Name: uv-vis spectroscopic characterization of nanomaterials in aqueous media
Uploaded: 2021-10-25T13:00:00.000+00:00
Duration: 5 min 16 s
Description: Watch this Scientific Journal Video about UV-Vis Spectroscopic Characterization of Nanomaterials in Aqueous Media at JoVE.com

ACQ would like to thank The National Council for Science and Technology (CONACyT) in Mexico for funding her PhD studies. All authors acknowledge support from the European Union Horizon 2020 Programme (H2020) under grant agreement no 720952, project ACEnano (call NMBP-26-2016).

1. Delivery of the AuNP samples:
<ol>
	<li>Prepare aliquots of 5 mL of Au colloid dispersions with sizes of 5, 20, 40, 60, and 100 nm including a 50 &#181;g/mL sample of &#8216;unknown size&#8217; (See Table of Materials for more specific details about the nanomaterials used).</li>
	<li>Send the samples in 7 mL polystyrene containers with gel packs to each participating laboratory to maintain a suitable temperature during the shipping. Store the samples at 4 &#176;C immediately. 
	NOTE: The &#8216;unknown size&#8217; sample must present a size of 80 nm; this information should be known by the partner distributing the material, but not disclosed to the other partners.</li>
</ol>
2. Calibration of the spectrophotometer:
<ol>
	<li>Turn on the UV-Vis spectrometer for at least 20 min to allow the lamp to heat up. 
	NOTE: Refer to the Table of Materials for the model and brand of the spectrophotometer used.</li>
	<li>In the software, select the option&#160;Spectrum scan from the mode window, which displays the operating modes.</li>
	<li>Adjust the parameter settings in Instrument | Settings and parameters in the software before proceeding with measurements: Measurement Mode | Spectrum scan, Data Mode | ABS, Start wavelength of 680 nm, End Wavelength of 380 nm, Scan Speed of 400 nm/min, Sampling interval of 0.5, Slit Width of 1.5, and Path Length of 10.</li>
	<li>After the parameters have been set, fill two cuvettes (3 mL; polystyrene) with 1 mL of ultrapure water (UPW) (18.2 M&#183;&#937;&#183;cm). Place the cuvettes in the reference cell holder (rear) and the sample cell holder (front) to cover the light path (See Table of Materials for the specific brand and model of the cuvettes used). 
	NOTE: Make sure the cuvettes are positioned and aligned correctly to cancel the noise effect and other environmental effects that are not sample-related.</li>
	<li>Close the UV-Vis instrument cover and continue with the blank calibration by selecting Blank from the command bar. The baseline correction is performed by running a reference with the two cuvettes filled with 1 mL of UPW placed in the sample holders. For alternative protocols used by other partners, please see Supplementary Information (SI).</li>
</ol>
3. Preparation of the samples
<ol>
	<li>Take a subsample of 500 &#181;L for each AuNP of 5, 20, 40, 60, 100 nm, and the unknown size, and prepare a dilution with 500 &#181;L of UPW.</li>
	<li>Place the diluted suspensions in 1 mL cuvettes; the total dilution ratio should be 1:1 and final concentration 25 &#181;g/mL. 
	NOTE: The diluted sample must be prepared immediately before the UV-Vis measurement.</li>
</ol>
4. Measurement of the nanoparticle dispersions
<ol>
	<li>After the blank calibration has been performed, and a fresh sample has been prepared, replace one of the blank cuvettes in the sample cell holder (front) with the AuNP dispersion sample; the other reference cuvette filled with 1 mL of UPW must be left untouched. 
	NOTE: Use a new disposable cuvette for different samples to avoid cross-contamination between samples. When using quartz cuvettes, rinse the sample cuvette with UPW between samples.</li>
	<li>Select the option Measure/Start from the command bar to run the spectrum scans for each diluted AuNP dispersion. Three spectrum scan runs should be obtained for each AuNP sample, including the unknown size sample. 
	NOTE: Ensure that the blank cuvette remains in the reference cell holder when running a measurement.</li>
</ol>
5. Reporting results
<ol>
	<li>Extract the raw experimental data for each measurement in a spreadsheet-compatible file by selecting File menu and clicking Export report (*.csv) file.</li>
	<li>Note the maximum absorption wavelength (Absmax) and lambda (&#955;max) for each of the UV-Vis readings and record them in the provided template. 
	NOTE: The predesigned template was provided to the ACEnano partners to automatically calculate the wavelengths&#8217; average standard deviations by setting the appropriate calculation formula in the workbook. For further details and access to the template, see Supplementary Information (SI).</li>
	<li>In the workbook, plot a calibration curve with the average of the &#955;max (y-axis) against the known nanoparticle size (nm) (5, 20, 40, 60, and 100 nm). For example, in the spreadsheet, create the calibration curve by selecting in the command bar Data | Insert Graph | Scatter Plot | Add Trendline | Polynomial Curve (Power 2).</li>
	<li>Include the polynomial equation for the calibration curve: select Trendline options | Display Equation On Chart from the command bar (Figure 1).</li>
	<li>Finally, to calculate the unknown size of the AuNP sample, isolate the polynomial equation from the calibration curve to fit the mean value for the unknown &#955;max, using a derivation of the quadratic formula (Figure 1). The calculated size can be included in the template to complete a full summary of the data for consistency, faster interpretation, and evaluation of the results (see SI).</li>
</ol>
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61764/61764fig01.jpg" /> 
Figure 1: Calibration curve to calculate the size of the unknown sample.&#160;The plot represents the wavelengths (&#955;max) and the size of the AuNPs used to plot the calibration. The plot shows only one calibration curve from one partner. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61764/61764fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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The authors declare that they have no competing interests.

Several methods are available for the characterization of nanoscale-related properties (e.g., analytical ultracentrifugation (AUC), Scanning Electron Microscopy/Transmission Electron Microscopy (SEM/TEM), and Dynamic Light Scattering (DLS)10,11). However, these techniques lack the simplicity of UV-Vis to obtain primary results in the characterization of NMs12,13. UV-Vis is a common instrument even in not-so-well equipped laboratories, making it an unbeatable tool for the characterization of NMs6. When characterizing NMs, it is important to consider the limitations, strengths, and weaknesses of the techniques to be applied. In the UV-Vis spectrometer, the light beam passes through the sample compartment resulting in absorption values; as a result, external vibrations, outside light, contaminants, and the user&#8217;s performance may interfere with the measurement and results4,12. Similarly, when plotting a calibration curve to determine the size of an unknown sample, it is important to register all the measurements needed to construct the calibration, as missing factors may contribute to variations among measurements and users.
For example, the high variation in the overall Absmax mean of the unknown sample might be linked to differences between the laboratories due to the dependence between the beam intensity, position, and the instrument itself17,18. Furthermore, the missing data for the 100 nm size from laboratory 5, due to a contamination problem, may also contribute to the high differences between the results, as the missing data may have affected the calibration curve and the plotted polynomial equation used to calculate the size of the unknown AuNP suspension. Certainly, reproducibility between protocols and laboratories can be complicated, as many factors might contribute to the lack of consistency in laboratory activities, resulting in researchers being occasionally unable to reproduce findings from other labs, which may lead to slower scientific progress, wasted time, money, and resources19. The successful characterization of physicochemical properties of NMs, particularly size, requires an easy-to-execute method by all participating laboratories, which can mostly be addressed by following a systematic and conceptual replication, such as the creation of an SOP, instrument training, and avoiding the use of misidentified or cross-contaminated samples15,19.
Similarly, the quality and stability of the colloid suspension are also important factors to consider, as changes in their physicochemical properties may lead to different outcomes. Therefore, to ensure their stability for longer periods, nanoparticle suspensions should be stored in the dark at 4 &#176;C. Likewise, during the shipping process, the aliquoted samples should be kept cold, as long periods at room temperature may lead to significant aggregation20. Additionally, to overcome failures in NM characterization, it is necessary to provide access to the original data, protocols, and key research materials between collaborating labs, especially, when assessing the proficiency, consistency, and reliability through an ILC15. Making these factors clear and accessible is key to achieving a successful NM characterization by any laboratory or equipment. Disregarding these aspects might result in a lack of reproducibility, accuracy, and misleading or erroneous results15. Although UV-Vis spectroscopy has been demonstrated to be the gold standard in NM characterization, it can be exploited in many other fields as it allows quantitative determination of an extended dynamic range of solutions in both inorganic and organic compounds6,21.
Besides, UV-Vis can be easily combined with other tools to measure a large variety of attributes, thereby improving the quality of any analysis22. Based on these features, UV-Vis is widely used in many areas such as in the biopharmaceutical field by measuring UV-Vis spectra in high concentration protein solutions, in environmental control when comparing similarities between contaminants and their product-related impurities in real time, in industrial wastewater treatments plants as part of regulations for wastewater color determination and acceptability level22,23. Certainly, as technology progresses and more advanced features and experience become available in spectrophotometry, further broadening of the applications and parameters that can be measured using this technique will occur22. For example, in field applications, on-line UV-Vis spectrometry is a valuable tool for monitoring numerous parameters in real time and in various types of liquids, which is an exceptional feature among online sensor systems22.
The ILC described here was designed as a test of the SOP developed for UV-Vis amongst six participating labs involved in the H2020 ACEnano project. The analysis of the results demonstrated that an ILC provides valuable information to allow technical confidence in an internal method for NM characterization by each participant laboratory. Data collection in an established template confirmed consistency and faster interpretation of the results and provided a model for the estimation of the size of an unknown AuNP sample, which also displayed repeatability between results when sufficient points in the calibration curve were included. Furthermore, the results validated the effectiveness of UV-Vis for NM characterization as well as the importance of the creation of best practice protocols. Such an approach further provides an opportunity for the implemented procedure to contribute towards the development of a legislative framework through reproducible NM characterization protocols based on method selection and data interpretation that are relevant for accreditation regulators and research management bodies.

Nanomaterials (NMs) have become popular due to their unique properties in the nanoscale (1 to 100 nm), which differ from the properties of their bulk counterparts, either due to size-related or quantum effects (e.g., increased specific surface area by volume) along with distinct reactivity, optical, thermal, electrical, and magnetic properties1,2. The potential applications of NMs in society are diverse and widely related to fields such as health care, food industry, cosmetics, paints, coatings, and electronics3,4,5. Gold nanoparticles (AuNPs) are widely applied in nanotechnology (e.g., in health care, cosmetics, and electronic applications), mainly due to their simple fabrication, size-dependent optical features, surface functionalization potential, and physicochemical properties, which can be suitable for many key applications6,7.
Quality and reproducibility in the synthesis and characterization of NMs are extremely important for quality assurance, but also for the safe manufacture of nano-based products, especially due to the reactivity of NMs, notably in complex environments, where NM properties, such as size distribution and morphology, may undergo rapid changes8,9. Numerous methods are available to monitor nanoscale-related properties. For example, scanning/transmission electron microscopy (SEM/TEM) are techniques used to obtain high-resolution (down to sub-nanometer) optical and compositional information of NMs; atomic force microscopy (AFM) provides nanoscale resolution in the vertical (z axis) dimension; and X-ray diffraction (XRD) provides information on the atomic structure of NMs; all these methods can only be used on dry samples (powders)10,11. Techniques suitable for the characterization of NMs in liquid media include field flow fractionation (FFF), which allows the separation of large molecules, aggregates, and particles based on their size; dynamic light scattering (DLS); and nanoparticle tracking analysis (NTA)&#8212;two methods widely used to determine the size distribution profile of particles using Brownian motion&#8212;and ultraviolet-visible spectrophotometry (UV-Vis), which allows the assessment of NM characteristics such as size, aggregation state, and refractive index by a simple absorption measurement11,12,13. Although all these techniques allow NM characterization, their performance is dependent on instrument setup, instrument-related differences, complex methodology for sample preparation, and the user&#8217;s level of expertise. Moreover, most of the techniques do not allow real-time monitoring of NM size, sample integrity, or differentiation between dispersed or aggregated particles6. UV-Vis spectroscopy is a widely used technique that provides non-invasive and fast real-time evaluation of NM size, concentration, and aggregation state. Additionally, it is a simple and inexpensive process with minimal sample preparation, which makes this technique an essential tool that is extensively used in numerous laboratories within many disciplines and markets6,12,14. UV-Vis works by measuring the transmittance of electromagnetic radiation of a wavelength between 180 and 1100 nm through a liquid sample. The UV and VIS spectral ranges cover the wavelength range for the ultraviolet (170 nm to 380 nm), visible (380 nm to 780 nm), and near-infrared (780 nm to 3300 nm)4,14. The wavelength of light passing through the sample cell is measured; the intensity of light entering the sample is referred to as I0, and the intensity of the light emerging on the other side is designated as I114. The Beer-Lambert law reflects the relationship between A (absorbance) as a function of sample concentration C, the sample extinction coefficient &#1013;, and the two intensities14. Absorption measurements can be collected at a single wavelength or over an extended spectral range; the measured light transmittance is transformed into an absorbance measurement by following the Beer-Lambert law equation. The standard equation for absorbance is A = &#603;lc, where (A) is the amount of light absorbed by the sample for a given wavelength (&#603;) is the molar attenuation coefficient (absorbance/(g/dm3) (l) is the distance the light travels through the solution (cm), and (c) is the concentration per unit volume (g/dm3). The absorbance is calculated as the ratio between the intensity of a reference sample (I0) and the unknown sample (I), as described in the following equation14:
<img alt="Equation 1" src="/files/ftp_upload/61764/61764equ01.jpg" />
The simplicity of UV-Vis makes it an ideal technique to compare PTS of an established measurement protocol6,12,15. The objective of an ILC or PTS is to verify the performance and reproducibility of a method using an SOP15. This, in turn, provides a standardized approach for quick characterization of nanoparticle suspensions for other users.
To assess the proficiency, consistency, and reliability of the method presented here, six laboratories participated in an ILC as members of the Horizon 2020 ACEnano project (https://cordis.europa.eu/project/id/720952). The ILC involved UV-Vis characterization of standard Au colloid dispersions of different particle sizes (5&#8211;100 nm). An SOP was provided to all the involved laboratories to ensure the identical preparation of AuNP suspensions, evaluation, and reporting of results to contribute towards the development of an implementable and robust tiered approach in NM physicochemical characterization, data interpretation, and improvement of best practice protocols for industrial and regulatory needs8.

<table><tbody><tr><td>Absorption Ultra-Micro-cuvette, 200 &amp;micro;L</td><td>Hellma</td><td>105.201-QS</td><td></td></tr><tr><td>Cary 5000 spectrophotometer (Spectrophotometer C)</td><td>Agilent</td><td>Cary 5000</td><td></td></tr><tr><td>Gold nanoparticles 5 nm</td><td>BBI solutions</td><td>EM.GC5</td><td></td></tr><tr><td>Gold nanoparticles 20 nm</td><td>BBI solutions</td><td>EM.GC20</td><td></td></tr><tr><td>Gold nanoparticles 40 nm</td><td>BBI solutions</td><td>EM.GC40</td><td></td></tr><tr><td>Gold nanoparticles 60 nm</td><td>BBI solutions</td><td>EM.GC60</td><td></td></tr><tr><td>Gold nanoparticles 80 nm</td><td>BBI solutions</td><td>EM.GC80</td><td></td></tr><tr><td>Gold nanoparticles 100 nm</td><td>BBI solutions</td><td>EM.GC100</td><td></td></tr><tr><td>Agilent / HP 8453 (Spectrophotometer E)</td><td></td><td></td><td></td></tr><tr><td>Jenway 6800 spectrophotometer (Spectrophotometer A)</td><td>Jenway</td><td>UV6800</td><td></td></tr><tr><td>Polystyrene cuvette, 1.5 mL, micro 10 mm pathlength</td><td>Sigma</td><td>759015</td><td></td></tr><tr><td>Polystyrene cuvette, 3 mL (10 mm x 10 mm x 45 mm)</td><td>Sarstedt Inc</td><td>67.742</td><td></td></tr><tr><td>Semi-micro quartz cuvette, 1mL (1 mm x 10 mm x 45 mm)</td><td>Agilent</td><td>6610001</td><td></td></tr><tr><td>Ultrapure water (UPW) (18.2 M&amp;Omega;cm).&amp;nbsp;</td><td>/</td><td>/</td><td></td></tr><tr><td>UV-1800 spectrophotometer (Spectrophotometer B)</td><td>Shimadzu</td><td>UV1800</td><td></td></tr><tr><td>Varian Cary 50 spectrophotometer (Spectrophotometer D)</td><td>Agilent</td><td>Cary 50</td><td></td></tr></tbody></table>

uv-vis spectroscopic characterization of nanomaterials in aqueous media

The physicochemical characterization of nanomaterials (NMs) is often an analytical challenge, due to their small size (at least one dimension in the nanoscale, i.e. 1&#8211;100 nm), dynamic nature, and diverse properties. At the same time, reliable and repeatable characterization is paramount to ensure safety and quality in the manufacturing of NM-bearing products. There are several methods available to monitor and achieve reliable measurement of nanoscale-related properties, one example of which is Ultraviolet-Visible Spectroscopy (UV-Vis). This is a well-established, simple, and inexpensive technique that provides non-invasive and fast real-time screening evaluation of NM size, concentration, and aggregation state. Such features make UV-Vis an ideal methodology to assess the proficiency testing schemes (PTS) of a validated standard operating procedure (SOP) intended to evaluate the performance and reproducibility of a characterization method. In this paper, the PTS of six partner laboratories from the H2020 project ACEnano were assessed through an interlaboratory comparison (ILC). Standard gold (Au) colloid suspensions of different sizes (ranging 5&#8211;100 nm) were characterized by UV-Vis at the different institutions to develop an implementable and robust protocol for NM size characterization.

UV-Vis is one of the most popular techniques for nanoparticle characterization as it allows the user to obtain precise analysis of properties of NMs such as Absmax and &#955;max6,12. Results of the present study represent the UV-Vis characterization of AuNP dispersions through an ILC between six participating labs.
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/61764/61764fig02.jpg" /> 
Figure 2: Lambda and absorbance results.&#160;The figures show the plots for the results reported by each laboratory for different AuNP sizes. A) Lambda max results. B) Absorbance max results. Laboratory 5 was not able to report data for 100 nm due to sample contamination. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61764/61764fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Results for the &#955;max wavelengths showed close repeatability among the partners (Figure 2A). This was also the case for the calculated range, which was used to assess the difference between values, and which showed small differences ranging between 1.00 and 2.40 (&#955;max) for most of the AuNP sizes (Table 1). The overall &#955;max mean, calculated using the recorded mean for each laboratory for each AuNP size, similarly displayed low standard deviations for most of the sizes. The 100 nm size was the only exemption, as it displayed a high variation range (4.66 &#955;max) between partners, leading to a greater standard deviation (572 &#177; 2.00 nm) compared to other AuNP sizes (Table 1). It is important to mention that laboratory 5 was not able to perform any measurements for the 100 nm size particles, due to contamination issues that might have compromised the repeatability of the results.
In contrast, absorbance results (Absmax) exhibited a more scattered range of data values (Figure 2B) compared to &#955;max results. Despite the apparently higher variability of these results between laboratories, the analysis displayed overall means with lower standard deviations and unexpected inferior variation ranges (0.11&#8211;0.21 Absmax) between laboratories compared to the &#955;max results (Table 1).
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td rowspan="2">Value</td>
			<td colspan="6">AuNP (nm)</td>
		</tr>
		<tr>
			<td>5</td>
			<td>20</td>
			<td>40</td>
			<td>60</td>
			<td>100</td>
			<td>Unknown</td>
		</tr>
		<tr>
			<td>Range &#955;max</td>
			<td>1.45</td>
			<td>1.00</td>
			<td>3.00</td>
			<td>2.00</td>
			<td>4.66</td>
			<td>2.40</td>
		</tr>
		<tr>
			<td>Range Aumax</td>
			<td>0.12</td>
			<td>0.11</td>
			<td>0.13</td>
			<td>0.13</td>
			<td>0.12</td>
			<td>0.21</td>
		</tr>
		<tr>
			<td>Mean &#955;max</td>
			<td>517.7 &#177; 0.59</td>
			<td>524.6 &#177; 0.45</td>
			<td>527.8 &#177; 1.13</td>
			<td>535.3 &#177; 0.74</td>
			<td>572 &#177; 2.00</td>
			<td>549.7 &#177; 0.85</td>
		</tr>
		<tr>
			<td>Mean Aumax</td>
			<td>0.395 &#177; 0.048</td>
			<td>0.497 &#177; 0.050</td>
			<td>0.509 &#177; 0.057</td>
			<td>0.689 &#177; 0.055</td>
			<td>0.472 &#177; 0.051</td>
			<td>0.661 &#177; 0.101</td>
		</tr>
	</tbody>
</table>
Table 1: Lambda and Absorbance calculated range and means. The range and overall mean and standard deviation for each AuNP size are shown. Results were calculated using the reported mean for lambda and absorbance for each laboratory (six measurements), except for the 100 nm size for which only 5 measurements were used to calculate the values due to a sample contamination reported by laboratory 5.
The Z-score values were also calculated to note the distance of individual values from the overall mean. The analysis of Z-scores provided information about the confidence of the ILC results, as the scores are directly related to the population distribution by displaying, in a number of standard deviations, how far a data point is from the mean16. In the results, most of the laboratories showed positive Z-score values of 0.01&#8211;1.93 for &#955;max, indicating that most of the results were close to the mean and presented a normal distribution curve, as Z-scores greater than the absolute value of 2 and -2 are considered values that are distant from the mean and do not have a normal distribution16. The highest Z-score for Absmax was recorded for the 40 nm size reported by laboratory 1, with a value of 1.93 and an Absmax average of 530 &#177; 0, compared to the overall mean of 527.82 &#177; 1.13 (Figure 3A). The maximum Z-score value of 1.23 for &#955;max was reported by laboratory 3 along with a reported &#955;max of 0.454 &#177; 0 for 5 nm AuNP size compared to the overall mean of 0.395 &#177; 0.04. This was followed by the 60 nm AuNP with a Z-score of 1.18 and an &#955;max mean of 0.754 &#177; 0 compared to the overall average of 0.689 &#177; 0.05. The remaining sizes displayed Z-score values from -0.04 to -1.23 (Figure 3B).
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/61764/61764fig03.jpg" /> 
Figure 3: Lambda and Absorbance Z-scores.&#160;Z-scores were calculated using the results reported by each laboratory against the overall mean. A) Calculated Lambda max Z-scores. B) Calculated Absorbance max Z-scores. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61764/61764fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Results for the unknown sample showed that most of the partners calculated the size to be 76&#8211;80 nm. The mean of laboratories 1-4 and 6 was recorded as 78.02 &#177; 1.36 nm. Laboratory 5 reported a larger size of 109 nm, broadening the overall average and standard deviation up to 83.18 &#177; 12.70 nm, suggesting that this value was an outlier (Figure 4A). The Z-scores were calculated to be between -0.25 to -0.56 for all the laboratories; the only exception was for the unknown size reported by laboratory 6, which displayed the highest positive Z-score (2.03) compared to all the measurements, which can be considered as a value that is distant from the mean (Figure 4B).
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/61764/61764fig04.jpg" /> 
Figure 4: Unknown sample size and Z-scores.&#160;A) Reported size for each laboratory for the provided unknown sample. B) Calculated Z-scores for each individual result against the overall mean of 83.18 &#177; 12.70 nm. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61764/61764fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Supplementary Information (SI): <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61764/61764_R1_SI.docx" target="_blank">Please click here to download this File.</a>

Watch this Scientific Journal Video about UV-Vis Spectroscopic Characterization of Nanomaterials in Aqueous Media at JoVE.com

UV-Vis Spectroscopic Characterization of Nanomaterials in Aqueous Media

This study presents the benchmarking results for an interlaboratory comparison (ILC) designed to test the standard operating procedure (SOP) developed for gold (Au) colloid dispersions characterized by ultraviolet-visible Spectroscopy (UV-Vis), amongst six partners from the H2020 ACEnano project for sample preparation, measurement, and analysis of the results.

The physicochemical characterization of nanomaterials (NMs) is often an analytical challenge, due to their small size (at least one dimension in the ...

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Introduction to Molecular Spectroscopy

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9.4K Views.  University of Birmingham. UV-Vis-spectrofotometrie is een gevestigde, eenvoudig te gebruiken en goedkope techniek voor het meten van de grootte, concentratie, aggregatietoestand en brekingsindex van nanomaterialen. UV-Vis biedt niet-invasieve en snelle, realtime screening en evaluatie van nanomateriaalkenmerken met behulp van eenvoudige absorptiemetingen. UV-Vis is uitzonderlijk eenvoudig.De monsters vereisen weinig voorbereiding en de software heeft weinig variabelen en is gemakkelijk te gebruiken. Schakel de ...

Video: UV-Vis Spectroscopic Characterization of Nanomaterials in Aqueous Media

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 ÃÂ¢ÃÂÃÂ 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 ÃÂ¢ÃÂÃÂ 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, ÃÂÃÂ». Here, h is Planck'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" alt="Photon energy equation, \(E = \frac{hc}{\lambda}\), formula for calculating energy from wavelength." data-altupdatedat="1747830616000" data-alt="Energy equation \(E=\frac{hc}{\lambda}\), formula for photon energy calculation, physics concept.">
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" alt="ΔE equation for HOMO-LUMO gap in molecular absorption; energy-wavelength relation diagram." data-altupdatedat="1747830616000" data-alt="ÎE(HOMO-LUMO)=hc/Î» equation, diagram for molecular energy gap analysis, quantum chemistry.">
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" alt="Transmittance equation T=P/P0, mathematical formula for optical analysis." data-altupdatedat="1747830616000" data-alt="Transmittance equation T=P/P0 in optics formula for light transmission analysis.">
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" alt="Absorption equation A=log(P0/P)=-logT, formula for optical absorbance analysis." data-altupdatedat="1747830616000" data-alt="Absorbance equation, A=log(P0/P)=-logT, key in spectroscopic absorbance studies.">
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" alt="Beer-Lambert Law equation A=εcl, absorption spectroscopy formula, educational use." data-altupdatedat="1747830616000" data-alt="Beer-Lambert law formula \(A = \varepsilon cl\) describing light absorption, diagram for spectroscopy.">
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 ÃÂÃÂ»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. ...

JoVE Lab Manual

Chemistry

Fabricating a UV-Vis and Raman Spectroscopy Immunoassay Platform

Immunoassays are used to detect proteins based on the presence of associated antibodies. Because of their extensive use in research and clinical settings, a large infrastructure of immunoassay instruments and materials can be found. For example, 96- and 384-well polystyrene plates are available commercially and have a standard design to accommodate ultraviolet-visible (UV-Vis) spectroscopy machines from various manufacturers. In addition, a wide variety of immunoglobulins, detection tags, and blocking agents for customized immunoassay designs such as enzyme-linked immunosorbent assays (ELISA) are available.
Despite the existing infrastructure, standard ELISA kits do not meet all research needs, requiring individualized immunoassay development, which can be expensive and time-consuming. For example, ELISA kits have low multiplexing (detection of more than one analyte at a time) capabilities as they usually depend on fluorescence or colorimetric methods for detection. Colorimetric and fluorescent-based analyses have limited multiplexing capabilities due to broad spectral peaks. In contrast, Raman spectroscopy-based methods have a much greater capability for multiplexing due to narrow emission peaks. Another advantage of Raman spectroscopy is that Raman reporters experience significantly less photobleaching than fluorescent tags1. Despite the advantages that Raman reporters have over fluorescent and colorimetric tags, protocols to fabricate Raman-based immunoassays are limited. The purpose of this paper is to provide a protocol to prepare functionalized probes to use in conjunction with polystyrene plates for direct detection of analytes by UV-Vis analysis and Raman spectroscopy. This protocol will allow researchers to take a do-it-yourself approach for future multi-analyte detection while capitalizing on pre-established infrastructure.

Nanoparticle-based optical probes have been designed as a vehicle for detecting antigens using Raman and UV-Vis spectroscopy. Here we describe a protocol for preparing such probes for a UV-Vis/Raman spectroscopy immunoassay in such a way to incorporate future multiplexing capabilities.

Immunoassays are used to detect proteins based on the presence of associated antibodies. Because of their extensive use in research and clinical settings, ...

Biochemistry

Generation of Zerovalent Metal Core Nanoparticles Using n-(2-aminoethyl)-3-aminosilanetriol

In this work, a facile one-pot reaction for the formation of metal nanoparticles in a water solution through the use of n-(2-aminoethyl)-3-aminosilanetriol is presented. This compound can be used to effectively reduce and complex metal salts into metal core nanoparticles coated with the compound. By controlling the concentrations of salt and silane one is able to control reaction rates, particle size, and nanoparticle coating. The effects of these changes were characterized through transmission electron microscopy (TEM), UV-Vis spectrometry (UV-Vis), Nuclear Magnetic Resonance spectroscopy (NMR) and Fourier Transform Infrared spectroscopy (FTIR). A unique aspect to this reaction is that usually silanes hydrolyze and cross-link in water; however, in this system the silane is water-soluble and stable. It is known that silicon and amino moieties can form complexes with metal salts. The silicon is known to extend its coordination sphere to form penta- or hexa-coordinated species. Furthermore, the silanol group can undergo hydrolysis to form a Si-O-Si silica network, thereby transforming the metal nanoparticles into a functionalized nanocomposites.

Generation of Zerovalent Metal Core Nanoparticles Using n-2-aminoethyl-3-aminosilanetriol

A novel method for metal core nanoparticle synthesis using a water stable silanol is described.

In this work, a facile one-pot reaction for the formation of metal nanoparticles in a water solution through the use of ...

Functionalization and Dispersion of Carbon Nanomaterials Using an Environmentally Friendly Ultrasonicated Ozonolysis Process

Functionalization of carbon nanomaterials is often a critical step that facilitates their integration into larger material systems and devices. In the as-received form, carbon nanomaterials, such as carbon nanotubes (CNTs) or graphene nanoplatelets (GNPs), may contain large agglomerates. Both agglomerates and impurities will diminish the benefits of the unique electrical and mechanical properties offered when CNTs or GNPs are incorporated into polymers or composite material systems. Whilst a variety of methods exist to functionalize carbon nanomaterials and to create stable dispersions, many the processes use harsh chemicals, organic solvents, or surfactants, which are environmentally unfriendly and may increase the processing burden when isolating the nanomaterials for subsequent use. The current research details the use of an alternative, environmentally friendly technique for functionalizing CNTs and GNPs. It produces stable, aqueous dispersions free of harmful chemicals. Both CNTs and GNPs can be added to water at concentrations up to 5 g/L and can be recirculated through a high-powered ultrasonic cell. The simultaneous injection of ozone into the cell progressively oxidizes the carbon nanomaterials, and the combined ultrasonication breaks down agglomerates and immediately exposes fresh material for functionalization. The prepared dispersions are ideally suited for the deposition of thin films onto solid substrates using electrophoretic deposition (EPD). CNTs and GNPs from the aqueous dispersions can be readily used to coat carbon- and glass-reinforcing fibers using EPD for the preparation of hierarchical composite materials.

Here, a novel method for the functionalization and stable dispersion of carbon nanomaterials in aqueous environments is described. Ozone is injected directly into an aqueous dispersion of carbon nanomaterial that is continuously recirculated through a high-powered ultrasonic cell.

Functionalization of carbon nanomaterials is often a critical step that facilitates their integration into larger material systems and devices. In the ...

Environment

Dispersion of Nanomaterials in Aqueous Media: Towards Protocol Optimization

The sonication process is commonly used for de-agglomerating and dispersing nanomaterials in aqueous based media, necessary to improve homogeneity and stability of the suspension. In this study, a systematic step-wise approach is carried out to identify optimal sonication conditions in order to achieve a stable dispersion. This approach has been adopted and shown to be suitable for several nanomaterials (cerium oxide, zinc oxide, and carbon nanotubes) dispersed in deionized (DI) water. However, with any change in either the nanomaterial type or dispersing medium, there needs to be optimization of the basic protocol by adjusting various factors such as sonication time, power, and sonicator type as well as temperature rise during the process. The approach records the dispersion process in detail. This is necessary to identify the time points as well as other above-mentioned conditions during the sonication process in which there may be undesirable changes, such as damage to the particle surface thus affecting surface properties. Our goal is to offer a harmonized approach that can control the quality of the final, produced dispersion. Such a guideline is instrumental in ensuring dispersion quality repeatability in the nanoscience community, particularly in the field of nanotoxicology.

Here, we present a step-wise protocol for the dispersion of nanomaterials in aqueous media with real-time characterization to identify the optimal sonication conditions, intensity, and duration for improved stability and uniformity of nanoparticle dispersions without impacting the sample integrity.

The sonication process is commonly used for de-agglomerating and dispersing nanomaterials in aqueous based media, necessary to improve homogeneity and ...

A Technical Guide for Performing Spectroscopic Measurements on Metal-Organic Frameworks

Metal-organic frameworks (MOFs) offer a unique platform to understand light-driven processes in solid-state materials, given their high structural tunability. However, the progression of MOF-based photochemistry has been hindered by the difficulty in spectrally characterizing these materials. Given that MOFs are typically larger than 100 nm in size, they are prone to excessive light scatter, thereby rendering data from valuable analytical tools like transient absorption and emission spectroscopy nearly uninterpretable. To gain meaningful insights of MOF-based photo-chemical and physical processes, special consideration must be taken toward properly preparing MOFs for spectroscopic measurements, as well as the experimental setups that garner higher quality data. With these considerations in mind, the present guide provides a general approach and set of guidelines for the spectroscopic investigation of MOFs. The guide addresses the following key topics: (1) sample preparation methods, (2) spectroscopic techniques/measurements with MOFs, (3) experimental setups, (3) control experiments, and (4) post-run stability characterization. With appropriate sample preparation and experimental approaches, pioneering advancements toward the fundamental understanding of light-MOF interactions are significantly more attainable.

Here, we use a polymer stabilizer to prepare metal-organic framework (MOF) suspensions that exhibit markedly decreased scatter in their ground-state and transient absorption spectra. With these MOF suspensions, the protocol provides various guidelines to characterize the MOFs spectroscopically to yield interpretable data.

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Optimizing VIS-SWIR Spectroscopy for Biological Molecules

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.

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.

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Bioengineering

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

Determination Of NOx in Automobile Exhaust Using UV-VIS Spectroscopy

Source: Laboratories of Margaret Workman and Kimberly Frye - Depaul University
In the troposphere, ozone is naturally formed when sunlight splits nitrogen dioxide (NO2):
NO2 + sunlight &#8594; NO + O
O + O2 &#8594; O3
Ozone (O3) can go on to react with nitric oxide (NO) to form nitrogen dioxide (NO2) and oxygen:
NO + O3 &#8594; NO2 + O2
This results in no net gain of ozone (O3). However, with the anthropogenic production of ozone forming precursors (NO, NO2, and volatile organic compounds) through the combustion of fossil fuels, elevated levels of ozone in the troposphere have been found. Motor vehicle exhaust is a significant source of these ozone forming precursors: NO, NO2, and volatile organic compounds (VOCs). For example, mobile sources make up nearly 60% of NO + NO2 emissions.
At the high temperatures found in a car&#8217;s combustion chamber, nitrogen and oxygen from the air react to form nitric oxide (NO) and nitrogen dioxide (NO2):
N2(g) + O2 (g)&#8594; 2 NO(g)
2 NO(g) + O2(g)&#8594; 2 NO2(g)
The nitric oxide (NO) emitted in the car exhaust is gradually oxidized to nitrogen dioxide (NO2) in ambient air. This mixture of NO and NO2 is often referred to as NOx. When NOx reacts with volatile organic compounds in the atmosphere in the presence of sunlight, tropospheric ozone forms, as seen in this simplified chemical reaction:
NOx + VOCs + sunlight &#8594; O3 + other products
This noxious mixture of air pollution, which can include aldehydes, peroxyacetyl nitrates, ozone, VOCs, and NOx, is called photochemical smog. Ozone is the largest component of photochemical smog. This smog is found in all modern cities, but it&#8217;s found especially in cities with sunny, warm, dry climates and large numbers of motor vehicles. The yellow-brown color of smog in the air is due in part to the nitrogen dioxide present, since this gas absorbs visible light near 400 nm (Figure 1).
Short-term NO2 exposure (30 min to 1 day) leads to adverse respiratory effects in healthy people and increased respiratory symptoms in people with asthma. NOx reacts with ammonia and other compounds to form particulates. These small particles can penetrate the lungs and cause respiratory problems, including emphysema and bronchitis. Individuals who spend a lot of time on the road or who live near a roadway experience considerably higher exposure to NO2.
Due to the impact it has on human health and the environment, the U.S. Environmental Protection Agency (EPA) has classified NO2 as a criteria pollutant and has set the primary standard at 100 ppb (98th percentile of 1-h daily maximum concentrations, averaged over 3 years) and 53 ppb (annual mean). Considering that on-road vehicles account for approximately 1/3 of NOx emissions in the U.S., automobile emissions are therefore regulated through the Clean Air Act. The U.S. EPA established emission standards that automobile manufacturers must follow when producing cars. Currently, Tier 2 emission standards set that manufacturers must have fleet average NOx emissions of no more than 0.07 g/mile.
One way manufacturers meet this standard is by using catalytic converters on their cars. This device is placed between the engine and the tailpipe. The exhaust stream passes through the catalytic converter and is exposed to a catalyst. A reduction catalyst of platinum and rhodium is used to reduce the NOx concentration in the exhaust. When an NO or NO2 molecule in the exhaust contacts the catalyst, the nitrogen atom is grabbed off the molecule and held onto by the catalyst. The oxygen is freed and forms O2. The nitrogen atom on the catalyst binds with another nitrogen atom held on the catalyst to form N2.
Catalytic converters have greatly reduced the emissions of NOx from car exhaust &#8211; up to 80% reduction, when performing properly. However, they only work when they have reached a fairly high temperature. Therefore, when doing a cold start of a car, the catalytic converter is removing virtually no NOx. It isn&#8217;t until the catalytic converter reaches higher temperatures that it effectively removes the NOx from the exhaust stream. Catalytic converters do not work on diesel passenger cars due to the lean conditions under which they operate. In addition, the sulfur in diesel fuel also deactivates the catalyst. The NOx in diesel engines are reduced mainly through the exhaust gas recirculation (EGR) valve, which cools the temperature of the combustion gases. As a result, diesel cars generally emit more NOx than gasoline cars.
<img alt="Figure 1" src="/files/ftp_upload/10076/10076fig1.jpg" /> 
Figure 1. Characteristic coloration for smog in California in the beige cloud bank behind the Golden Gate Bridge. The brown coloration is due to the NOx in the photochemical smog.

Determination Of NOx in Automobile Exhaust Using UV-VIS Spectroscopy

Source: Laboratories of Margaret Workman and Kimberly Frye - Depaul University
In the troposphere, ozone is naturally formed when sunlight splits nitrogen ...

Environmental Sciences

Environmental Science

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-Vis Spectroscopic Characterization of Nanomaterials in Aqueous Media

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