Nitrogen/Protein Determination in Starch by

Flash Combustion using Large Sample Weight

as an Alternative to the Kjeldahl Method
Liliana Krotz and Guido Giazzi
Thermo Fisher Scientific, Milan, Italy
 Overview
Method
Purpose: To show the determination of Nitrogen / Protein in starch samples by flash
combustion at high sample weight. The sample is weighed in
via the Thermo ScientificTM
Methods: Starch samples were analyzed using an elemental analyzer with an amount of oxygen using th
automatic autosampler. complete combustion of th
capsule in the range of 100
Results: Data collected of Nitrogen / Protein from different starch samples are
discussed to assess the performance of the analyzer. After combustion, the prod
reactor filled with copper. W

Introduction
adsorbed by the No-Stop T
column and finally is detec

A complete report is autom

The production process in the starch industry, the protein content, calculated by the Xperience dedicated data
determination of nitrogen, is periodically monitored and tested for quality control. analysis. Eager Xperience
Because starch is used in the preparation of animal feeds, bakery products, puddings, balance to the sample tabl
instant meals, syrups and desserts, the determination of N/Protein is critical. the instrument.

It is therefore very important to have a method which allows the fast analysis of FIGURE 2. Nitrogen / Pro
N/Protein with an excellent reproducibility.

The Thermo ScientificTM FLASH 4000 Nitrogen/Protein Analyzer (Figure 1), based on
the dynamic flash combustion of the sample, satisfies all the requirements of modern
laboratories such as stability, accuracy, day-by-day reproducibility and high sample
throughput , and does not require sample digestion or toxic materials. This alternative
to the classical Kjeldahl method, based on Dumas (combustion) method, has been
developed and approved by different associations such as ASBC, AOAC, AACC,
AOCS, IFFO and ISO.

This paper presents data on Nitrogen/Protein determination of different starch samples

in a large range of concentrations (150 – 2500 ppm nitrogen), obtained with the
analyzer using large sample weight to demonstrate the validity of the method without
matrix effect. Data compared to the results obtained by the Kjeldahl method
demonstrates the validity of the system.

FIGURE 1. FLASH 4000 Nitrogen / Protein Analyzer.

Analytical conditions:

Left Furnace Temperature

Right Furnace Temperature
Oven Temperature:
Carrier Flow:
Reference Flow:
Standard:
Sample Weight:

Note: The oxygen amount

calculated automatically by
software.

*EDTA: EthyleneDiamineTe

Results
The starch samples analyz
The data obtained demons
indicating complete combu

The calibration of the FLAS

as calibration method.

Table 1 shows the nitrogen

0.25 % N, analyzed ten tim
obtained analyzing the sam
the data are comparable a
sample weight.

2 Nitrogen/Protein Determination in Starch by Flash Combustion using Large Sample Weight as an Alternative to the Kjeldahl Method

n in starch samples by flash
ental analyzer with an
nt starch samples are
n content, calculated by the
tested for quality control.
s, bakery products, puddings,
N/Protein is critical.
TABLE 1. Nitrogen data of sta
Method
Sample weight (mg)
The sample is weighed in a tin capsule and introduced into the combustion reactor
via the Thermo ScientificTM MASTM 4000 autosampler together with the correct 981.0
amount of oxygen using the Thermo ScientificTM OxyTuneTM function, insuring a 997.4
complete combustion of the sample. The samples are weighed directly in the tin
capsule in the range of 1000 - 2000 mg. 984.8
998.3
After combustion, the produced gases are carried by a helium flow to a second
reactor filled with copper. Water is trapped through a Peltier system while the CO2 is 1032.2
adsorbed by the No-Stop Twin traps. Then the nitrogen is swept through a GC 1013.4
column and finally is detected by a thermal conductivity detector (TCD) (Figure 2).
998.0
A complete report is automatically generated by the Thermo ScientificTM Eager 1017.3
Xperience dedicated data handling software, and is displayed at the end of the
analysis. Eager Xperience also allows the direct sample weight transfer from the 964.3
balance to the sample table, and the complete control of the analytical parameters of 987.6
the instrument.

ows the fast analysis of FIGURE 2. Nitrogen / Protein configuration

TABLE 2. Nitrogen data of sta

nalyzer (Figure 1), based on

the requirements of modern Sample weight (mg)
ducibility and high sample
xic materials. This alternative 797.7
bustion) method, has been
946.2
as ASBC, AOAC, AACC,
1101.6
1255.0
ion of different starch samples
gen), obtained with the 1401.4
alidity of the method without
1611.1
he Kjeldahl method
2001.5
987.7
802.7
Analytical conditions:
1025.0

Left Furnace Temperature : 950°C

Right Furnace Temperature : 840°C
Oven Temperature: 50°C Table 3 shows the Nitrogen / Pr
Carrier Flow: 300 ml/min 400 ppm N weighed in a range
Reference Flow: 300 ml/min The protein content was calcula
Standard: 500 mg EDTA* (9.59 %N) software using 6.25 as protein f
Sample Weight: 1000 - 2000 mg

Note: The oxygen amount necessary for complete combustion of samples is TABLE 3. Nitrogen / Protein da
calculated automatically by the OxyTune function present in the Eager Xperience 1.0 – 1.1 grams.
software.
N%
*EDTA: EthyleneDiamineTetraAcetic acid
0.0472
Results 0.0484

The starch samples analyzed were chosen on basis of their differing nitrogen content. 0.0430
The data obtained demonstrates the no-matrix effect in the determination of nitrogen, 0.0454
indicating complete combustion for all type of samples.
0.0427
The calibration of the FLASH 4000 was performed with EDTA (9.59 %N) using K factor 0.0419
as calibration method.
0.0441
Table 1 shows the nitrogen results obtained of a starch sample of approximately 0.0438
0.25 % N, analyzed ten times at about 1 gram. Table 2 shows the nitrogen data
obtained analyzing the same starch in a range from 700 mg to 2 grams. In both cases 0.0422
the data are comparable and no significant difference was observed changing the
0.0422
sample weight.

Thermo Scientific Poster Note • PN42212_PITTCON 2014_E_02/14S 3

 TABLE 1. Nitrogen data of starch (0.25 %N) at 1 gram. Table 4 shows the sequen
randomly to evaluate mem
content.
Sample weight (mg) N% Average N % RSD %
Table 5 shows the statistic
uced into the combustion reactor
981.0 0.2547 calculated automatically b
pler together with the correct
protein factor.
OxyTuneTM function, insuring a 997.4 0.2500 No memory effect was ob
are weighed directly in the tin
984.8 0.2518 the nitrogen.

998.3 0.2550 TABLE 4. Random sequ

by a helium flow to a second
h a Peltier system while the CO2 is 1032.2 0.2529 0.2527 1.0520
ogen is swept through a GC 1013.4 0.2514 Run Star
ctivity detector (TCD) (Figure 2).
998.0 0.2579
1
e Thermo ScientificTM Eager 1017.3 0.2502
s displayed at the end of the 2
ample weight transfer from the 964.3 0.2496
3 C
ntrol of the analytical parameters of 987.6 0.2538
4 D
5
TABLE 2. Nitrogen data of starch (0.25 %N) in the range of 700 mg to 2 grams.
6
7
Sample weight (mg) N% Average N % RSD % 8
9 D
797.7 0.2536
10 G
946.2 0.2530
11 G
1101.6 0.2528
12
1255.0 0.2600
13
1401.4 0.2556 0.2556 1.4550
14
1611.1 0.2571
15 C
2001.5 0.2637
16 C
987.7 0.2522
17
802.7 0.2536
18 D
1025.0 0.2541
19
°C
°C TABLE 5. Statistical dat
C Table 3 shows the Nitrogen / Protein data of a starch sample of approximately
ml/min 400 ppm N weighed in a range of 1000 – 1100 mg.
ml/min The protein content was calculated automatically by the Eager Xperience dedicated
Starch ID N
mg EDTA* (9.59 %N) software using 6.25 as protein factor.
0 - 2000 mg 0.0
TABLE 3. Nitrogen / Protein data of starch (0.04 %N) in the range of A 0.0
combustion of samples is
present in the Eager Xperience 1.0 – 1.1 grams. 0.0
0.0
N% RSD % Protein % RSD % 2 B 0.0
0.0
0.0472 0.2947 0.0
0.0484 0.2756 C 0.0
0.0430 0.2736 0.0
is of their differing nitrogen content.
ect in the determination of nitrogen, 0.0
0.0454 0.2640
ples. D 0.0
0.0427 5.0777 0.2636 5.0535 0.0
with EDTA (9.59 %N) using K factor 0.0419 0.3022 0.0
0.0441 0.2685 E 0.0
arch sample of approximately 0.0
0.0438 0.2841
ble 2 shows the nitrogen data 0.0
m 700 mg to 2 grams. In both cases 0.0422 0.2667 F
0.0
nce was observed changing the
0.0422 0.2619 0.0
G
0.0

4 Nitrogen/Protein Determination in Starch by Flash Combustion using Large Sample Weight as an Alternative to the Kjeldahl Method
1 gram. Table 4 shows the sequence of analysis of starch samples in trace level analyzed Table 6 shows the N/Prot
randomly to evaluate memory effect when changing the sample type and nitrogen liquid sample was adsorb
content. the tin capsule and the sa
Average N % RSD %
Table 5 shows the statistical data of the relative samples. The protein content was TABLE 6. Nitrogen / Pro
calculated automatically by the Eager Xperience dedicated software using 6.25 as
protein factor.
N%
No memory effect was observed indicating complete combustion and conversion of
the nitrogen. 0.0355
TABLE 4. Random sequence of starch samples at trace level. 0.0352
0.0366
0.2527 1.0520
0.0380
Run Starch ID Weight (mg) N% Protein %
0.0350
1 A 1000.4 0.0177 0.1107 0.0354

2 B 1002.5 0.0404 0.2528

An overlay of chromatogr
3 C 1051.5 0.0162 0.1015 the FLASH 4000 analyzin
4 D 1050.4 0.0380 0.2378 chromatogram is obtaine
173015 uV/sec while the
5 B 1099.6 0.0404 0.2525 glucose at 1 gram giving
he range of 700 mg to 2 grams.
6 A 1100.9 0.0179 0.1120
FIGURE 3. Overlay of ch
7 E 1000.9 0.0356 0.2228
Average N % RSD % 8 E 1003.9 0.0354 0.2212
9 D 1050.7 0.0382 0.2387
10 G 1050.8 0.0191 0.1196
11 G 1100.9 0.0188 0.1174
12 E 1100.3 0.0365 0.2280
13 F 1101.2 0.0143 0.0914
0.2556 1.4550
14 F 999.9 0.0150 0.0935
15 C 1050.9 0.0167 0.1046
16 C 1100.1 0.0162 0.1014
17 B 1102.1 0.0403 0.2519
18 D 1051.8 0.0387 0.2419
19 A 1051.0 0.0178 0.1115 TABLE 7. Nitrogen / Pro

TABLE 5. Statistical data of starch samples at trace level. Sample

ch sample of approximately
.
by the Eager Xperience dedicated N
Starch ID N% RSD % Protein % RSD %
1 0.2
0.0177 0.1107
2 0.0
%N) in the range of A 0.0179 0.5618 0.1120 0.5886
3 0.0
0.0178 0.1115

Protein % RSD % 2 B
0.0404
0.0404 0.1430
0.2528
0.2525 0.1816
Conclusion
0.0403 0.2519 The FLASH 4000 analyze
0.2947 0.0162 0.1015 determination due to:.

0.2756 C 0.0167 1.7638 0.1046 1.7750  Excellent reproduc

0.2736 0.0162 0.1014  No memory effect

0.0380 0.2378  Nitrogen determina
0.2640
D 0.0382 0.9414 0.2387 0.8998 effect.
0.2636 5.0535 0.0387 0.2419  Combustion metho
0.3022 0.0356 0.2228 ISO, etc).
0.2685 E 0.0354 1.6352 0.2212 1.5872
AOAC is a trademark of The Ass
0.0365 0.2280
0.2841 Association of Cereal Chemists.
0.0143 0.0914 trademark of The American Soci
0.2667 F 3.3787 3.3285 Fish Oil Organization. ISO is a tr
0.0150 0.0935 of IMERYS MINERALS CALIFOR
0.2619 0.0191 0.1196 its subsidiaries
G 1.1194 1.3128 This information is not intended t
0.0188 0.1174 intellectual property rights of othe

Thermo Scientific Poster Note • PN42212_PITTCON 2014_E_02/14S 5

amples in trace level analyzed Table 6 shows the N/Protein data obtained of a slurry starch sample analysis. The
g the sample type and nitrogen liquid sample was adsorbed by the inert material Chromosorb (WAW 30/60 mesh) into
the tin capsule and the sample weight used for analysis was 1.0 – 1.5 grams.

mples. The protein content was TABLE 6. Nitrogen / Protein data of slurry starch.
dicated software using 6.25 as
N% RSD % Protein % RSD %
e combustion and conversion of
0.0355 0.2216
at trace level. 0.0352 0.2200
0.0366 0.2291
3.1947 3.1968
0.0380 0.2373
N% Protein %
0.0350 0.2186
0.0177 0.1107 0.0354 0.2214

0.0404 0.2528
An overlay of chromatograms is shown in Figure 3 to demonstrate the performance of
0.0162 0.1015 the FLASH 4000 analyzing samples at about 100 ppm nitrogen at 1 gram. The black
0.0380 0.2378 chromatogram is obtained from sample F (about 150 ppm N) with a Nitrogen Area of
173015 uV/sec while the red chromatogram is the blank analysis obtained with
0.0404 0.2525 glucose at 1 gram giving a Nitrogen Area of 34223 uV/sec.
0.0179 0.1120
FIGURE 3. Overlay of chromatograms
0.0356 0.2228
0.0354 0.2212
0.0382 0.2387
0.0191 0.1196
0.0188 0.1174
0.0365 0.2280
0.0143 0.0914
0.0150 0.0935
0.0167 0.1046
0.0162 0.1014
0.0403 0.2519
0.0387 0.2419
0.0178 0.1115 TABLE 7. Nitrogen / Protein data comparison

ace level. Sample FLASH 4000 Kjeldahl Method

N% Protein % N% Protein %
Protein % RSD %
1 0.2527 1.5794 0.2504 1.5650
0.1107
2 0.0404 0.2525 0.0399 0.2494
0.1120 0.5886
3 0.0358 0.2237 0.0360 0.2250
0.1115
0.2528
0.2525 0.1816
Conclusion
0.2519 The FLASH 4000 analyzer demonstrates the best solution for Nitrogen / Protein
0.1015 determination due to:.
0.1046 1.7750  Excellent reproducibility and accuracy.
0.1014  No memory effect when changing the sample and content of nitrogen.
0.2378  Nitrogen determination in a wide range from trace to high content without matrix
0.2387 0.8998 effect.
0.2419  Combustion method is approved by official organizations (AOAC, AACC,AOCS,
0.2228 ISO, etc).
0.2212 1.5872
AOAC is a trademark of The Association of Official Analytical Chemists. AACC is a trademark of The American
0.2280 Association of Cereal Chemists. AOCS is a trademark of The American Oil Chemists' Society. ASBC is a
0.0914 trademark of The American Society of Brewing Chemists. IFFO is a trademark of The International Fishmeal and
3.3285 Fish Oil Organization. ISO is a trademark of The International Standards Organization. Chromosorb is a trademark
0.0935 of IMERYS MINERALS CALIFORNIA, INC. All other trademarks are the property of Thermo Fisher Scientific and
0.1196 its subsidiaries
1.3128 This information is not intended to encourage use of these products in any manners that might infringe the
0.1174 intellectual property rights of others.

6 Nitrogen/Protein Determination in Starch by Flash Combustion using Large Sample Weight as an Alternative to the Kjeldahl Method
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