Potentiometric titration for determination of amylose ...

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Aug 17, 2011 - Potentiometric titration method was adapted from the method of Schoch (1964). 2.3.1. Reagents i. Potassium iodate solution for titration (0.0005 ...
Food Chemistry 130 (2012) 1142–1145

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Potentiometric titration for determination of amylose content of starch – A comparison with colorimetric method Derek X. Duan a,1, Elizabeth Donner a, Qiang Liu a,⇑, Damon C. Smith b, François Ravenelle b,2 a b

Guelph Food Research Centre, Agriculture and Agri-Food Canada, 93 Stone Road West, Guelph, Ontario, Canada N1G 5C9 Labopharm Inc., 480 Boul. Armand-Frappier, Laval, Québec, Canada H7V 4B4

a r t i c l e

i n f o

Article history: Received 18 October 2010 Received in revised form 4 May 2011 Accepted 31 July 2011 Available online 17 August 2011 Keywords: Amylose Auto-titrator Iodine Potentiometric Starch

a b s t r a c t Amylose, the amount of which varies significantly depending on the source, is one of the key components of starch. The proportion of the essentially linear amylose and the highly branched amylopectin greatly influences the physico-chemical properties of starch. In this study, potentiometric titration using an autotitrator and colorimetric methods were compared for determining amylose contents of a variety of starch samples. Potentiometric titration results for starches from a variety of botanical sources were within the reported literature ranges while the colorimetric method seemed to overestimate amylose content. Crown Copyright Ó 2011 Published by Elsevier Ltd. All rights reserved.

1. Introduction Starch is a natural polysaccharide that has been widely studied for many years in the food industry as well as for use in non-food applications (Ao et al., 2007; Dumoulin, Alex, Szabo, Cartilier, & Mateescu, 1998; Liu, Weber, Currie, & Yada, 2003; Ravenelle & Rahmouni, 2006). It is well known that starch is composed of two types of macromolecules: the essentially linear amylose and the highly branched amylopectin. Many of the physicochemical properties of starch, such as solubility, swelling ability, crystallinity, retrogradation and texture, which impact its intended use, are dependent on the ratio of amylose and amylopectin (Jane et al., 1999). More importantly, amylose-to-amylopectin ratio is a major factor affecting the formation of resistant starch (Xie, Liu, & Cui, 2006). A number of techniques, including potentiometric titration, iodine colorimetric determination, concanavalin A precipitation, high performance size exclusion chromatography (HPSEC) and differential scanning calorimetry (DSC) have been used for amylose determination (Zhu, Jackson, Wehling, & Geera, 2008). Iodine colorimetric determination method is the most frequently used method due to its simplicity and long history of usage

⇑ Corresponding author. Tel.: +1 519 780 8030; fax: +1 519 829 2600. E-mail address: [email protected] (Q. Liu). Present address: Eyegenix LLC, 1946 Young Street, Suite 288, Honolulu, HI 96826, USA. 2 Present address: Theratechnologies Inc., 2310, Boul. Alfred-Nobel, Montréal, Québec, Canada H4S 2B4. 1

in the food industry. However, this method requires a reliable pure amylose from different sources to construct a standard curve, which is problematic at times. Moreover, iodine also binds to long amylopectin chains and intermediate size polymers, albeit with a much lower affinity, which would affect the accuracy of the results (Himmelsbach, Barton, McClung, & Champagne, 2001). It has been found in our laboratory that iodine colorimetric determination gave higher amylose content values than expected. In this study, potentiometric titration using an auto-titrator and colorimetric methods were compared for determining amylose content of a variety of starch samples in order to find a more precise and efficient way for amylose content determination. 2. Materials and methods 2.1. Materials Potato starch was isolated from potatoes received from Agriculture and Agri-Food Canada Research Centres in Charlottetown, Prince Edward Island, Fredericton, New Brunswick, and Lethbridge, Alberta, following the isolation procedure of Liu et al. (2003). Dried starch was passed through a 125 lm sieve. All other starch samples including rice, wheat, normal corn, waxy corn starch, and potato amylose were obtained from Sigma Aldrich (St. Louis, MO, USA); tapioca and high amylose corn starch (Hylon V and VII) were obtained from National Starch and Chemical Company (Bridgewater, NJ, USA). Other reagents and chemicals were obtained from Sigma Aldrich (St. Louis, MO, USA).

0308-8146/$ - see front matter Crown Copyright Ó 2011 Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.foodchem.2011.07.138

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2.3.3. Apparatus An auto-titrator (Mettler Toledo DL50) equipped with a DM140 platinum electrode, DV1010 burette and LabX light titration software (version 2.1) (Mettler–Toledo Inc., Columbus, OH) was used for the titration of dispersed starch solutions at room temperature (21 °C). Titrant addition was based on the kinetics of the titration curve (dynamic addition), and measurement of potential was equilibrium controlled. Recognition of the equivalence point (EQP) was set by adjusting the threshold to appropriate values. Termination of the titration occurred after one equivalence point was reached, or the maximum volume of the burette (10.0 mL) was dispensed. The software reported the volume of titrant at EQP, iodine affinity (IA, %), and the content of amylose (%).

2.2. Amylose/iodine complex colorimetric method The apparent amylose content was measured based on the colorimetric method of Williams, Kuzina, and Hlynka (1970). Briefly, the starch samples were first dispersed in 0.5 N KOH, and after dilution and neutralisation with 0.1 N HCl, an iodine reagent was added and the absorbance of the blue complex was measured at 625 nm with a spectrophotometer. A standard curve was constructed using mixtures of potato amylose and waxy corn starch (100% amylopectin), maintaining the level of amylose at 20%. The reported values are means of duplicate measurements.

2.3. Potentiometric titration method 2.3.4. Calculation The iodine, which binds to amylose, was produced by the reaction of the titrant KIO3 with potassium iodide and hydrochloric acid, as shown in the following scheme:

Potentiometric titration method was adapted from the method of Schoch (1964).

KIO3 þ 5KI þ 6Hþ ! 3I2 þ 6Kþ þ 3H2 O

2.3.1. Reagents i. ii. iii. iv. v.

Iodine affinity was calculated from the volume of titrant (VEQ, mL) at EQP using the equation:

Potassium iodate solution for titration (0.0005 N) HCl (1 N) KOH (0.5 N) KI (0.4 N) Ascorbic acid (as primary standard to check the titer of potassium iodate solution)

IAð%Þ ¼

T  VEQ  c  z  M  100 m

where T is the titer of the titrant, c is the nominal concentration of the titrant (0.0005 N), z is the stoichiometric factor of the I2 production reaction (3), M is the molecular weight of I2 (254 g/mol), and m is the dry mass of the sample (mg). Titer of the titrant was determined using ascorbic acid as a primary standard. Briefly, 1–2 mg of ascorbic acid was added to a 100 mL titration beaker, in addition to 5 mL of 1 N HCl, 5 mL of 0.4 N KI and 50 mL of distiled water. The mixture was titrated with 0.0005 N potassium iodate until the equivalence point was reached. Titer was calculated using the following equation:

2.3.2. Sample Preparation A starch sample (20–50 mg, equivalent to 5–10 mg of pure amylose depending on the amylose content of the starch) was weighed into a 50 mL beaker. Ten millilitres of 0.5 N KOH was added, and the mixture was stirred magnetically at room temperature for 20 min or until complete dispersion of the starch. The dispersed starch solution was transferred to a 100 mL titrator beaker, using 50 mL of distilled water to quantitatively transfer the solution. 10 mL of 1 N HCl and 5 mL of 0.4 N potassium iodide solution were added to the beaker, which was installed on the auto-titrator for titration with 0.0005 N potassium iodate.



m  1000 ¼ titer VEQ  c  M  z

where m is the mass of ascorbic acid (g), VEQ is the volume of titrant at the equivalence point (consumption, mL), c is the nominal con-

60

370 Equivalence Point (EQP)

50

360

40

30 340 20 330 10 320

1st derivative 0

310

-10

inflection point signal 300

-20 0

1

2

3

4

5

6

7

8

Volume of titrant (mL) Fig. 1. Titration profile of ascorbic acid with potassium iodate.

9

10

1st derivative (mV/mL)

Potential (mV)

350

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value for potato amylose is 19.9%, corn 19%, tapioca 18.6% and wheat 19.9% (Schoch, 1964). Rice amylose has been reported to have an iodine affinity of 16.7% (Hamori & Kallay, 1972).

Equivalence Volume (VEQ, mL)

10 y = 0.112x + 0.243 R2 = 0.9995

9 Amylose 8 y = 0.3203x + 0.2534 R2 = 0.997

7 6

Amyloseð%Þ ¼

Potato Starch

IAstarch  100 IAamylose

5 4

2.3.5. Validation of the method A series of validation experiments on the precision, linearity, and determination limit of the method were carried out.

3 2 1 0 0

10

20

30

40

50

60

70

80

3. Results and Discussion

Sample mass (mg) Fig. 2. Linearity of the titration method with potato amylose and potato starch.

A typical titration profile of ascorbic acid with potassium iodate is shown in Fig. 1. The sharp peak of the 1st derivative of the potential indicated the equivalence point of the reaction. It should be noted that, unlike the potentiometric titration method by Schoch (1964), which determined the amount of bound iodine by extrapolation of bound iodine vs. free iodine, the consumed/bound iodine is directly calculated from the volume of titrant at the EQP (equivalence point). The titer of potassium iodate solution was found to be independent of the mass of the ascorbic acid standard used, and had a value of 1.01 ± 0.03, n = 18.

10 9 8 7

RSD (%)

3.1. Titration of ascorbic acid with potassium iodate

6 5 4 3 2 1

3.2. Validation of the titration method

0 0

10

20

30

40

50

60

70

80

Sample mass (mg) Fig. 3. Relative standard deviation of amylose content of potato starch as a function of sample mass. The determination limit was 3 mg of amylose (or 15 mg of potato starch) which could be titrated with good precision (RSD 61%).

centration of the titrant (0.0005 N), M is the molecular weight of ascorbic acid (176.12 g/mol), and z is the equivalent number (3) of the I2 production reaction. Amylose content was calculated from the ratio of iodine affinity of the starch to the iodine affinity of pure amylose. The literature

Linearity of the method was tested using potato amylose (1– 16 mg) and potato starch (6–75 mg). The method had excellent linearity as shown in Fig. 2, with r2 values of 0.9970 and 0.9995 for amylose and potato starch, respectively. The determination limit of the method, i.e. the smallest amount of amylose that could be titrated with good precision, was found to be around 3 mg of amylose (equivalent to 15 mg of potato starch, as shown in Fig. 3). When the amount of amylose was lower than the determination limit, 3 mg, relative standard deviation increased to above 1%, possibly due to the detection limit of the electrode. On the other hand, it should be noted that as sample mass

Table 1 Amylose content of potato starch by potentiometric and colorimetric methods.a

a b

Potato growing location

Potato variety

Amylose content of starch by potentiometric methodb (%, w/w)

Apparent amylose content of starch by colorimetric method (%, w/w)

Charlottetown, PEI

Goldrush Norland Yukon Gold Organic Goldrush Organic Norland Organic Yukon Gold

21.3 ± 0.1 19.2 ± 0.0 20.9 ± 0.3 21.0 ± 0.1 18.7 ± 0.3 20.3 ± 0.3

33.0 ± 0.1 31.4 ± 0.0 33.1 ± 0.2 32.5 ± 0.3 30.8 ± 0.3 31.7 ± 0.0

Fredericton, NB

15058–15 15060–03 F01013 F04012 F04054 F04056 F62008 H08675–21

19.1 ± 0.0 22.1 ± 0.2 20.2 ± 0.2 19.1 ± 0.3 20.2 ± 0.1 23.5 ± 0.1 21.4 ± 0.1 22.6 ± 0.0

29.8 ± 0.1 34.9 ± 1.5 31.3 ± 0.6 34.9 ± 0.1 32.6 ± 0.3 36.6 ± 0.0 33.7 ± 0.1 36.4 ± 0.4

Lethbridge, AB

AC Stampede Russet CV92028–1 CV92056–4 FV12486–2 V1102–1

21.4 ± 0.1 23.2 ± 0.6 24.9 ± 0.0 21.3 ± 0.1 23.1 ± 0.2

31.9 ± 0.5 35.2 ± 0.3 38.9 ± 0.3 32.5 ± 0.3 34.8 ± 0.2

Values denote means ± standard deviations, n = 2. Literature value of amylose content of potato starch is 21–24% (Williams et al., 1970).

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D.X. Duan et al. / Food Chemistry 130 (2012) 1142–1145 Table 2 Amylose content of commercial starches by potentiometric titration and colorimetric method, with a comparison to literature values. Starch

Amylose content by potentiometric methoda (%, w/w)

Amylose content by colorimetric methoda (%, w/w)

Literature valuesb

Corn, normal Corn, Hylon V Corn, Hylon VII Corn, waxy Potato Rice Tapioca Wheat

22.4 ± 1.2 55.1 ± 0.6 66.5 ± 0.6 1.0 ± 0.2 26.1 ± 0.5 15.2 ± 0.4 21.3 ± 0.3 22.7 ± 0.1

25.7 ± 0.3 41.3 ± 5.6 54.3 ± 0.6 6.3 ± 1.7 30.3 ± 0.2 18.0 ± 1.2 26.1 ± 0.1 30.4 ± 0.5

19–24% 50% 70%