improved methods for the determination of hydroxypropylation in

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

alicia@inti.gov.ar / eelopez@inti.gov.ar/ Fax +(54) 11 4 753 5749