Embed Size (px)
Citation preview
5.1 5.0 4.9 4.8 4.7 4.6 4.5
0
1x108
2x108
3x108
4x108
a.u.
ppm (water)
2.0 1.5 1.0 0.5 0.0
0.00
2.50x106
5.00x106
7.50x106
1.00x107
10% solution of HCl (conc.)in D
2O a.u.
ppm (sample+standard)
Introduction
Large quant i t ies of s tarch are c h e m i c a l l y a n d / o r p h y s i c a l l y m o d i f i e d t o o b t a i n d e s i r e d p r o p e r t i e s f o r d i f f e r e n t a p p l i c a t i o n s . O n e i m p o r ta n t 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 l i fe, freeze/thaw stability, cold storage stability, cold water swelling and reconstituting properties to a formulated product.
I t i s e s s e n t i a l t o s t u d y t h e relationships between modification procedure, functional properties and structure to enable control of t h e d e r i v a t i s a t i o n p r o c e s s . Therefore, it is of great importance to reliably determine parameters like the hydroxypropyl level (% HP) in modified starches.
S e v e r a l m e t h o d s h a v e b e e n reported, including a colourimetr. method, IR and NMR.
Abstract / Resumen
Improved methods using h igh-resolution NMR were evaluated as r a p i d m e a s u r e m e n t s f o r determin ing the hydroxypropyl content of modified starch.
Six samples of modified starch were measured and the results were compared with the results of other k n o w n m e t h o d s l i k e I R , c o l o u r i m e t r i c a n d t h e F o o d -Chemical-Codex.
The d i ffe ren t advan tages and disadvantages of the mentioned methods were e labora ted and
Métodos opt imizados probados usando RMN de alta resolusión fueron evaluados para una medida rápida en la determinación del contenido de grupos hidroxipropilo en almidones modificados.
S e i s m u e s t r a s d e a l m i d o n e s modificados fueron analizadas y los r e s u l t a d o s o b t e n i d o s f u e r o n comparados con los resultados conocidos de otros métodos tales como IR, métodos colorimétricos y m é t o d o s d e l F o o d - C h e m i c a l -Codex.
Materials and Methods
NMR measurements
NMR in Dimetylsulfoxid:
! 10 mg of modified starch (not dried) + 1 ml of DMSO-d in 25 ml test-tube 6
! 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 D O) as internal standard2
! 0.5 ml of suspension transferred into NMR-tube
! shaked / not shaked (see Figure 5)
! s p e c t r a s a c q u i r e d o n 4 0 0 M H z -B R U K E R - U l t r a S h i e l d - N M R -Spectrometer
! see Figure 1
NMR in hydrochloric acid:
! 50 mg modified starch + 1 ml 10 % HCl (conc. HCl with D O)2
! solution heated in water bath for 15 minutes + cooling down
! + internal standard (acetic acid, 50 mg in 1 ml D O)2
! measurement see above (Figure 1)
NMR in trifluor-acetic acid:
! Instead of 10 % HCl (conc. HCl with D O) 2
20 % solution of trifluor-acetic acid (100 % for spectroscopy in D O) was used2
0.25 mg of starch + 0.5 mg of KBr mixed, tr i turated and transferred to a die for pressing
! die evacuated for 5 min2 ! pressed under a load of 100 kg/cm for 5
2min, then 200 kg/cm for 10 min
Colourimetric method
IR measurements
! spectra acquired using a NICOLET-IMPACT 400D (32 scans, background-corrected)
! see Figures 2 and 4
! spectrophotometric determination of hydroxypropyl-group was carried out us ing the method of Johnson (see References)
! method involves hydrolysis of the group to p ropy lene g l yco l wh i ch i n t u rn ( c o n c e n t r a t e d s u l f u r i c a c i d ) i s dehydrated to propionaldehyde and the enolic form of allyl alcohol
! p r o d u c t s a r e m e a s u r e d spectrophotometrically after reaction with ninhydrin (purple color)
! see Figure 3
Food-Chemical-Codex
! method was performed using the Food-Chemical-Codex (see References)
540 560 580 600 620 6400.25
0.30
0.35
0.40
0.45
0.50
0.55
0.60
0.65
wavelength (nm)
a.u.
3020 3000 2980 2960
-2.0x10-4
0.0
2.0x10-4
4.0x10-4
a.u.
wavenumber (cm-1)
4000 3000 2000 10000.0
0.5
1.0
1.5
2.0
2.5
3.0
a.u.
wavenumber (cm-1)
6 5 4 3 2 1 0
0.0
2.0x106
4.0x106
6.0x106
8.0x106
1.0x107
FIDRES 0.126314 HzAQ 3.9584243 secDW 60.400 usecDECIM 16DIGMOD digitalDDR 2TE 300.0 KNUC1 1HPL1 0.40 dB
F2 - Processing ParametersSI 32768SF 400.1300000 MHzWDW EMLB 0.30 HzPC 1.00SSB 0GB 0
F2 - Acquisit ion ParametersINSTRUM spectPROBHD 5 mm QNP 1H/15N/13C/31PPULPROG zg30TD 65536NS 16SWH 8278.146 HzFW 93750.00 HzRG 35.925DWOV 3.775 usecDIGTYP SADC+DR 18DE 6.00 usecD1 1.00000000 secP1 9.00 usecSFO1 400.1324710 MHzAQ_mod DQDSOLVENT D2ODS 2
sample (-O-CH
2-CH(-CH
3)-OH)
standard (CH
3COOH)
a.u.
ppm
Figure 1 (NMR):
The % HP was calculated using the
integrals of the signal of the methyl-
protons of the acetic acid (around 1.5
ppm) and the signal of the methyl-
protons of the hydroxypropyl-group
(around 0.5 ppm).
Figure 2 (IR):
-1The shoulder at 2974 cm (C-H stretch of the
methy l -g roup) i s charac te r i s t i c fo r the
hydroxypropyl-group.
Figure 4 (IR):
The average of 2-4 spectras were two times
automatical ly smoothed and the second
derivative of the absorbance was calculated. -1The area of the peak centered at 2974 cm
minus the area of a “blank” (unmodified starch)
i s p r o p o r t i o n a l t o t h e a m o u n t o f
hydroxypropylation (areas obtained by drawing
a horizontal baseline, see Figure).
Figure 3 (colourimetric):
The figure shows the spectra of the resulted
product (ninhydrin reaction) of the Johnson
(References) method. The maximum is clearly
v is ib le at 590 nm and the absorbence is
p r o p o r t i o n a l t o t h e a m o u n t o f
hydroxypropylation.
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 NMR-
tubes well when using DMSO-d as solvent.6
Figure 8:
This f igure shows good agreement
between the methods using F CCOOH 3
and HCl, although the % HP values
obtained with HCl were slightly higher
(reason unknown).
Figure 9:
The p lo t (see F igure) shows good
agreement between the NMR and the IR
method. Note that the IR method delivers
areas only.
Figure 10:
T h e % H P v a l u e s o b ta i n e d w i t h t h e
colourimetric method were all significant
lower than the values obtained with the NMR
method (assumed reason: the critical 3 min-
heating-period at 100 ºC, References).
Figure 11:
The ag reemen t be tween t he 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.
0 1 2 3 4 50
1
2
3
4
5
%H
P(N
MR
inH
Cl)
% HP (NMR in F3CCOOH)
R=0.99518
0 1 2 3 4 50.000
0.002
0.004
0.006
0.008
0.010
Are
ain
a.u.
(IR
)
% HP (NMR in F3CCOOH)
R=0.99518
0 1 2 3 4 50
1
2
3
4
5
R=0.94733
% HP (NMR in F3CCOOH)
%H
P(c
olou
rimet
ric)
0 1 2 3 4 50
1
2
3
4
5
R=0.96824
%H
P(F
CC
)
% HP (NMR in F3CCOOH)
Figure 6 / 7:
This figures show two parts of the NMR-
spectrum of the F CCOOH and the HCl 3
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.
5.1 5.0 4.9 4.8 4.7 4.6 4.5
0
1x108
2x108
3x108
4x108
ppm (water)
a.u.
2.0 1.5 1.0 0.5 0.0
0.00
2.50x106
5.00x106
7.50x106
1.00x107
20% solution of F
3CCOOH (100%)
in D2O
ppm (sample+standard)
a.u.
Conclusions
For fast and approximate determination: NMR method in DMSO-d 6
(shaked) is sufficient
! For accurate and fast analysis: NMR in F CCOOH is the recommended 3
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
!
References
! Stahl H., McNaugh R.P. A rapid nuclear magnetic resonance method for determining hydroxypropyl group in modified starch. Ceral Chemistry 47: 345-350 (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.
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.
0.0
0.2
0.4
0.6
0.8
1.0
**
NMR in NMR in DMSO F
3CCOOH shaked not shaked
%H
Psa
mpl
e/%
HP
sam
ple,
max 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
m e t h o d s . T h e d i s a d v a n ta g e s o f t h e
colourimetric and the FCC method are their
experimental times.
method
standard
deviation / mean
approx.
experiment time
NMR in F3CCOOH
5.8 %
0.5 h
NMR in HCl
3.5 %
0.5 h
NMR in DMSO
> 15 %
0.3 h
IR
11.4 %
0.5 h
colourimetric
5.3 %
5.0 h
FCC
> 15 %
12 h
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 IndustrialAv. Gral. Paz e/ Constituyentes y Albarellos, San Martin (1650), Buenos Aires, ARGENTINA
[email protected] / [email protected]/ Fax +(54) 11 4 753 5749
