UV-1900i/UV-2600i/UV-2700i UV-VIS Spectrophotometers
Quantitative Determination of Surface Functional
Application Groups of Cellulose Nanofibers Using Toluidine
News Blue O Adsorption Method
Kazumi Kawahara1, Jun Araki2
1 Shimadzu Corporation, 2 Faculty of Textile Science and Technology, Shinshu University
User Benefits
Simple quantitative determination of the surface functional groups of cellulose nanofibers (CNF) is possible with a small sample
amount on the order of a few mg1) per measurement.
Quantitation results with high repeatability and low dependence on individual skill can be obtained.
Due to the extremely short time of one measurement with a UV-Vis spectrophotometer, it is possible to conduct quantitative
analyses of 50 specimens in about 2.5 hours*.
* Here, 2.5 hours means the actual measurement time with the UV-Vis spectrophotometer, and does not include the time required for sample preparation procedure and time spent
in cell insertion/removal before and after measurements.
Introduction Fig. 2 shows the sample preparation procedure in the TBO
Cellulose is one of the polysaccharides which are the main adsorption method. The CNF suspension used in this
components of plant cell walls. Among the various types of experiment was a commercially-available viscous product.
cellulose, those with a fiber diameter of 4 to 100 nm, length of
several μm, and aspect ratio of 100 or more are called cellulose
nanofibers (CNF), and have attracted attention as a leading-edge
biomass material. In addition to the light weight and high Sample Preparation Procedure
strength of CNF, they also have other excellent functions,
① Dilute the CNF suspension with water to a sample
including a high gas barrier property, adsorption and
transparency, and as a plant-derived material, the environmental concentration of 2.5 wt/%, and stir for 10 min using
impacts of production and disposal are small. In the future, use in ultrasonic, a shaker, or the like so as to obtain a
automotive components, electronic materials, packaging uniform aqueous suspension.
materials and other applications are expected.
② Add 0.1 ml of the uniform CNF aqueous suspension
The surface of nanocellulose materials can be modified with prepared in ①, 2.9 ml of pure water, and 2 ml of a
surface functional groups such as the sulfate groups and carboxy
TBO solution to a 5 ml microcentrifuge tube.
groups to add various functions. In water, the ionic parts of these
surface functional groups function as charged groups, improving ③ Shake for 2 h using a shaker.
water dispersibility. Conventionally, the conductometric titration
method had been used in quantitative analysis of these surface ④ Conduct centrifugal separation at 3500 rpm or higher
charged groups. Although this is a general-purpose technique, it for 10 min using a centrifugal separator to sufficiently
has a number of problems, including ① a large amount (several precipitate the suspended solids.
hundred mg) of sample material is required, ② the measurement
time is long, ③ visual confirmation is necessary, and ④ the results
differ depending on the analyst. Therefore, a simple method which
does not depend on the skill of the individual analyst has been
needed to solve these problems. This article introduces an
example of a quantitative analysis of surface functional groups by
the toluidine blue O (TBO) adsorption method using a Shimadzu
ultraviolet-visible light (UV-Vis) spectrophotometer. This
experiment was carried out with the cooperation of Prof. Jun
Araki of Shinshu University.
Dye-adsorbed CNF aqueous suspension
Toluidine Blue O (TBO) Adsorption Method after centrifugal separation
TBO is a basic dye which is used in staining various types of ⑤ Using a micropipette, take 0.45 ml of the supernatant
specimens such as cell substrates. Fig. 1 shows the structure of TBO. in ④, and dilute 10x with 4.05 ml of pure water.
H3 C N -
Cl
H2 N + CH3
S N
CH3
Fig. 1 Structural Formula of TBO
Quantitative analysis of surface functional groups is possible by
adsorbing the basic dye TBO on anionic surface groups (here, the
carboxy groups) on the surface of CNF1), 2), 3). If this method is used,
the amount of sample material necessary for quantitative analysis is
very small, being on the order of a few mg1). Furthermore, because a
CNF aqueous suspension after
UV-Vis spectrophotometer is used, the time required for one
10x dilution of the supernatant
measurement is short, and quantitative analysis of 50 specimens in
around 2.5 hours is possible. It should be noted that this time is the
time required for the actual measurement with the UV-Vis Fig. 2 Sample Preparation Procedure
spectrophotometer, and does not include the time necessary for Using Toluidine Blue O Adsorption Method
the sample preparation procedure and time spent in
insertion/removal of the cells before and after measurements.
Application
News
Measurement of Spectrum of Dye-Adsorbed Quantification of Surface Functional Groups
CNF Supernatant Solution The surface carboxy groups were quantified by calculation using
The supernatant of the CNF aqueous suspension sample Eq. (1) 1).
prepared by the protocol described above was measured using 𝐴𝐴
𝑐𝑐𝑇𝑇𝑇𝑇𝑇𝑇 × 2 × 10−3 − ( 𝑤𝑤 × 5 × 10−2)
a Shimadzu UV-1900i UV-Vis spectrophotometer. The surface of 𝜀𝜀𝑤𝑤
𝑆𝑆𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶 =
the CNF measured in this experiment was modified with the 𝑐𝑐𝐶𝐶𝐶𝐶𝐶𝐶 × 𝑣𝑣 × 10−3
carboxy groups by the 2,2,6,6 tetramethylpiperidine1-oxyl free
radical (TEMPO) oxidation method using the TEMPO, which is a 𝐴𝐴
𝑐𝑐𝑇𝑇𝑇𝑇𝑇𝑇 × 0.2 − ( 𝑤𝑤 × 5)
stable nitroxyl radical. Fig. 3 shows the appearance of the 𝜀𝜀𝑤𝑤
instrument, and Table 1 gives the measurement conditions. Fig. 4 = × 104 … (1)
𝑐𝑐𝐶𝐶𝐶𝐶𝐶𝐶 × 𝑣𝑣
shows the absorption spectrum of the supernatant solution of the
TEMPO-oxidized CNF (TOCN). Since there is a possibility that the where, SCOOH: concentration of surface carboxy groups of CNF,
TBO used in quantitation may be adsorbed on quartz glass, CTBO: concentration of TBO solution at time of mixing, εw: molar
which is used in measurements, use of disposal-type cells is absorption coefficient, CCNF: concentration of CNF suspension, v:
recommended if multiple measurements are to be conducted. volume of CNF suspension, Aw: absorbance at 628 nm. Since this
sample contained only the carboxy groups, the total number of
surface charged groups corresponds to the total carboxy groups.
It may be noted this calculation was done using Excel®, which
enables quantification simply by inputting the absorbance
value and concentrations of the CNF suspensions1).
Table 2 shows the carboxy group concentration SCOOH calculated
by using the above-mentioned equation and the absorbance
value at 628 nm measured by the UV-Vis spectrophotometer.
The measurements and calculations were repeated 5 times. The
Fig. 3 Appearance of UV-1900i coefficient of variation (CV) in the table is one index showing
the variation of data. Here, it was found that the CV of the
Table 1 Measurement Conditions carboxy group concentration was 0.0324.
Instrument : UV-1900i Table 2 Quantification Results
Measurement Wavelength Range : 400 - 900 nm
Data Interval : 1 nm Absorbance value SCOOH
Repetition No.
Scanning Speed : High at 628 nm (Abs.) (mmol/g)
Slit Width : 1 nm
Light Source Switching Wavelength : 340 nm 1 0.557 2.098
2 0.568 2.050
0.80
3 0.554 2.112
4 0.550 2.129
0.60
5 0.592 1.944
Absorbance (Abs.)
Average value 2.067
0.40
Standard deviation (SD) 0.067
0.20 Coefficient of variation (CV)* 0.0324
* Coefficient of variation = Standard deviation / Average value
0.00
400.00 500.00 600.00 700.00 800.00 900.00
Wavelength (nm) Conclusion
Fig. 4 Absorption Spectrum of TEMPO-Oxidized CNF Supernatant Solution Quantitative determination of the surface functional groups of
CNF was carried out using the toluidine blue O (TBO) adsorption
method. Simple and quick quantification of the carboxy groups
From Fig. 4, it can be understood that the supernatant solution was possible by using a UV-Vis spectrophotometer and a TBO
shows its absorption maximum at 628 nm. It is possible to solution. Higher efficiency in quality control of CNF-based
quantify the surface functional groups by using the absorbance products can be expected by using this technique.
value at this 628 nm wavelength.
<Acknowledgement>
The authors wish to thank Principal Investigator Dr. Mitsuharu
Matsumoto of Kyodo Milk Industry Co., Ltd. for his generous
cooperation, including providing the samples used in these
measurements.
<References>
1) Jun Araki(2021), Cellulose vol. 28, 7707–7715(2021).
[Link]
2) Jun Araki(2022), Cellulose vol. 29, 8489–8490(2022)
3) Jun Araki, Minami Nakajima (2022), Cellulose vol. 30, 849–855
(2023)
01-00510-EN First Edition: Sep. 2023
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