Some rules are summarized from the equation as follows:

surfactant concentration below its CMC. ii) me microspheres size also decreases when the stirring speed increases. But the degree is controlled by the surfactant concentration. iii) The higher viscosity ratio of oil phase to starch solution re- sults in larger microspheres; while higher density ratio of oil phase to starch solution results in smaller ones.

[2] Hop& H., H. Lussi, and H. Gerspacher: Makromol. Chem. 78

[3] Arshady. R.: Polymer Eng. and Sci. 29 (1989), 1747-1758. [4] Kartha, K. P. R., and H. C. Srivastava: StarchIStarke 37 (1985)

[5] Yuan, H . G. , G . Kalfas, and W: H. Ray: Rev. Macromol. Chem.

i) The microspheres size decreases with the increase of the (1964) 37-46.

297-306.

Phys. C31 (283) (1991), 215-299.

Address of authors: Yu Jiugao and Liu Jie. Department ofchemistry, Tianjin University, Tianjin 300072, P.R. China.

Bibliography [I] Hop& H., H. Lussi, and H . Gerspacher: Makromol. Chem. 78

(1964), 24-37. (Received: October 19, 1993).

lmwoved Method for the Determination of the Total Adipyl Content in Acetylated Adipyl Cross-linked Starches

Peter Sanders and Kommer Brunt, Groningen (The Netherlands)

An improved gas chromatographic determination of the adipyl con- tent in acetylated adipyl cross-linked starches using pimelic acid as in- ternal standard is described. The modifications provide an analytical protocol in which the amount of organic solvent (ethyl acetate) needed for each determina- tion was reduced tenfold and the capacity of the analysis was increa- sed threefold. Compared to the original method the accuracy of the improved deter- mination is the same, but the repeatability is about three times better. The standard deviation based on 55 independent duplicates, is 13 ppm adipic acid. The adipic acid concentration in the analyzed sam- ples varied from 0-1630 ppm.

Verbesserte Methode zur Bestimrnung des Gesarnt-Adipyl-Ge- haltes in acetylierten adipinvernetzten StYlrken. Eine verbesserte gaschromatographische Bestimmung des Adipingehaltes in acetylier- ten adipinvernetzten Starken unter Verwendung von Pimelinsaure als innerer Standard wird beschrieben. Die Modifizierungen liefern einen analytischen Bericht, in dem die Menge des benotigten organ- ischen Losungsmittel (Ethylacetat) fur jede Bestimmung auf ein Zehntel reduziert und die Kapazitat der Analyse auf das Dreifache er- hoht wurde. Verglichen mit der Originalmethode ist die Genauigkeit mit der verbesserten Bestimmung die gleiche, jedoch ist die Wieder- holbarkeit dreimal besser. Die Standardabweichung, basierend auf 55 unabhangigen Duplikaten, betragt 13 ppm Adipinsaure. Die Adipin- saurekonzentration in den analysierten Proben variierte zwischen 0 und 1630 ppm.

1 Introduction

Acetylated adipyl cross-linked starch is a modified starch used in food applications. The adipyl content in these cross-linked starches can be determined by a gas chromatographic method as described by Mitchell et a1 [l]. According to this method the sample is saponified with alkali in the presence of an internal standard glutaric acid (pentanedioic acid). During the saponifi- cation the adipyl group is hydrolyzed from the starch and forms free adipate. After acidifying the hydrolysate, the result- ing adipic acid (hexanedioic acid) and the internal standard, glutaric acid, are extracted with ethyl acetate. After removal of the ethyl acetate, the organic acids are silylated to their corre- sponding trimethyl silylesters. These are quantified by gas chromatography using a packed column with silicone oil as the active phase. This method is laborious and uses large quantities of ethyl acetate (300 ml per determination). In this paper some modifications and improvements of the method are described. For environmental, and economic rea- sons, and for improved efficiency, efforts were made to mini- aturize the analytical method, especially with respect to the amount of organic solvent needed for each determination.

Provided that the samples of cross-linked starch to be ana- lyzed are homogeneous, the sample weight can be decreased considerably. Consequently, the amount of organic solvent can be decreased, resulting in a considerable reduction of time needed for the evaporation to dryness of the ethyl ace- tate extracts. Moreover, we propose pimelic acid (heptane- dioic acid) as an internal standard instead of glutaric acid. The solubility of pimelic acid in water, and its extraction behavi- our, is more simular to adipic acid than glutaric acid. The improved methodology results in a reduction of about 90% in the use of ethyl acetate needed for each determination, and in an increase of about 200% in the daily capacity of anal- ysis.

2 Experimental Section 2.1 Chemicals

The following chemicals were used: - concentrated hydrochloric acid (Merck, Darmstadt), - sodium hydroxide (Merck, Darmstadt), - ethyl acetate (Merck, Darmstadt),

starchlstiirke 46 (1994) Nr. 7, S. 255-259 0 VCH Verlagsgesellschaft mbH, D-69451 Weinheim, 1994 0038-9056/94/0707-0255$05.00+.25/0 255

- adipic acid (hexanedioic acid) (Merck, Darmstadt), - glutaric acid (pentanedioic acid) (Merck, Darmstadt), - pimelic acid (heptanedioic acid) (Merck, Darmstadt), - 3-methyladipic acid (Sigma), - acetonitrile (Lab-Scan), - bis-(trimethylsily1)-trifluoroacetamide (BSTFA) which in-

- nitrogen gas (Hoek Loos).

2.2 Apparatus

cludes 1% trimethylchlorosilane (TMCS) (Pierce),

glass reaction tubes (100xl6mm) with screw cap fitted with PTFE covered rubber seals were used for the saponifi- cation, the extraction, the evaporation, and the silylation of the sample respectively the analyte. home made rotary shaker, adjustable Finn pipettes 0.2-1.0 ml, waterbath adjusted to 3OoC, Pierce Reacti-Vap 111 evaporation device to evaporate to dryness simultaneously the sample solutions in 27 tubes with a stream of nitrogen, ultrasonic bath, Carlo Erba Vega gas chromatograph equipped with a cold on-column injection system, and a flame ionisation detec- tor (temperature 3OO0C, hydrogen pressure 0.5 bar, air pres- sure 1.0bar). The separation was performed on a WCOT fused silica CP-sil 5CB capillary column (length 50 m, in- ternal diameter 0.32mm, film thickness 0.12pm) with he- lium as the carrier gas (pressure 0.7 bar). During the sepa- ration the temperature of the column oven was pro- grammed as follows: after injection the temperature was kept constant at 130°C for 1 min, then the temperature was raised at S°C/min up to 190”C, immediately followed by a fast temperature rise of 25’C/min to 290OC. The tempera- ture was kept at 290°C for 5 min, and then the oven was cooled down to 13OOC in order to get the instrument ready for the next injection. The retention times of adipic and pi- melic acid derivates are 10.3 min and 12.2 min, respective- ly.

2.3 Method

The investigations as described in this paper have resulted in the following protocol for the determination of the total adi- pyl content in acetylated adipyl cross-linked starches.

Sample preparation

50mg of the acetylated adipic cross-linked starch sample is weighed accurately in a glass reaction tube, and 1.5ml dis- tilled water, and 1.0 ml aqueous solution containing 0.05 mg pimelic acid/ml are added. The reaction tube is shaken to dis- perse the sample and 2.5 ml of 4 M sodium hydroxide solution are added. Agitation of the reaction tube is continued in order to dissolve the starch sample. The reaction tube is closed and the adipyl-starch ester bond is saponified by continuously ro- tating the tube during at least 5 min. Then 1.0 ml of concen- trated hydrochloric acid is added and the mixture is homoge- nized. 5 ml of ethyl acetate are added, the tube is closed, and shaken vigorously for at least 1 min to extract the adipic and pi- melic acid into the ethyl acetate. After phase separation the (upper) ethyl acetate layer is transferred with a glass Pasteur pipette into a clean glass reaction tube. The ethyl acetate ex- traction of the aqueous solution is repeated three times and the ethyl acetate fractions are collected. These collected frac- tions are evaporated to dryness with a nitrogen stream in a Pierce Reacti-Vap Evaporator at a temperature of 30°C in a wa- terbath. Then 0.3 ml of acetonitrile is added to the dry residue,

and the reaction tube is placed in an ultrasonic bath for sev- eral min to dissolve the residue. 0.3 ml of BSTFA/lO/o TMCS solution is added, and the mixture is homogenized again in the ultrasonic bath for several minutes. After a reaction time of at least 30min in a waterbath at a temperature of 3OoC, 0 . 3 ~ 1 of the reaction mixture is injected in the capillary gas chromatograph.

Calibration

~

256 starchlstarke 46 (1994) Nr. 7, S 255-259

Four 50 mg samples of waxy corn starch are weighed into four glass reaction tubes. 1.0 ml aqueous pimelic acid solution con- taining 0.05mg pimelic acid/ml is added into each tube fol- lowed by the addition of 0.00,0.25,0.50, and 1.00 ml aqueous adipic acid solution, containing 0.05 mg adipic acidlml, into the respective tubes. The volume is adjusted to 2.50 ml with distilled water and the procedure as described in the sample preparation section, beginning with .. “The reaction tube is shaken to disperse the sample ..”, is carried out.

3 Results and Discussion In this paper two improvements are described with respect to the published method for the determination of the total adipyl content in acetylated adipyl cross-linked starches [ 11. These improvements concern firstly the use of pimelic acid instead of glutaric acid as internal standard, and secondly a miniaturi- zation of the analytical method; a full discussion follows.

3.1 Internal standard

An internal standard can only be used succesfully when the physico-chemical properties of the analyte and the internal standard compound are simular. When using an internal standard in an analytical method the amount of analyte is usually calculated with the so-called rela- tive response factor. This factor is defined as the quotient be- tween the ratio of the peak area of the analyte and the internal standard and the ratio of the weights of the analyte and the in- ternal standard. For standard solutions of mixtures of adipic acid and glutaric acid in ethylacetate, a relative response factor of 1.02 k 0.02 has been measured. This value is in good agreement with the theoretical expected value of 1.00, calculated by using the ef- fective carbon number [2] of the trimethylsilyl derivatives of both alkanedioic acids. In a single ethyl acetate extraction of an aqueous standard mixture of adipic and of glutaric acid, the measured relative response factor appears to be 1.14 f 0.02. This increase in the value of the relative response factor indicates that there is a difference in the partition coefficients of the acids. It can be concluded that at least the extraction of the glutaric acid from the aqueous phase is incomplete. However it might also be that the extraction of both acids with the ethylacetate is in- complete. Due to the high solubility of glutaric acid of 639 g/l in water [3] compared to adipic acid (14 g/l) [3], the partition coefficients for the ethyl acetate-water extraction for both compounds differ. Instead of glutaric acid, pimelic acid is a potential internal standard for the determination of adipic acid. The solubility of pimelic acid in water is about 50 g/l[3], which is closer to adip- ic acid than glutaric acid. Therefore the extraction behaviour of pimelic acid was compared to the extraction behaviour of adipic acid and glutaric acid. By successive ethyl acetate ex- tractions of an acidified solution of waxy corn starch spiked with a known amount of the alkanedioic acid, the different ac- ids were tested for their suitability as an internal standard.

The acidified aqueous solution was extracted four times. After each extraction the content of alkanedioic acid in the organic phase was measured by gas chromatography. For this specific determination 3-methyladipic acid was used as internal stan- dard of which a known amount was added to the ethyl acetate after the extraction. In this way the extraction of the two different alkanedioic ac- ids was compared to the extraction behaviour of adipic acid. The results are presented in Figure 1. It is obvious that the ex-

%

80 -

60 -

LO -

Extraction efficiency

u pimelic acid X-x adipic acid . +-+ glutaric acid

number extractions

I

1 2 i 2 Figure 1. Extraction efficiency of the aqueous solutions of the alkane- dioic acids in relation to the number of ethyl acetate extractions.

traction of pimelic acid is better than the extraction of adipic acid and glutaric acid. After a threefold extraction with ethyl acetate the pimelic acid is fully extracted, while adipic acid was 94% extracted, and glutaric acid 83%. For a complete ex- traction of adipic acid a fourfold ethyl acetate extraction is needed, and for glutaric acid at least a sixfold extraction. These extraction experiments were also performed at elevated (4OOC) and lower (OOC) temperatures, which established that the relative extraction efficiency of the three different alkane- dioic acids was hardly affected by temperature. Figure 1. demonstrates clearly that following the original pro- tocol, and performing the ethyl acetate extraction in triplicate, will result in an incomplete extraction of both the adipic acid and the internal standard glutaric acid. It is beyond dispute that the most reliable results will be obtained when the ana- lyte and the internal standard are both fully extracted. There- fore we decided to use pimelic acid as the internal standard, and to apply four successive extractions instead of three.

3.2 Miniaturization

For environmental reasons and to improve efficiency, we aimed to miniaturize the analytical method, especially with re- spect to reducing the amount of organic solvent needed for each determination. Due to the rapid evolution in modern analytical instrumentation including the increase in sensitiv- ity of detectors, it is possible to decrease the sample weight considerably provided that the samples to be analyzed are ho- mogeneous. The most laborious and time consuming steps in the analytical procedure are the extractions with ethyl acetate

followed by the complete evaporation of the organic phase. By decreasing the sample weight from the original 1 g to just 50mg, the saponification and the extractions can be carried out in small volumes of a few ml in screw-cap glass reaction tubes. Total extraction of the analyte and the internal standard pime- lic acid into the organic phase was achieved by four successive extractions with 5 ml of ethyl acetate. Moreover by performing the extractions in closed reaction tubes placed in a rack, it is possible to do 20-30 extractions simultaneously. 27 samples can be evaporated to dryness in a nitrogen steam simultane- ously by applying a Reacti-Vap Evaporator (Pierce). Then the adipic acid and the pimelic acid in the residue are dissolved in acetonitrile (instead of pyridine), derivatized with the BSTFAITMCS reagent to form their corresponding trimeth- ylsilyl derivatives, and then separated and quantified by capil- lary gas chromatography (Figure 2).

Figure 2. Chromatogram of a calibration standard to which 1.00 ml adipic acid and 1.00 ml pimelic acid solution was added. Retention time of the adipic acid derivative is 10.3 min and of pimelic acid de- rivative 12.2 min.

A calibration graph based on standard addition of adipic acid to waxy corn starch is used for quantification.

3.3 Statistical evaluation

Accuracy

In 12 acetylated adipyl cross-linked corn starches, the adipate content was determined using the original method and the im- proved method as described in this paper. A complication in this comparison of the methods is that in the original method both the analyte adipic acid and the internal standard glutaric acid are extracted incompletely by the triple extraction with ethyl acetate. It is known that in the case of incomplete extrac- tion of compounds with different partition coefficients, the volume ratio of the aqueous and organic phase becomes criti-

starch/st&ke 46 (1994) Nr. 7, S. 255-259 257

cal concerning the absolute amount of compound extracted. Therefore, special care was taken in order to ensure that for each of the three extractions of the aqueous phase with ethyl acetate, exactly the same volume ratio in the aqueous and or- ganic phase was used. In the improved method this precau- tion was not necessary because both compounds (the analyte, and the internal standard pimelic acid), will be fully extracted by the four extractions with ethyl acetate. Figure 3 shows the

I % rdipic acid

I 0.10

0.05

/ r’

/ % adipic acid Improwd method

1 -

0.05 0.10 0.15

Figure 3. Correlation between the analytical results of the original method and the improved version for the determination of the total adipyl content in acetylated adipyl cross-linked corn starches.

correlation between the analytical results of the original and of the improved method. By linear regression a slope was cal- culated of 1.02 k 0.01 and an intercept of-0.002 f 0.001. Both the slope and the intercept did not differ significantly (t-test, p = 0.05) from the ideal theoretical value of 1.00 and 0.00. It can thus be concluded that the analytical results obtained with both methods did not differ significantly.

Precision

The repeatability of the improved method was tested by ana- lyzing 18 acetylated adipyl cross-linked wheat starches and 37 acetylated adipyl cross-linked corn starches as independent duplicates. All samples were analyzed on two different days by the same person. The duplicate difference were used to cal- culate the standard deviation in the improved analytical me- thod. The results are summarized in Table 1.

Table 1. Repeatability of the Improved Method for Determination of the Total Adipate Content in Starch Samples, Based on Independent Duplicates. Total Adipate Content is Expressed in ppm in the Starch as is.

Sample Number range in total duplicate Standard matrix of adipate content differences deviation

samples rnin. max. min. max.

Wheat 18 4 1542 0 12 4 Corn 37 0 1631 2 75 16 Wheat and corn 55 0 1631 0 75 13

The overall standard deviation in the determination calculated for the wheat and corn samples together is 13ppm. This ap- pears to be three times less than we had established before for the original method with glutaric acid as internal standard (sd = 40ppm). Also a test for the within laboratory reproducibility was carried out. Therefore 8 samples of acetylated adipyl cross-linked wheat starches were analyzed in duplicate by two different per- sons with a time interval of several months. In the meantime the samples were stored in the dark in closed bottles. The re- sults are summarized in Table 2. Evaluation of these data with

Table 2. Reproducibility Test for the Improved Method for Determina- tion of the Total Adipyl Content by Analyzing Acetylated Adipyl Cross-linked Starches in Duplicate by two Different Persons.

Sample Person 1 Person 2

x1 x2 mean dupl y, y2 mean dupl diff. diff.

LAO 19 1 00 1 LAO 19 1005 LA019101 1 LAO 19 10 12 LAO 19 10 13 LAO 19 10 14 LA019 1017 LA019 1018

670 1110

85 195 161 399 637

1508

675 1115

79 198 163 397 612

1515

673 1113

82 197 162 398 625

1512

5 5 6 3 2 2

25 7

655 1073

76 20 I 179 393 672

1536

664 1109

74 197 168 402 67 1

1548

660 9 1091 36

75 2 199 4 174 11 398 9 672 1

1542 12

two-way analysis of variances showed that there is no signifi- cant difference (p = 0.05) between the duplicate means of the corresponding samples analyzed by both persons. The calcu- lated least significant difference (lsd) appeared to be 18 ppm. The standard deviations in the determination as performed by person 1 and 2 were rescpectively 7 and lOppm, and did not differ significantly from each other (F-test, p = 0.05). Because the corresponding duplicate means in both series did not dif- fer significantly, one can combine the two duplicates for each sample, to give one determination in fourfold, and to calculate the pooled standard deviation based on the analysis of 8 sam- ples in fourfold. This resulted in a standard deviation of 15 ppm (degree of freedom 24), which is indicative for the re- producibility of the determination.

4 Closing Remarks As mentioned earlier in the discussion on the miniaturization, acetonitrile was used instead of pyridine as solvent for prepar- ing the trimethylsilyl esters of the acids. It should be noted that at temperatures less than about 2OoC a phase separation of the acetonitrile and the BSTFA/TMCS solution might oc- cur. Therefore it is essential to have a temperature of 30°C dur- ing the derivatization. Instead of applying an elevated tem- perature during the derivatization, it is also possible to double the volume of acetonitrile to prevent phase separation. How- ever, this has the disadvantage of diluting the sample. The improved precision of the miniaturized method is mainly based on the improved extraction procedure of both alkane- dioic acids. With the new proposed method a relative response value of 0.984 f 0.009 (n = 8) is obtained for the adipic acid which is in good agreement with the theoretical expected value of 1.00, calculated on the basis of the effective carbon number [2]. Our results showed that both the analyte adipic acid, and the internal standard pimelic acid, are extracted com- pletely by the four ethyl acetate extractions. Prolonged investigations showed that the run time of the chro- matograms could be shortened considerably by the use of a

258 starchhtarke 46 (1994) Nr. 7, S . 255-259

shorter column (10m length) and a simple adaptation of the temperature program. A fast separation was obtained by rais- ing the temperature directly with 25OC/min. to 29OOC instead of applying first the relatively slow temperature raise of 5OC/ min. to 190OC. The chromatographic peaks are still very well separated while the chromatographic run time is decreased by about 10 min (Figure 4). With this modified procedure the re-

V)PZ0025RLYI 60 DO

50 00

4 0 00

30 00

20 00

10 00

0 00

1

0 1 2 3 4 5 6 7

Figure 4. Chromatogram of an acetylated adipyl cross-linked corn starch sample, measured with the modified temperature program and 10 m column. Retention time of the adipic acid derivative is 3.2 min and of pimelic acid derivative 3.7min.

tention times of adipic acid and pimelic acid become 3.2 and 3.7 min instead of 10.3 and 12.2 min, respectively. This change in chromatographic conditions does not affect the perform- ance of the determination with regard to standard deviation, reproducibility and repeatability.

5 Conclusions

This study leads to the following conclusions: 1. The use of pimelic acid as internal standard is preferred to

glutaric acid. 2. The total adipic acid content in acetylated adipyl cross-

linked starches can be determined well with the described improved method.

3. There is no significant difference in the accuracy of the original and the improved method of analysis. The repeat- ability of the improved method is about three times better than the original method.

4. Advantages of the miniaturized method with respect to the original method are: - increase in daily analysis capacity of about 8 samples to about 25 samples, -considerable decrease in the consumption of organic sol- vent per determination; instead of 300 m1,just 20ml ethyl acetate is needed per determination, - improved repeatability.

Acknowledgement

The authors are indebted to Mr. G. A . Mitchellfor the constructive dis- cussions concerning the adipate determination and his critical re- viewing of the manuscript.

Bibliography [ l ] Mitchell, G. A., M. J. Vanderbist, and R R Meert: Gas-liquid Chro-

matography of Adipate Content of Acetylated Distarch Adipate. J. Assoc. Off. Anal. Chem. 65 (1982), 238-240.

[2] Scanlon, J. Z, and D. E. Willis: Calculation of Flame Ionization Detector Relative Response Factors Using the Effective Carbon Number Concept. J. Chromatogr. Sci. 23 (1985), 333-340.

[3] The Merck Index, Merck & Co., Inc., Rahway, N. J., USA 1989, pp 27,703, and 1180.

Address of authors: Dr. K. Brunt (corresponding author) and P. San- ders, Department of Analytical Chemistry and Raw Materials, Nether- lands Institute for Carbohydrate Research TNO, Rouaanstraat 27, 9723 CC Groningen, The Netherlands.

(Received: December 27, 1993).

starch/stiirke 46 (1994) Nr. 7, S. 255-259 259