 1 2 3</td><td></td></tr><tr><td>UV/VIS/SWIR Spectrophotometer</td><td>On Line Instrument Systems</td><td>Olis Cary 14</td><td></td></tr><tr><td>Weigh Boats</td><td>Amazon</td><td>&amp;nbsp;B07M5RMNPF</td><td></td></tr></tbody></table>

characterization of biological absorption spectra spanning the visible to the short-wave infrared

For noninvasive light-based physiological monitoring, optimal wavelengths of individual tissue components can be identified using absorption spectroscopy. However, because of the lack of sensitivity of hardware at longer wavelengths, absorption spectroscopy has typically been applied for wavelengths in the visible (VIS) and near-infrared (NIR) range from 400 to 1,000 nm. Hardware advancements in the short-wave infrared (SWIR) range have enabled investigators to explore wavelengths in the ~1,000 nm to 3,000 nm range in which fall characteristic absorption peaks for lipid, protein, and water. These molecules are difficult to visualize in the VIS-NIR and can provide label-free sources of biological contrast. Furthermore, lower SWIR absorption has been observed for melanin, the primary chromophore responsible for skin pigmentation. In vivo&#160;optical devices like clinically standard pulse oximeters have been found to have reduced accuracy in people with darkly pigmented skin, possibly because of the stronger melanin absorption in the VIS range. Thus, error associated with skin pigmentation could be reduced by using devices operating in the SWIR. Optical instrument design is facilitated by the understanding of the absorption properties of core tissue components from the VIS to the SWIR range. This article describes protocols and instrumentation for obtaining VIS-SWIR absorption spectra of common tissue absorbers: oxygenated hemoglobin, deoxygenated hemoglobin, melanin, water, and lipid.

Data are available via the WUSTL Digital Research Materials Repository (DRMR) doi:&#160;<a href="https://doi-org.remotexs.ntu.edu.sg/10.7936/6RXS-108249">https://doi-org.remotexs.ntu.edu.sg/10.7936/6RXS-108249 </a>
Spectral characterization of water 
Water, one of the most common biological absorbers, has characteristic, highly absorbing peaks in the NIR and SWIR. These characteristic peaks are so strong that when measuring the absorbance of water, it must often be diluted with deuterated water to prevent saturation. The difference in absorbance between water and deuterated water is shown in Figure 3, highlighting more than an order of magnitude increase in water absorbance over deuterated water. Strong water absorption in the SWIR also makes it difficult to obtain the absorption spectra of other biological absorbers that contain water in the sample or that use water as the solvent and reference. As a solution to this problem, deuterated water can be used instead of water. Deuterated water acts in a similar manner to water while being a much weaker SWIR absorber, making it an ideal replacement for water in SWIR spectroscopy measurements.
To measure the absorption spectrum of water, water was used as the sample and deuterated water as the reference. To prevent saturation in the SWIR, the sample was diluted with deuterated water in the SWIR region. The resulting water absorption spectrum shows low absorption in the VIS, which increases at longer wavelengths. Characteristic absorption peaks can be seen at approximately 750 nm, 970 nm, 1,200 nm, and 1,450 nm (Figure 4). The resulting water spectrum has an SNR of 3536.6 and was compared to Hale and Querry&#39;s water absorption spectrum20, showing a similar absorption curve from ~500 nm to 1,600 nm. However, from 400 to 500 nm, the absorption curves differ, with Hale and Querry showing a higher absorbance value at lower wavelengths. This difference could be due to residual small particles in the water that will have higher scattering at shorter wavelengths compared to longer wavelengths3 and may invalidate the assumptions of no scattering in Beer-Lambert law-based absorption measurements.
Spectral characterization of melanin 
Melanin, the skin&#39;s primary optical absorber, was spectrally characterized using powdered melanin as the sample and DMSO as the solvent and reference. The resulting spectrum has a SNR of 172 and was compared to eumelanin data from Sarna et al.21,22 and Crippa et al.23 (Figure 5). Eumelanin is the primary pigment responsible for the color of skin and, therefore, provides the most accurate data for how melanin will impact optical devices. The spectrum collected&#160;using the protocol described herein shows high correlation to these literature values.
Spectral characterization of corn oil 
To investigate the absorption spectrum of a lipid, corn oil was chosen due to its minimal optical scattering when compared to other lipids and its known similarity in chemical composition to animal fats1. To take the absorption spectrum of corn oil, corn oil was used as the sample with air as the reference. As seen in Figure 6, the corn oil spectrum contains characteristic peaks at approximately 930 nm, 1,210 nm, and 1,410 nm. The resulting corn oil spectrum has a SNR of 10363.1 and was overlaid with Cao et al.&#39;s corn oil spectrum1, showing a strong correlation.
Spectral characterization of oxygenated and deoxygenated hemoglobin 
While the characterization of the absorption of oxygenated and deoxygenated hemoglobin is one of the most important feats in biomedical optics, their absorption spectra spanning the VIS to the SWIR remains poorly understood. One of the most challenging aspects of determining the absorption spectrum of oxygenated and deoxygenated hemoglobin in the SWIR is removing the water in blood, which overwhelms the relatively small hemoglobin absorption. To disentangle the absorbance of hemoglobin and water, hemoglobin was first isolated from human whole blood using centrifugation and the supernatant was removed and replaced with deuterated water. Deuterated water was also used as the reference and solvent that hemoglobin was reconstituted in to cause hypotonic burst. As shown in Figure 3, deuterated water has a much lower optical absorption than water, making it an ideal solvent and reference for SWIR absorption measurements. The resulting oxygenated hemoglobin spectrum spans the VIS to the SWIR with a SNR of 23118.7 and matched very closely Prahl&#39;s published oxygenated hemoglobin spectrum4&#160;(Figure 7). In the VIS, the oxygenated hemoglobin spectrum shows high absorption with characteristic absorption peaks at approximately 415 nm and a doublet peak from approximately 540 to 575 nm. In the NIR, the oxygenated hemoglobin spectrum shows a characteristic peak spanning a wider range of wavelengths, approximately 800 nm to 1,100nm. In the SWIR, the absorption of hemoglobin is low. However, from approximately 1,400 nm to 1,600 nm, the hemoglobin spectrum shows higher absorption with peaks, likely due to water in hemoglobin that was unable to be fully removed during the centrifugation process.
For deoxygenated hemoglobin, sodium dithionite was added to the hemoglobin solution to introduce dissociation of dioxygen from oxygenated hemoglobin and converting the sample to deoxygenated hemoglobin. The resulting spectrum has an SNR of 9813.9 and was compared against Prahl&#39;s deoxygenated hemoglobin spectrum and shows a very strong correlation between 400 nm and 1,000 nm4&#160;(Figure 8). The resulting deoxygenated hemoglobin spectrum shows characteristic absorption peaks at approximately 430 nm, 560 nm, and 760 nm. After this, the absorption of deoxygenated hemoglobin drops off but contains smaller absorption peaks around 1,200 nm and from 1,400 nm to 1,600 nm. The absorbance between 1,400 and 1,600 nm is likely due to water that was not fully removed from the hemoglobin sample during the centrifugation process.
Comparison of biological absorbers from the VIS-SWIR 
The full VIS-SWIR spectra from all the biological absorbers characterized in this protocol are shown in Figure 9, scaled based on their biological concentration in tissue2, which highlight the distinct spectral differences between biological absorbers. For instance, melanin, oxygenated hemoglobin, and deoxygenated hemoglobin have strong, characteristic absorption in the VIS that generally decreases with wavelength, whereas water and lipid show characteristic peaks in the NIR and SWIR. In biological tissue, the combination of decreases in melanin and hemoglobin as well as decreases in optical scattering provide increased optical penetration depth in the NIR and SWIR. Additionally, the NIR and SWIR&#39;s reduced melanin absorption enable optical investigation of oxygenated and deoxygenated hemoglobin, lipid, and water with minimal effects due to skin pigmentation.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/67403/67403fig01.jpg" /> 
Figure 1: Previously published spectra of common biological absorbers across the UV to the SWIR. The oxygenated and deoxygenated hemoglobin spectra were plotted using data from Prahl4. The melanin spectrum was plotted using data from Sarna21 and Crippa23. The lipid spectrum was plotted using data from van Veen24. The water spectrum was plotted using data from Hale and Querry20.&#160;Abbreviation: SWIR = short-wave infrared. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/67403/67403fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/67403/67403fig02.jpg" /> 
Figure 2: Photo of the supernatant and red blood cell pellet from centrifuged whole blood. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/67403/67403fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/67403/67403fig03.jpg" /> 
Figure 3: Absorbance spectrum comparison of water and deuterated water collected with identical acquisition settings in log (left) and linear (right) absorbance scale. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/67403/67403fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/67403/67403fig04.jpg" /> 
Figure 4: Comparison of water absorbance spectrum acquired using methods described herein versus Hale and Querry20. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/67403/67403fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/67403/67403fig05.jpg" /> 
Figure 5: Comparison of melanin absorbance spectrum acquired using methods described herein versus Sarna et al.21,22 and Crippa et al.23. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/67403/67403fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/67403/67403fig06.jpg" /> 
Figure 6: Comparison of corn oil absorbance spectrum acquired using methods described herein versus Cao et al.1. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/67403/67403fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/67403/67403fig07.jpg" /> 
Figure 7: Comparison of oxygenated hemoglobin absorbance spectrum acquired using methods described herein versus Prahl4. Spectral features from 1,400 to 1,600 nm likely attributed to residual water in the sample. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/67403/67403fig07large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 8" class="xfigimg" src="/files/ftp_upload/67403/67403fig08.jpg" /> 
Figure 8: Comparison of deoxygenated hemoglobin absorbance spectrum acquired using methods described herein versus Prahl4. Spectral features from 1,400 to 1,600 nm likely attributed to residual water in the sample. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/67403/67403fig08large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 9" class="xfigimg" src="/files/ftp_upload/67403/67403fig9v3.jpg" /> 
Figure 9: VIS-SWIR spectra of common biological absorbers. Abbreviation: VIS-SWIR = visible-short-wave infrared. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/67403/67403fig9v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Characterization of Biological Absorption Spectra Spanning the Visible to the Short-Wave Infrared

Biological samples have distinct optical characteristics in the short-wave infrared (SWIR) compared to the visible (VIS) and near-infrared (NIR) wavelength ranges. However, recording pure SWIR absorption spectra is challenging and rarely conducted for biological molecules. This article presents a method to characterize the VIS-SWIR absorption spectra of biological absorbers.

For noninvasive light-based physiological monitoring, optimal wavelengths of individual tissue components can be identified using absorption spectroscopy. ...

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872 Views.  Washington University in St. Louis. Biological samples have distinct optical characteristics in the short-wave infrared (SWIR) compared to the visible (VIS) and near-infrared (NIR) wavelength ranges. However, recording pure SWIR absorption spectra is challenging and rarely conducted for biological molecules. This article presents a method to characterize the VIS-SWIR absorption spectra of biological absorbers.

Video: Characterization of Biological Absorption Spectra Spanning the Visible to the Short-Wave Infrared

Beer's Law

Absorbance and Fluorescence
When light hits a substance, it is either absorbed, transmitted, or reflected. Typically, a substance interacts with a range of wavelengths of light, each one interacting with the molecules or atoms differently. A substance may absorb a specific range of wavelengths, reflect another range of wavelengths, and transmit the other wavelengths of light.
When a molecule absorbs light, the energy is used in four different ways: (1) translation, which causes the molecule to change its molecular velocity; (2) vibration, which causes the distance between the molecules to change rapidly; (3) rotation, which causes the atoms to rotate around the bonds in a molecule; and (4) electron excitement, which causes electrons to transition to higher energy levels.
<h4>Energy Levels</h4>
In 1913, Niels Bohr proposed a model for the hydrogen atom in which electrons travel around the nucleus in fixed, circular orbits, called stationary states. The energy associated with each orbit, or stationary state, exists only at fixed, discrete energies. Only when an electron moves to another orbit is energy absorbed or emitted. The electron is never in-between states. This change only occurs if the absorbed or emitted energy equals the difference between the two energy states.
In Bohr&#8217;s model, the quantum number n represents the electron&#8217;s energy. When an electron occupies the lowest possible energy state, it is said to occupy the ground state, which is n = 1. When an electron absorbs a photon, whose energy equals the difference between the first and second states, the electron becomes excited and transitions from the ground state to the excited state, where n = 2. If the energy of the photon equals the difference between the first and third states, the electron transitions to the third state, or n = 3, and so on.
Electrons can spontaneously return to the ground state or any other lower, excited state. When this happens, the excess energy that was gained from excitation is released in the form of an emitted photon. The energy of the photon is equal to the difference between the two energy states and corresponds to different wavelengths of light.
<h4>Absorption and Emission Spectra</h4>
While most substances absorb or emit the maximum amount of light at one wavelength, they also tend to absorb or emit light at a range of wavelengths. This range of wavelengths is called a spectrum. The energy of the absorbed light is quantified and visualized using an absorption spectrum, while the energy of the emitted light is quantified and visualized using an emission spectrum.
Absorption and emission spectra are measured using a spectrophotometer, which is a device that transmits light through a sample and then measures both the wavelength and intensity of light that passes through it. Inside the spectrophotometer is either a diffraction grating or a prism, which separates incoming light into its component wavelengths. The different wavelengths are then transmitted through the sample, and the intensity is recorded on a linear charge-coupled device (CCD) detector. The CCD is an integrated circuit etched onto a silicon surface that forms light-sensitive elements called pixels. The CCD collects and sorts the diffracted light and reads it back at an absorption wavelength.
When measuring the absorbance of a sample, the solute is usually dissolved in a solvent and placed in a container known as a cuvette. Then, the sample is placed inside the spectrophotometer, and the intensity of the transmitted light is measured along with the wavelengths of light to obtain an absorbance spectra. As expected, the intensity of the transmitted light is lower than when there is no sample present inside the spectrophotometer.
This is because the transmitted light is absorbed by the sample, the cuvette, and the solvent. Before measuring samples, the spectrophotometer must be calibrated with a &#8216;blank&#8217;. A blank is a cuvette that only contains the solvent that is used to dissolve the solute. The spectrophotometer is calibrated so that the total absorbance due to the cuvette and solvent is subtracted from the sample&#8217;s measured absorbance. This allows us to record the absorbance that is only attributed to the species of interest.
The absorbance is often measured at one wavelength, the maximum absorbance wavelength. However, the absorption can also be measured at a range of wavelengths to acquire the absorption spectrum. For this, the sample is exposed to a range of wavelengths of incident light, and the absorption is recorded at each wavelength. If the sample emits light, the emission spectrum is measured similarly, except the incident wavelength is fixed at the wavelength of maximum absorbance. The instrument then measures the intensity of emitted light over a range of wavelengths.
<h4>Beer-Lambert Law</h4>
The absorbance of a sample at the wavelength of maximum absorbance provides information about the sample, namely its concentration. The Beer-Lambert Law is an equation that relates transmittance to sample concentration. The transmittance, or intensity of transmitted light, is the fraction of original light that passes through the sample, I, divided by the intensity of the incident light, I0.
<img src="https://www-jove-com.remotexs.ntu.edu.sg/CDNSource/lm/labs/49/49_Concepts_1.jpg" />
The Beer-Lambert Law states that the optical absorbance, A, of a species in solution is related to the negative log of the transmittance.
<img src="https://www-jove-com.remotexs.ntu.edu.sg/CDNSource/lm/labs/49/49_Concepts_2.jpg" />
An alternative version of the Beer-Lambert Law states that the optical absorbance, A, of a species in solution is linearly proportional to the concentration, c, of that species when the wavelength, &#955;, and pathlength, l, are held constant.
<img src="https://www-jove-com.remotexs.ntu.edu.sg/CDNSource/lm/labs/49/49_Concepts_3.jpg" />
The molar attenuation coefficient, &#949;, is a measure of how strongly a species absorbs light at a given wavelength. The greater the molar attenuation coefficient, the greater the absorbance. The pathlength, l, is the distance that the light travels through the sample, which is the width of the cuvette. Standard cuvettes have a pathlength of 1 cm.
This linear relationship between absorbance and concentration is a powerful tool that is used to determine the concentration of an unknown sample based on its absorbance. To do this, a standard curve is generated using a gradient of known concentrations of the solute. The absorbance at the peak absorbance wavelength, &#955;max, is measured for each concentration.
By plotting concentration versus absorbance, a linear relationship is observed that corresponds to the Beer-Lambert equation. The slope of this line is equal to the product of the pathlength and the molar attenuation coefficient. Using this calculated linear function, if the absorbance of the unknown sample is known, the concentration can easily be determined.
If the sample being analyzed is a reaction at equilibrium, Beer&#8217;s Law can be used to determine the equilibrium concentration of a product or reactant if the absorbance is measured at &#955;max specific to that product or reactant. Once the concentration is known, you can determine the equilibrium concentrations of the remaining reactants and products and then solve for the equilibrium constant Keq.
<h4>References</h4>
<ol>
	<li style="font-size:17px">Kotz, J.C., Treichel Jr, P.M., Townsend, J.R. (2012). Chemistry and Chemical Reactivity. Belmont, CA: Brooks/Cole, Cengage Learning.</li>
	<li style="font-size:17px">Silderberg, M.S. (2009). Chemistry: The Molecular Nature of Matter and Change. Boston, MA: McGraw Hill, Boston.</li>
	<li style="font-size:17px">Harris, D.C. (2015). Quantitative Chemical Analysis. New York, NY: W.H. Freeman and Company.</li>
</ol>

Beer-Lambert Law and Equilibrium Constant Determination - Concept

Absorbance and Fluorescence
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UV-Vis Spectroscopy of Dyes

Absorbance
When light interacts with a substance, a portion of the light is absorbed while the rest is either reflected or transmitted through it. Substances that we perceive as having a color reflect light in the visible range. The color of the substance that we are able to see depends on which wavelength of light is reflected. A substance that we perceive as blue reflects light in the blue range (430 &#8211; 480 nm) of the visible spectrum. According to the color wheel, the same substance absorbs light that is complementary to the reflected light. So, the blue substance absorbs light in the orange region (590 &#8211; 630 nm) of the visible spectrum. Not all compounds absorb in the visible region, and as a result, they appear as colorless to the human eye.
Light is defined by its energy, E, and its wavelength, &#955;. Here, h is Planck&#39;s constant, and c is the speed of light.
<img src="https://www-jove-com.remotexs.ntu.edu.sg/CDNSource/lm/labs/70/70_Concepts_1.jpg" />
The wavelength of light is inversely proportional to its energy. So, higher energy light has a shorter wavelength.
<h4>Dyes</h4>
Different colored dyes vary in the wavelength of light that they absorb. Most dyes are conjugated compounds with alternating double and single bonds and typically absorb light in the visible region.
The conjugated part of the dye molecule can be very short, meaning that there is a low degree of conjugation and few alternating double and single bonds, or long, meaning that there is a high degree of conjugation with many alternating double and single bonds. These alternating double bonds do not necessarily only have to be between two carbons. These conjugated bonds can include the carbonyl groups and the double bonds between carbon and oxygen. The degree of conjugation determines the wavelength of light the compound absorbs. For example, compounds with a high degree of conjugation absorb a longer wavelength than compounds with a lower degree of conjugation.
Based on molecular orbital theory, delocalized electrons occupy molecular orbitals. The highest occupied molecular orbital, or HOMO, is the highest energy orbital with an electron. The lowest unoccupied molecular orbital, or LUMO, is the lowest energy orbital with no electron. Molecules with little or no conjugation typically have a large energy gap between the HOMO and LUMO. However, conjugated molecules have a smaller energy gap between the HOMO and LUMO.
To excite an electron from a lower energy level to a higher energy level, or from the HOMO to LUMO, the molecule must absorb light with energy equal to the energy gap between the two orbitals. For this reason, molecules with a large energy gap require higher energy light, such as UV light, to excite an electron. Dyes, however, have a smaller energy gap and require lower energy light, such as visible light, to excite an electron.
<img src="https://www-jove-com.remotexs.ntu.edu.sg/CDNSource/lm/labs/70/70_Concepts_2.jpg" />
For this reason, molecules with a large energy gap require higher energy light, such as UV light, to excite an electron. Dyes, however, have a smaller energy gap and require lower energy light, such as visible light, to excite an electron.
Recall that the energy of light is inversely proportional to the wavelength. So, higher energy light has shorter wavelengths than lower energy light that has longer wavelengths.
<h4>Spectrophotometer</h4>
Experimentally, light absorbance is measured using a UV-Visible (UV-Vis) spectrophotometer. This instrument utilizes a light source that is transformed by a monochromator into specific wavelengths of light that will pass through a sample and into a detector at the other end. The samples must be in a liquid, so a solvent is required if the organic compound is a solid. This solution is held in a sample holder known as a cuvette. Depending on the sample, the cuvette may be made of quartz crystal, glass, or plastic, and has a particular pathlength. This pathlength is the distance the light must travel through the sample. Since the solvent will also absorb light, a sample blank of the solvent alone is required. Therefore, when the instrument captures the absorbance spectrum of the sample compound, it can subtract the background spectrum of the solvent to display absorbance caused by only the sample. Transmittance, T, is the fraction of the original light that passes through the sample.
<img src="https://www-jove-com.remotexs.ntu.edu.sg/CDNSource/lm/labs/70/70_Concepts_3.jpg" />
Here, P0 is the irradiance, or the energy per second per unit area, of the light beam prior to striking the sample. P is the irradiance of the light beam striking the detector. P is typically lower than P0, as some of the light is absorbed by the sample.
Absorbance, A, is defined as the negative log of transmittance.
<img src="https://www-jove-com.remotexs.ntu.edu.sg/CDNSource/lm/labs/70/70_Concepts_4.jpg" />
Absorbance has a value range between 0 (no absorption) and 2 (99% absorption). When no light is absorbed, P0 is equal to P, and the transmittance is equal to one. Thus, absorbance is zero. If 90% of the light is absorbed, then 10% is transmitted and T is equal to 0.1. This results in an absorbance equal to 1. If 99% of the light is absorbed, then 1% is transmitted (T= 0.01), and absorbance is equal to 2.
The spectrum obtained is a plot of the absorbance versus the wavelength. For a UV-Vis spectrophotometer, this range is between 200 and 800 nm.
<h4>Beer-Lambert Law</h4>
Transmittance and absorbance of a particular compound is related to the concentration of the compound in solution. This relationship is described by the Beer-Lambert law.
<img src="https://www-jove-com.remotexs.ntu.edu.sg/CDNSource/lm/labs/70/70_Concepts_5.jpg" />
The absorbance of the sample is equal to the product of the concentration of the compound, the path length, and the molar attenuation coefficient. This coefficient is unique to each compound and will vary by wavelength. However, if the wavelength is held constant, the molar attenuation coefficient will be the same irrespective of changes in concentration. The wavelength that corresponds to the highest absorbance of the sample, known as &#955;max, also will have the largest molar attenuation coefficient.
<h4>References</h4>
<ol>
	<li style="font-size:17px">Silberberg, M.S. (2012). Chemistry: The Molecular Nature of Matter and Change. Boston, MA: McGraw Hill.</li>
	<li style="font-size:17px">Harris, D.C. (2015). Quantitative Chemical Analysis. New York, NY: W.H. Freeman and Company.</li>
</ol>

Ultraviolet-Visible (UV-Vis) Spectroscopy of Dyes - Concept

Absorbance
When light interacts with a substance, a portion of the light is absorbed while the rest is either reflected or transmitted through it. ...

Improving Infrared Spectroscopy Characterization of Soil Organic Matter with Spectral Subtractions

Soil organic matter (SOM) underlies numerous soil processes and functions. Fourier transform infrared (FTIR) spectroscopy detects infrared-active organic bonds that constitute the organic component of soils. However, the relatively low organic matter content of soils (commonly &#60; 5% by mass) and absorbance overlap of mineral and organic functional groups in the mid-infrared (MIR) region (4,000-400 cm-1) engenders substantial interference by dominant mineral absorbances, challenging or even preventing interpretation of spectra for SOM characterization. Spectral subtractions, a post-hoc mathematical treatment of spectra, can reduce mineral interference and enhance resolution of spectral regions corresponding to organic functional groups by mathematically removing mineral absorbances. This requires a mineral-enriched reference spectrum, which can be empirically obtained for a given soil sample by removing SOM. The mineral-enriched reference spectrum is subtracted from the original (untreated) spectrum of the soil sample to produce a spectrum representing SOM absorbances. Common SOM removal methods include high-temperature combustion (&#39;ashing&#39;) and chemical oxidation. Selection of the SOM removal method carries two considerations: (1) the amount of SOM removed, and (2) absorbance artifacts in the mineral reference spectrum and thus the resulting subtraction spectrum. These potential issues can, and should, be identified and quantified in order to avoid fallacious or biased interpretations of spectra for organic functional group composition of SOM. Following SOM removal, the resulting mineral-enriched sample is used to collect a mineral reference spectrum. Several strategies exist to perform subtractions depending on the experimental goals and sample characteristics, most notably the determination of the subtraction factor. The resulting subtraction spectrum requires careful interpretation based on the aforementioned methodology. For many soil and other environmental samples containing substantial mineral components, subtractions have strong potential to improve FTIR spectroscopic characterization of organic matter composition.

SOM underlies many soil functions and processes, but its characterization by FTIR spectroscopy is often challenged by mineral interferences. The described method can increase the utility of SOM analysis by FTIR spectroscopy by subtracting mineral interferences in soil spectra using empirically obtained mineral reference spectra.

Soil organic matter (SOM) underlies numerous soil processes and functions. Fourier transform infrared (FTIR) spectroscopy detects infrared-active organic ...

Environment

Ultraviolet-Visible (UV-Vis) Spectroscopy

Source: Laboratory of Dr. B. Jill Venton - University of Virginia
Ultraviolet-visible (UV-Vis) spectroscopy is one of the most popular analytical techniques because it is very versatile and able to detect nearly every molecule. With UV-Vis spectroscopy, the UV-Vis light is passed through a sample and the transmittance of light by a sample is measured. From the transmittance (T), the absorbance can be calculated as A=-log (T). An absorbance spectrum is obtained that shows the absorbance of a compound at different wavelengths. The amount of absorbance at any wavelength is due to the chemical structure of the molecule.
UV-Vis can be used in a qualitative manner, to identify&#160;functional groups or confirm the identity of a compound by matching the absorbance spectrum. It can also be used in a quantitative manner, as concentration of the analyte is related to the absorbance using Beer&#39;s Law. UV-Vis spectroscopy is used to quantify the amount of DNA or protein in a sample, for water analysis, and as a detector for many types of chromatography. Kinetics of chemical reactions are also measured with UV-Vis spectroscopy by taking repeated UV-Vis measurements over time. UV-Vis measurements are generally taken with a spectrophotometer. UV-Vis is also a very popular detector for other analytical techniques, such as chromatography, because it can detect many compounds.
Typically, UV-Vis is not the most sensitive spectroscopy technique, because not a lot of light is absorbed over a short path length. Other spectroscopy techniques such as fluorescence have higher sensitivity, but they are not as generally applicable, as most molecules are not fluorescent. UV-Vis has a similar sensitivity to other absorbance measurements, such as infrared spectroscopy.

Ultraviolet-Visible UV-Vis Spectroscopy: Principle and Uses

Source: Laboratory of Dr. B. Jill Venton - University of Virginia
Ultraviolet-visible (UV-Vis) spectroscopy is one of the most popular analytical ...

JoVE Science Education

Analytical Chemistry

Molecular Spectroscopy: Absorption and Emission

Molecules possess discrete energy levels called quantum states. Unlike atoms, which have simpler energy levels, molecules possess additional rotational and vibrational energy levels.&#160; Each energy level is separated by an energy gap, with the gaps between adjacent electronic, vibrational, and rotational levels varying significantly. The three types of energy levels in a diatomic molecule are shown in Figure 1.
<img alt="Figure1" src="/files/ftp_upload/13024/13024_PT_Figure_1.svg" style="text-align: center; height: 400px;" />
Figure 1: The three types of energy levels in a diatomic molecule.
A molecule can absorb energy in the form of a photon from electromagnetic radiation and use that energy to excite the molecule into a higher energy state. During this process, changes can occur in the rotation around a bond, the frequency of bond vibration, or the transition of an electron from its ground state (the lowest energy state) to an excited state (a higher energy level). When the excited-state molecule returns to its ground state, it emits radiation. The energy of the absorbed or emitted photon is equivalent to the energy gap between the two energy levels involved in the transition. Therefore, each transition is dependent on the wavelength or frequency of the radiation.
Due to the varying magnitudes of energy gaps, the wavelengths of radiation absorbed during these transitions differ. For example, the energy consumed or released during the transition of a specific rotation in a molecule is in the range of microwave radiation. In contrast, the energy of infrared radiation corresponds to changes in bond vibrations. Furthermore, photons in the UV&#8211;visible range can excite an electron to a different orbital, particularly in the case of molecules with conjugated double bonds.

Molecules possess discrete energy levels called quantum states. Unlike atoms, which have simpler energy levels, molecules possess additional rotational ...

JoVE Core

Unit 1

Introduction to Molecular Spectroscopy

KEY TERMS AND CONCEPTS

Spectrophotometry: Introduction

Spectrophotometry is the quantitative measurement of the absorption, reflection, diffraction, or transmission of electromagnetic radiation through a material as a function of the intensity and wavelength of the radiation. A spectrophotometer is a device used to measure the change in the radiation intensity caused by its interaction with the material.
The essential components of a spectrophotometer include a source of electromagnetic radiation, a slot for placing a material to be analyzed, and a detector to measure the intensity of the incident radiation from the source and the radiation exiting from the material (transmitted, reflected, or diffracted radiation). The difference between the intensities of incident radiation and transmitted radiation reaching the detector is used to calculate the intensity of radiation absorbed by the material. The sample is exposed to radiation of different wavelengths, often one at a time. The spectrometer provides a series of data with absorption details in each attempt, a process known as scanning. Since the material energy levels are finite, absorption occurs only at specific wavelengths.
The plot of absorbed radiation against the corresponding wavelength is known as the absorption spectrum. Similarly, the plots of reflected, diffracted, and transmitted radiation as a function of wavelength are the reflection, diffraction, and transmission spectra, respectively. These spectra provide information about the specific wavelength at which the interaction between the material and radiation occurs.
The wavelength range of radiation determines the type of transition that occurs in the material. For example, UV&#8211;vis spectra show UV&#8211;vis radiation absorption by the material. The absorbed radiation causes an electronic excitation in the material. In contrast, infrared spectra show the excitation of vibration levels of a specific bond in the material. The specific absorption wavelength details the molecule&#39;s chemical structure inside a range of wavelengths. For example, the specific wavelength of infrared radiation absorbed by a hydroxyl bond in a molecule is different from the wavelength of radiation absorbed by the carbonyl bond. So, infrared spectra can be used to identify functional groups in the material.
Unlike other chemical characterization methods like titration, spectroscopy is a nondestructive characterization technique, and sample material can be recovered after analysis.

Spectrophotometry is the quantitative measurement of the absorption, reflection, diffraction, or transmission of electromagnetic radiation through a ...

IR Spectrum

When infrared (IR) radiation passes through a molecule, the bonds stretch or bend by absorbing the radiation. This absorption creates the molecule&#39;s absorption spectrum, which is the plot of its percentage transmittance versus wavenumber.
Transmittance is defined as the ratio of the radiant power passing through a sample to that from the radiation&#39;s source. Multiplying the transmittance by 100 gives the percent transmittance (%T), which varies between 100% (no absorption) and 0% (complete absorption). When IR radiation passes through a molecule, 100% transmittance indicates that the molecule has not absorbed any energy. Conversely, lower percentage transmittance values suggest that the molecule has absorbed some energy, allowing us to obtain the IR spectrum. Each downward-pointing peak in the spectrum is called an absorption band. Usually, the position of the absorption band in the IR spectrum is reported using wavenumber or frequency values. In the IR spectrum, wavenumber values range from 400 to 4000 cm&#8722;1.
In the IR spectrum, signals on the left side correspond to higher-energy and higher-frequency radiations, whereas those on the right side denote low-energy, low-frequency radiations. Based on shape and intensity, these signals are categorized as strong, medium, weak, broad, or sharp.
The IR spectrum can be divided into two main regions. The functional group region or diagnostic region is the left two-thirds portion of the spectrum. The absorption bands corresponding to most functional groups can be observed in this area. The right-hand side of the spectrum is referred to as the fingerprint region. Similar to the fingerprint of individuals, this region is unique to each compound. The diagnostic region of an infrared spectrum is dominated by bond stretching vibrations, while the fingerprint region is characterized by more complex vibrations like bond rocking, bending, and twisting. Although two compounds may contain the same functional group, they will show a unique pattern in the fingerprint region due to the differences in their chemical environment.

When infrared (IR) radiation passes through a molecule, the bonds stretch or bend by absorbing the radiation. This absorption creates the molecule&#39;s ...

Molecular Vibrational Spectroscopy

Ultraviolet and Visible (UV&ndash;Vis) Spectroscopy: Overview

Ultraviolet&#8211;visible (UV&#8211;visible or UV&#8211;Vis) spectroscopy is an analytical technique that investigates the interaction between matter and UV&#8211;Vis light within the electromagnetic spectrum. This method is widely used for its versatility, simplicity, and relatively quick data acquisition, making it valuable for both qualitative and quantitative analysis. When UV&#8211;Vis radiation passes through a material,&#160; molecules absorb light depending on the energy required for electronic transitions. As a result of energy absorption, atoms or molecules pass from the ground state to an excited state or higher energy state. If a molecule absorbs UV light, a UV spectrum is generated; if it absorbs visible light, a visible spectrum is produced.
When continuous radiation is transmitted through a material, some wavelengths are absorbed by the molecules, resulting in a decrease in the intensity of transmitted light. This selective absorption leads to the formation of an absorption spectrum that displays peaks corresponding to specific wavelengths absorbed by a molecule. The UV wavelength range of light is from 180 to 400 nanometers, as visible light has a wavelength ranging from 400 to 780 nanometers.&#160; This spectrum, which displays gaps corresponding to absorbed wavelengths, reveals information about the molecule&#39;s chemical structure.
In qualitative analysis, UV&#8211;Vis can be used to identify functional groups or confirm the identity of a compound by matching its absorbance to the absorbance of a known compound. Compared to other spectroscopy techniques, such as fluorescence, which has a higher sensitivity, UV&#8211;Vis is moderately sensitive but widely accessible due to its straightforward instrumentation and ease of use. UV&#8211;Vis instruments typically consist of components such as a light source, sample holder, monochromator or filter for wavelength selection, and detector. The path length, or the distance light travels through the sample, is an important factor in absorbance measurements, as it affects the amount of light absorbed. However, UV&#8211;Vis does have a similar sensitivity to other absorbance measurements, such as infrared spectroscopy.

Ultraviolet&#8211;visible (UV&#8211;visible or UV&#8211;Vis) spectroscopy is an analytical technique that investigates the interaction between matter and ...

UV&ndash;Vis Spectroscopy: Molecular Electronic Transitions

In Ultraviolet&#8211;Visible (UV&#8211;Vis) spectroscopy, the absorption of electromagnetic radiation is used to probe the electronic structure of molecules. This technique provides insights into molecular electronic transitions, particularly the movement of electrons between different molecular orbitals. Radiation is absorbed if the energy of the electromagnetic radiation passing through the molecule is precisely equal to the energy difference between the excited and ground states. During this process, the electrons in a molecule transit from the highest occupied molecular orbital (HOMO) to the lowest unoccupied molecular orbital (LUMO), moving to a higher energy state. This transition and the types of orbitals involved are central to understanding how molecules interact with UV&#8211;Vis light. The energy differences between the HOMO and the LUMO are referred to as the bandgap, which typically ranges from 125 to 650 kJ/mol.
A group of atoms in a molecule responsible for absorbing radiation is called a chromophore. When a chromophore undergoes structural changes, both the energy and intensity of the molecule&#39;s absorption change. Molecules with different chemical structures have band gaps and absorb radiation at different wavelengths, producing different absorption spectra.
In molecular orbital theory, the energy levels of orbitals influence the types of electronic transitions that can occur. Typically, the lowest-energy occupied molecular orbitals are the &#963; orbitals, corresponding to &#963; bonds. Next in energy are the &#960; orbitals, followed by nonbonding (n) orbitals that contain an unshared pair of electrons. The highest energy levels are found in unoccupied or antibonding orbitals (&#960;* and &#963;*). Certain restrictions, known as selection rules, must be considered during electron transitions. One important selection rule states that transitions involving a change in an electron&#39;s spin quantum number are not allowed. Such transitions are called &#34;forbidden&#34;&#160;transitions. A common example is a transition from the n to &#960;* orbitals, which are often seen in molecules containing lone pairs.

In Ultraviolet&#8211;Visible (UV&#8211;Vis) spectroscopy, the absorption of electromagnetic radiation is used to probe the electronic structure of ...

UV&ndash;Vis Spectrum

When light passes through a substance, a portion of the light is absorbed while the remaining light is reflected or transmitted. If the molecule absorbs light between the wavelengths of 180&#8211;400 nm range, the UV spectrum is obtained, and if it absorbs light in the 400&#8211;780 nm wavelength range, the visible spectrum is obtained.&#160;&#160;&#160;&#160;&#160;
The UV&#8211;Vis spectrum of a molecule is the plot of its absorbance versus wavelength. The plot is drawn by taking molar absorptivity (&#949;) or log &#949; on the y-axis (ordinate) and wavelength on the x-axis (abscissa). Absorbance values represent light absorption by the molecule and cannot exceed 100 percent. When the absorbance is plotted against wavelength, the wavelength at which the molecule shows the maximum absorbance is called &#955;max. Since absorption occurs at a broad wavelength, it is often called an absorption band rather than a single peak.
Peaks in the UV&#8211;Vis spectrum vary in height and width based on molecular structure, electronic transitions, and solvent interactions.
<ol style="list-style-type: circle;">
	<li>Molecular Structure: Different structures, like conjugated double bonds (alternating single and double bonds), have smaller energy gaps between their molecular orbitals. This means they absorb light at longer wavelengths, often in the visible range. As a result, the structure of the molecule can shift the peak to higher or lower wavelengths and change its intensity.</li>
	<li>Electronic Transitions: Various electron transitions (e.g., &#960; &#8594; &#960;*, n &#8594; &#960;*) absorb light differently, influencing peak strength. Molecules with &#960; (pi) bonds or nonbonding (n) electron pairs can exhibit multiple types of transitions, leading to different peaks or bands in the spectrum.</li>
	<li>Solvent Effects:&#160; The solvent in which the molecule is dissolved can also affect the spectrum. When a molecule interacts with a solvent, it can alter the molecule&#39;s electronic environment, sometimes stabilizing or destabilizing certain molecular orbitals. This solvent effect can cause shifts in the peak position and may alter the peak&#39;s intensity.</li>
</ol>
These factors often result in broadened bands instead of single peaks, reflecting the molecule&#39;s complex behavior in different conditions.

When light passes through a substance, a portion of the light is absorbed while the remaining light is reflected or transmitted. If the molecule absorbs ...

Characterization of Biological Absorption Spectra Spanning the Visible to the Short-Wave Infrared

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