Enhance Your UV-Vis Spectroscopy With a Wide Dynamic Range
App Note / Case Study
Last Updated: June 3, 2024
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Published: November 2, 2023
Credit: iStock
UV-Vis spectroscopy is a well-established tool for routine quantitative analytical methods. To achieve the highest quality results, it is important to choose a spectrophotometer with a wide linear dynamic range, thus enabling the accurate quantification of low- and high-concentration samples.
This application note highlights a spectrophotometer that offers advanced photometric performance in a routine system for the direct measurement of liquid and solid samples.
Download this application note to learn more about a spectrophotometer that is:
- Ideal for analyzing low- and high-concentration samples
- Intuitively designed for easy sample loading irrespective of a user’s experience
- Equipped with a powerful xenon flash lamp, eliminating the need for daily warmup
Technical Overview
Abstract
Measuring sample concentrations of analytes by ultraviolet visible (UV-Vis)
spectroscopy is a well-established approach used in many laboratories. With its
wide linear dynamic range, the Agilent Cary 3500 Flexible UV-Vis spectrophotometer
offers best-in-class research performance in a routine bench-top UV-Vis
spectrophotometer. Instrument performance was investigated by analyzing
potassium permanganate solutions ranging from 0.03 to 490 mg/L.
Agilent Cary 3500 Flexible UV-Vis
Spectrophotometer: Benefits of a
Wide Dynamic Range
Enabling fast, easy, routine quantitative
measurements of low to high concentration samples
Figure 1. The Agilent Cary 3500 Flexible UV-Vis spectrophotometer offers superior photometric
performance in a routine system for direct measurements of liquid and solid samples. It is ideal for
analyzing low to high-concentration samples.
2
Introduction
The Agilent Cary 3500 Flexible UV-Vis is a double-beam
spectrophotometer equipped with a powerful xenon flash
lamp eliminating daily warmup. The lamp comes with a
10-year warranty (for Cary 3500 instruments purchased from
Agilent or participating partners) that drastically reduces the
frequency and cost of lamp replacement for ultimate peace
of mind. The Cary 3500 Flexible UV-Vis spectrophotometer
offers advanced photometric performance in a routine
system for direct measurements of liquid and solid samples.
For added flexibility, the instrument includes a large sample
compartment that can accommodate a range of accessories
for the analysis of a variety of sample sizes and types.
The linear dynamic range (LDR) of the Cary 3500 Flexible
UV-Vis goes beyond 6 Abs, providing best in class research
performance in a routine benchtop UV-Vis spectrophotometer.
The wide LDR allows for direct measurements of highly
absorbing samples, minimizing sample preparation time, and
increasing the efficiency and accuracy of the analysis.
In addition to an extended LDR, the Cary 3500 Flexible UV-Vis
is configured for routine and flexible use. The system has
a large sample compartment to facilitate sample handling
and user access, while maintaining a small footprint.
The Cary 3500 Flexible UV-Vis is also equipped with a custom
cell holder base that can accommodate both 1 cm and longer
pathlength cells. The cell holder base is designed so that cells
can be loaded quickly in a fixed position, ensuring accurate
placement of samples irrespective of user-experience
or expertise.
This technical overview demonstrates how high (up to
490 mg/L), and low (0.03 mg/L) concentration solutions
of potassium permanganate (KMnO4
) were measured
using the Cary 3500 Flexible UV-Vis. The required LDR for
the measurements was achieved by switching between a
standard 1 cm pathlength cell and a 10 cm pathlength cell.
Changing the variable pathlength cell holder to the desired
pathlength is quick and easy, with no tools required. Once in
position, no further alignment of the cell holder is necessary,
allowing many different sample types to be analyzed with
speed, accuracy, and confidence.
Experimental
Standard KMnO4
solutions
– A series of aqueous KMnO4
solutions of known
concentration from 0.03 to 500 mg/L were prepared in
20 mL volumetric flasks through appropriate dilution using
distilled water.
– A standard 1 cm pathlength cell was used to measure
KMnO4
solutions at 5, 50, 100, 200, 300, 350, 400, 420,
460, 490, and 500 mg/L.
– A rectangular 10 cm pathlength cell was used to measure
the absorbance of the KMnO4
solutions at 0.03, 0.04, 0.05,
0.1, 0.3, 0.5, 0.8, 1, 2, 3, 5, 8, and 10 mg/L.
Note: It is important to rinse the sample cell with each
solution between measurements. Make sure that the
optical faces are dry and free from fingerprints before each
measurement using lint-free tissues.
Instrumentation
The Cary 3500 Flexible UV-Vis spectrophotometer
was fitted with the standard 1 cm cell holder
(part number G9886-68005) and the variable pathlength
rectangular cell holder (part number G9886-68003).
Depending on the concentration of the KMnO4
standard
solutions, the standards were added to either 1 or 10 cm
pathlength quartz cells. Absorbance measurements for
KMnO4
solutions were acquired using the Agilent Cary UV
Workstation software, version 1.4. Instrument operating
parameters are shown in Table 1.
Parameter Setting
Wavelength Range (nm) 400 to 750
Data Interval (nm) 0.5
Signal Averaging Time (s) 1.0
Baseline Distilled water
Table 1. Experimental parameters for the Agilent Cary 3500 Flexible
UV-Vis spectrophotometer.
3
Results and discussion
Analysis of high concentration samples using a standard
1 cm pathlength cell
The LDR of the Cary 3500 Flexible UV-Vis spectrophotometer
was determined by measuring 5, 50, 100, 200, 300, 350, 400,
and 420 mg/L of KMnO4
solutions in a 1 cm standard cell. The
UV-Vis spectra of the eight standard solutions are shown in
Figure 2A. Figure 2B shows the calibration plot of absorbance
taken at 525 nm versus concentration (mg/L). An excellent
linear calibration graph up to 6.6 Abs with a correlation
coefficient of R2
= 0.9995 was obtained. These results
confirm that quantitative analysis of high concentration
KMnO4
solutions up to 420 mg/L (6.60 Abs) is achievable with
the Cary 3500 Flexible UV-Vis using a standard 1 cm cell.
However, by increasing the signal averaging time to
10 seconds, higher absorbance measurements were
achievable for KMnO4
solutions up to 490 mg/L (Figure 3A).
Corresponding calibration curve is shown in Figure 3B and
has an excellent linear trend with a correlation coefficient of
R2
= 0.9997.
Figure 2. (A) Spectra of aqueous KMnO4
solutions measured using a 1 cm standard pathlength cell and signal averaging time of 1 second. (B) Calibration plot of
absorbance (at 525 nm) versus concentration (mg/L) with excellent linearity, as shown by the correlation coefficient of R2
= 0.9995.
0
1
2
3
4
5
6
7
8
400 450 500 550 600 650 700 750
Absorbance
Wavelength (nm)
A
y = 0.0156x – 0.0296
R² = 0.9995
0 50 100 150 200 250 300 350 400 450
Absorbance
Concentration (mg/L)
0
1
2
3
4
5
6
7
8 B
Figure 3. Analysis of high concentrations of aqueous KMnO4
solutions up to 7 Abs using a standard 1 cm pathlength cell and signal averaging time of 10
seconds. (A) The UV-Vis spectra of the eight standard solutions (B) Calibration plot of absorbance (at 525 nm) versus concentration (mg/L) with excellent linearity
(R2
= 0.9997).
Wavelength (nm)
0
1
2
3
4
5
6
7
8
400 450 500 550 600 650 700 750
Absorbance
A
Concentration (mg/L)
Absorbance
y = 0.0148x + 0.0595
R² = 0.9997
0
1
2
3
4
5
6
7
8
0 50 100 150 200 250 300 350 400 450 500
B
4
Analysis of low concentration samples using a 10 cm
pathlength cell
The Cary 3500 Flexible UV-Vis spectrophotometer is equipped
with a specially designed cell holder base that is designed to
accommodate cell holders with different pathlengths. The cell
holder base allows the use of a variable pathlength holder for
rectangular cells (2, 4, 5, and 10 cm) and a variable pathlength
holder for cylindrical cells (2, 4, 5, and 10 cm). The variable
pathlength cell holders come with predefined positions
to ensure the optimized setting is used for the desired
cell pathlength.
Figure 4A shows the variable pathlength rectangular cell
holder adjusted to support the 10 cm pathlength cell. The cell
holder is adjusted by sliding the carriage ends over the guided
rail of the cell holder base at the correct pathlength and
tightening the thumbscrews. No further alignment of the cell
holder is necessary. Low concentration KMnO4
solutions, less
than 10 mg/L, were analyzed using the 10 cm long pathlength
cell, as shown in Figure 4B.
Qualitative and quantitative analysis of low concentration
KMnO4
solutions in the range of 0.03 to 10 mg/L are
shown in Figure 5. The inset of Figure 5A shows that the
spectral profiles of the spectra obtained for the lowest
concentration samples (0.10, 0.05, and 0.03 mg/L) were
identical to the profiles of the more concentrated samples.
The corresponding calibration plot shown in Figure 5B has
excellent linearity (R2 = 0.9996). The lowest concentration
range of the calibration curve (inset of Figure 5B)
0.0
0.4
0.8
1.2
1.6
400 450 500 550 600 650 700 750
Absorbance
Wavelength (nm)
0.000
0.003
0.006
0.009
0.012
0.015
450 500 550 600
A
y = 0.1491x – 0.0055
R² = 0.9996
0.0
0.4
0.8
1.2
1.6
0246 8 10
Absorbance
Concentration (mg/L)
B
R² = 0.9999
0.00
0.02
0.04
0.06
0.08
0.0 0.1 0.2 0.3 0.4 0.5
Figure 5. (A) Spectra of standard KMnO4
solutions measured using a rectangular 10 cm pathlength cell. The inset shows the spectra obtained for the low
concentration standards 0.10, 0.05, and 0.03 mg/L. (B) Linear calibration plot of absorbance (at 525 nm) versus concentration (mg/L) with a correlation coefficient
of R2
= 0.9996. The inset shows the calibration plot for the lowest concentration range of 0.03 to 0.50 mg/L.
Figure 4. (A) The variable pathlength rectangular cell holder for the Agilent Cary 3500 Flexible UV-Vis spectrophotometer. Changing the variable pathlength cell
holder to the desired pathlength is quick and easy, requiring no tools or further alignment of the cell holder. (B) The Cary 3500 Flexible UV-Vis spectrophotometer
with a 10 cm pathlength cell filled with a KMnO4 solution.
A B
5
demonstrates the excellent photometric linearity
(R2
of 0.9999) of the Cary 3500 Flexible UV-Vis from 0.03 to
0.5 mg/L.
Limit of quantification
The limit of quantification (LOQ) is defined as the lowest
possible concentration of an analyte in a test sample that can
be quantified by an analytical method. The standard deviation
method as described by Equation 1 was used to determine
the quantification limit of KMnO4
.
Equation 1.
LOQ = 10 × σ/S
Where σ is the standard deviation of an appropriate number
of blank sample measurements and S is the slope of the
calibration curve.1
Note: According to the guidelines given by the International
Council for Harmonization of Technical Requirements for
Pharmaceuticals for Human Use (ICH), the estimation of σ
may be carried out in different ways. The standard deviation
of the blank is one such method that involves calculating the
standard deviation by analyzing an appropriate number of
blank samples.
The LOQ of KMnO4
was determined using the Cary 3500
Flexible UV-Vis spectrophotometer fitted with a standard
1 cm pathlength cell and a rectangular 10 cm pathlength cell.
Corresponding calibration curves are shown in Figures 2B
and 5B. To calculate the standard deviation (σ) of the blank,
sample measurements for each pathlength, absorbance
spectra were acquired during 10 replicate determinations
of the blank, and absorbance values were taken at 525 nm.
As can be seen in Table 2, the LOQs for KMnO4
for the 1 and
10 cm pathlength cells were calculated to be 0.029 and
0.003 mg/L, respectively. The LOQ demonstrates that
extremely low concentration samples, such as residual
and impurity absorptions, can be measured using the Cary
3500 Flexible UV-Vis spectrophotometer with a 10 cm
pathlength cell.
Scan
Absorbance of Blank at 525 nm
1 cm Pathlength 10 cm Pathlength
1 0.000063 0.000205
2 0.000079 0.000205
3 0.000171 0.000199
4 0.000175 0.000200
5 0.000176 0.000188
6 0.000188 0.000190
7 0.000192 0.000187
8 0.000188 0.000182
9 0.000190 0.000177
10 0.000188 0.000022
Standard Deviation (σ) 0.000046 0.000052
Slope of the Calibration Curve (S) 0.0156 0.1491
LOQ (mg/L) 0.029 0.003
Table 2. Determination of limit of quantification using 1 and 10 cm
pathlength cells.
Conclusion
The Agilent Cary 3500 Flexible UV-Vis is an innovative double
beam UV-Vis spectrophotometer that offers advanced
photometric performance over a wide LDR—ideal for routine
UV-Vis measurements. It is a double beam instrument
equipped with a powerful xenon flash lamp. The xenon lamp
has a 10-year warranty ensuring uninterrupted operation while
avoiding expensive and time-consuming lamp replacements.
The benchtop Cary 3500 Flexible UV-Vis includes a large
sample compartment to facilitate easy sample handling and
user access. It is also equipped with a specially designed cell
holder base that can accommodate variable pathlength cell
holders. Switching and positioning cell holders to achieve
the desired pathlength can be done quickly and accurately
without the need for tools, minimizing time-consuming
accessory handling.
The quantitative analysis of aqueous KMnO4
solutions
demonstrated the excellent linear dynamic range, beyond
6 Abs, for the Cary 3500 Flexible UV-Vis spectrophotometer.
High and low concentrations of aqueous KMnO4
samples
were quantified accurately using a standard 1 cm cell and a
10 cm long pathlength cell, respectively. The LOQ for KMnO4
was estimated to be 0.003 mg/L with 10 cm pathlength,
demonstrating the sensitivity of the Cary 3500 Flexible
UV-Vis spectrophotometer for analyzing extremely low
concentration samples.
The Cary 3500 Flexible UV-Vis measures highly absorbing
liquid or solid samples directly over a wide LDR, minimizing
sample preparation time and increasing the sensitivity and
accuracy of the analysis.
www.agilent.com
DE82183607
This information is subject to change without notice.
© Agilent Technologies, Inc. 2023
Printed in the USA, April 3, 2023
5994-5840EN
References
1. ICH Q2, Validation of Analytical Procedures: Text and
Methodology, November 2005.
Further information
– Cary 3500 Flexible UV-Vis Spectrophotometer
– Cary UV Workstation software
– Data Integrity Options for GMP Facilities for the Agilent
Cary 3500 UV-Vis Spectrophotometer Series
– UV-Vis Spectroscopy & Spectrophotometer FAQs
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