IMPROVED METHODS for the DETERMINATION of HYDROXYPROPYLATION in MODIFIED STARCH USING NMR and
COMPARISON with CLASSICAL METHODS
Roland Gamsjaeger, Eduardo E. López* and Alicia Lagomarsino* Johannes Kepler University Linz, Altenbergerstrasse 69, 4020 Linz, AUSTRIA
* Centro de Investigación y Desarrollo en Química y Petroquímica, Instituto Nacional de Tecnologia Industrial Av. Gral. Paz e/ Constituyentes y Albarellos, San Martin (1650), Buenos Aires, ARGENTINA alicia@inti.gov.ar / eelopez@inti.gov.ar/ Fax +(54) 11 4 753 5749
Abstract / Resumen
Materials and Methods
Improved methods using highresolution NMR were evaluated as rapid measurements for determining the hydroxypropyl content of modified starch.
Six samples of modified starch were measured and the results were compared with the results of other known methods like IR, colourimetric and the FoodChemical-Codex.
The different advantages and disadvantages of the mentioned methods were elaborated and
Métodos optimizados probados usando RMN de alta resolusión fueron evaluados para una medida rápida en la determinación del contenido de grupos hidroxipropilo en almidones modificados.
Seis muestras de almidones modificados fueron analizadas y los resultados obtenidos fueron comparados con los resultados conocidos de otros métodos tales como IR, métodos colorimétricos y métodos del Food-ChemicalCodex.
Introduction
Large quantities of starch are chemically and/or physically modified to obtain desired properties for different applications. One important product group is hydroxypropylated starch that has a wide spectrum of application areas, mainly in food and food-related products. Use of hydroxypropylated starch gives improved shelf life, freeze/thaw stability, cold storage stability, cold water swelling and reconstituting properties to a formulated product.
It is essential to study the relationships between modification procedure, functional properties and structure to enable control of the derivatisation process. Therefore, it is of great importance to reliably determine parameters like the hydroxypropyl level (% HP) in modified starches.
Several methods have been reported, including a colourimetr. method, IR and NMR.
a. u. a. u.
Figure 1 (NMR):
Figure 2 (IR):
The % HP was calculated using the integrals of the signal of the methylprotons of the acetic acid (around 1.5 ppm) and the signal of the methylprotons of the hydroxypropyl-group (around 0.5 ppm).
The shoulder at 2974 cm-1 (C-H stretch of the methyl-group) is characteristic for the hydroxypropyl-group.
3.0 2.5
1.0x107 8.0x106 6.0x106 4.0x106 2.0x106
standard (CH COOH)
3
F2 - Acquisition Parameters
FIDRES 0.126314 Hz
INSTRUM spect
AQ 3.9584243 sec
PROBHD5 mm QNP 1H/15N/13C/31P DW 60.400 usec
PULPROG zg30
DECIM 16
TD 65536
DIGMOD digital
NS 16
DDR 2
SWH 8278.146 Hz
TE 300.0 K
FW 93750.00 Hz
NUC1 1H
RG 35.925
PL1 0.40 dB
DWOV 3.775 usec
DIGTYP SADC+
F2 - Processing Parameters
DR 18
SI 32768
DE 6.00 usec
SF 400.1300000 MHz
D1 1.00000000 sec
WDW EM
P1 9.00 usec
LB 0.30 Hz
SFO1 400.1324710 MHz
PC 1.00
AQ\_mod DQD
SSB 0
SOLVENT D2O
GB 0
DS 2
sample
(-O-CH -CH(-CH )-OH)
2
3
2.0 1.5 1.0 0.5 0.0
4000
3000
2000
1000
wavenumber (cm-1)
0.0
6543210
ppm
Figure 3 (colourimetric):
The figure shows the spectra of the resulted product (ninhydrin reaction) of the Johnson (References) method. The maximum is clearly visible at 590 nm and the absorbence is proportional to the amount of hydroxypropylation.
Figure 4 (IR):
The average of 2-4 spectras were two times automatically smoothed and the second derivative of the absorbance was calculated. The area of the peak centered at 2974 cm-1 minus the area of a “blank” (unmodified starch) is proportional to the amount of hydroxypropylation (areas obtained by drawing a horizontal baseline, see Figure).
a. u. a. u.
0.65 0.60 0.55 0.50 0.45 0.40 0.35 0.30 0.25
540 560 580 600 620 640
wavelength (nm)
4.0x10-4 2.0x10-4
0.0
-2.0x10-4 3020 3000 2980 2960
wavenumber (cm-1)
Figure 8: This figure shows good agreement between the methods using F3CCOOH and HCl, although the % HP values obtained with HCl were slightly higher
Figure 9: The plot (see Figure) shows good agreement between the NMR and the IR method. Note that the IR method delivers areas only.
(reason unknown).
Figure 5:
The differences (*) are all significant at a level of 0.01 (t-test). Therefore the DMSO method is an approximate method and it is strongly recommended to shake the NMRtubes well when using DMSO-d6 as solvent.
5
4
R=0.99518
3
0.010 0.008 0.006
R=0.99518
NMR measurements NMR in Dimetylsulfoxid: ! 10 mg of modified starch (not dried) + 1
ml of DMSO-d6 in 25 ml test-tube ! placed in a boiling-water bath for 5-10
min ! cooling down to room-temperature ! + 30 mg of solution of acetic acid (10-20
mg in 1 ml D2O) as internal standard ! 0.5 ml of suspension transferred into
NMR-tube ! shaked / not shaked (see Figure 5) ! spectras acquired on 400 MHz-
BRUKER-UltraShield-NMRSpectrometer ! see Figure 1
NMR in hydrochloric acid: ! 50 mg modified starch + 1 ml 10 % HCl
(conc. HCl with D2O) ! solution heated in water bath for 15
minutes + cooling down ! + internal standard (acetic acid, 50 mg in
1 ml D2O) ! measurement see above (Figure 1)
NMR in trifluor-acetic acid: ! Instead of 10 % HCl (conc. HCl with D2O)
20 % solution of trifluor-acetic acid (100 % for spectroscopy in D2O) was used
IR measurements 0.25 mg of starch + 0.5 mg of KBr mixed, triturated and transferred to a die for pressing ! die evacuated for 5 min ! pressed under a load of 100 kg/cm2 for 5
min, then 200 kg/cm2 for 10 min ! spectra acquired using a NICOLET-
IMPACT 400D (32 scans, backgroundcorrected) ! see Figures 2 and 4
Colourimetric method
! spectrophotometric determination of hydroxypropyl-group was carried out using the method of Johnson (see References)
! method involves hydrolysis of the group to propylene glycol which in turn (concentrated sulfuric acid) is dehydrated to propionaldehyde and the enolic form of allyl alcohol
!products are measured spectrophotometrically after reaction with ninhydrin (purple color)
! see Figure 3
Food-Chemical-Codex
! method was performed using the FoodChemical-Codex (see References)
Area in a.u.(IR)
% HP (NMR in HCl)
sample, max
sample
%HP / %HP
1.0
0.8
*
0.6
*
0.4
0.2
0.0
NMR in
NMR in DMSO
F CCOOH shaked not shaked 3
Figure 6 / 7:
This figures show two parts of the NMRspectrum of the F3CCOOH and the HCl method respectively. Comparing the figures, a clear difference in the relation of the signal of the sample-protons to the signal of the water-protons can be seen. Note also the slight differences in the chemical shift.
2.0 4x108
ppm (sample+standard)
1.5
1.0
0.5
0.0 1.00x107
2
1
0
0
1
2
3
4
5
% HP (NMR in F CCOOH) 3
Figure 10:
The % HP values obtained with the colourimetric method were all significant lower than the values obtained with the NMR method (assumed reason: the critical 3 minheating-period at 100 ºC, References).
5
4
R=0.94733
3
2
1
0.004
0.002
0.000
0
1
2
3
4
5
% HP (NMR in F CCOOH) 3
Figure 11: The agreement between the NMR method and the FCC is worse than with other methods. In addition the dispersion (see also Table 1) of the FCC method is high.
5
4
R=0.96824
3
2
1
% HP (FCC)
Table 1:
This table summarizes the dispersion and the experimental time of all used methods. The dispersion of the DMSO method and the FCC are both too high to get exact % HP values, the dispersion of the IR method is acceptable but also high in comparison to the other methods. The disadvantages of the colourimetric and the FCC method are their experimental times.
method
standard
approx.
deviation / mean experiment time
NMR in F3CCOOH NMR in HCl
5.8 % 3.5 %
0.5 h 0.5 h
NMR in DMSO IR
> 15 % 11.4 %
0.3 h 0.5 h
% HP (colourimetric)
3x108
20% solution of
7.50x106
F CCOOH (100%)
0
0
1
2
3
4
5
3
2x108
in D O 2
5.00x106
% HP (NMR in F CCOOH) 3
0
0
1
2
3
4
5
% HP (NMR in F CCOOH) 3
colourimetric FCC
5.3 % > 15 %
5.0 h 12 h
a. u.
a. u.
1x108
2.50x106
0.00 0 5.1 5.0 4.9 4.8 4.7 4.6 4.5
ppm (water)
2.0 4x108
ppm (sample+standard)
1.5
1.0
0.5
0.0 1.00x107
3x108 2x108 1x108
10% solution of HCl (conc.) in D O
2
7.50x106 5.00x106 2.50x106
0.00 0 5.1 5.0 4.9 4.8 4.7 4.6 4.5
ppm (water)
a. u.
Conclusions
! For fast and approximate determination: NMR method in DMSO-d6 (shaked) is sufficient
! For accurate and fast analysis: NMR in F3CCOOH is the recommended method, only disadvantage: expensive equipment (NMR in HCl: worse resolution because of water-signal)
! IR method: alternative to NMR, fast, less experimental effort, but high dispersion and secondary standards necessary
! Colourimetric method: critical heating period, high experiment time, large amount of sulfuric acid, high dispersion, but inexpensive
Acknowledgements
The authors thank Leonardo Nardini and Pablo Rouge for their general assistance during this work, Leandro Santos for his help with the NMR-measurements, and all other colleagues of Fármacos and Cequipe who made this work possible.
References
! Stahl H., McNaugh R.P. A rapid nuclear magnetic resonance method for determining hydroxypropyl group in modified starch. Ceral Chemistry 47: 345350 (1970).
! Forrest B., Cove L. Identification and quantification of hydroxypropylation of starch by FTIR. Die Staerke 44(5): 179-183 (1992).
! Johnson D.P. Spectrophotometric determination of the hydroxypropylgroup in starch ethers. Analytical Chemistry 41: 859-860 (1969).
! Food-Chemical-Codex III (FCC III). Pages 126-129, 514-515.
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