Nitrogen/Protein Determination in Starch by Flash Combustion using Large Sample Weight

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
Purpose: To show the determination of Nitrogen / Protein in starch samples by flash
combustion at high sample weight.
Methods: Starch samples were analyzed using an elemental analyzer with an
automatic autosampler.
Results: Data collected of Nitrogen / Protein from different starch samples are
discussed to assess the performance of the analyzer.
Method
The sample is weighed in
via the Thermo ScientificTM
amount of oxygen using th
complete combustion of th
capsule in the range of 100
Introduction
After combustion, the prod
reactor filled with copper. W
adsorbed by the No-Stop T
column and finally is detec
The production process in the starch industry, the protein content, calculated by the
determination of nitrogen, is periodically monitored and tested for quality control.
Because starch is used in the preparation of animal feeds, bakery products, puddings,
instant meals, syrups and desserts, the determination of N/Protein is critical.
A complete report is autom
Xperience dedicated data
analysis. Eager Xperience
balance to the sample tabl
the instrument.
It is therefore very important to have a method which allows the fast analysis of
N/Protein with an excellent reproducibility.
FIGURE 2. Nitrogen / Pro
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
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
amount of oxygen using the Thermo ScientificTM OxyTuneTM function, insuring a
complete combustion of the sample. The samples are weighed directly in the tin
capsule in the range of 1000 - 2000 mg.
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
adsorbed by the No-Stop Twin traps. Then the nitrogen is swept through a GC
column and finally is detected by a thermal conductivity detector (TCD) (Figure 2).
n content, calculated by the
tested for quality control.
s, bakery products, puddings,
N/Protein is critical.
A complete report is automatically generated by the Thermo ScientificTM Eager
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
balance to the sample table, and the complete control of the analytical parameters of
the instrument.
ows the fast analysis of
FIGURE 2. Nitrogen / Protein configuration
981.0
997.4
984.8
998.3
1032.2
1013.4
998.0
1017.3
964.3
987.6
TABLE 2. Nitrogen data of sta
nalyzer (Figure 1), based on
the requirements of modern
ducibility and high sample
xic materials. This alternative
bustion) method, has been
as ASBC, AOAC, AACC,
Sample weight (mg)
797.7
946.2
1101.6
1255.0
ion of different starch samples
gen), obtained with the
alidity of the method without
he Kjeldahl method
1401.4
1611.1
2001.5
987.7
802.7
Analytical conditions:
Left Furnace Temperature :
Right Furnace Temperature :
Oven Temperature:
Carrier Flow:
Reference Flow:
Standard:
Sample Weight:
1025.0
950°C
840°C
50°C
300 ml/min
300 ml/min
500 mg EDTA* (9.59 %N)
1000 - 2000 mg
Note: The oxygen amount necessary for complete combustion of samples is
calculated automatically by the OxyTune function present in the Eager Xperience
software.
*EDTA: EthyleneDiamineTetraAcetic acid
Table 3 shows the Nitrogen / Pr
400 ppm N weighed in a range
The protein content was calcula
software using 6.25 as protein f
TABLE 3. Nitrogen / Protein da
1.0 – 1.1 grams.
N%
Results
0.0472
The starch samples analyzed were chosen on basis of their differing nitrogen content.
The data obtained demonstrates the no-matrix effect in the determination of nitrogen,
indicating complete combustion for all type of samples.
0.0430
The calibration of the FLASH 4000 was performed with EDTA (9.59 %N) using K factor
as calibration method.
0.0419
0.0484
Table 1 shows the nitrogen results obtained of a starch sample of approximately
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
the data are comparable and no significant difference was observed changing the
sample weight.
0.0454
0.0427
0.0441
0.0438
0.0422
0.0422
Thermo Scientific Poster Note • PN42212_PITTCON 2014_E_02/14S 3
TABLE 1. Nitrogen data of starch (0.25 %N) at 1 gram.
Sample weight (mg)
uced into the combustion reactor
pler together with the correct
OxyTuneTM function, insuring a
are weighed directly in the tin
by a helium flow to a second
h a Peltier system while the CO2 is
ogen is swept through a GC
ctivity detector (TCD) (Figure 2).
e Thermo ScientificTM Eager
s displayed at the end of the
ample weight transfer from the
ntrol of the analytical parameters of
N%
981.0
0.2547
997.4
0.2500
984.8
0.2518
998.3
0.2550
1032.2
0.2529
1013.4
0.2514
998.0
0.2579
1017.3
0.2502
964.3
0.2496
987.6
0.2538
Average N %
RSD %
°C
°C
C
ml/min
ml/min
mg EDTA* (9.59 %N)
0 - 2000 mg
combustion of samples is
present in the Eager Xperience
797.7
0.2536
946.2
0.2530
1101.6
0.2528
1255.0
0.2600
1401.4
0.2556
1611.1
0.2571
2001.5
0.2637
987.7
0.2522
802.7
0.2536
1025.0
0.2541
0.2527
1.0520
with EDTA (9.59 %N) using K factor
arch sample of approximately
ble 2 shows the nitrogen data
m 700 mg to 2 grams. In both cases
nce was observed changing the
Average N %
RSD %
0.2556
C
4
D
5
6
7
8
9
D
10
G
11
G
1.4550
13
14
15
C
16
C
17
18
D
19
Protein %
0.2947
0.0484
0.2756
0.0430
0.2736
0.0454
0.2640
5.0777
3
12
0.0472
0.0427
Star
2
0.2636
0.0419
0.3022
0.0441
0.2685
0.0438
0.2841
0.0422
0.2667
0.0422
0.2619
TABLE 5. Statistical dat
Starch ID
A
TABLE 3. Nitrogen / Protein data of starch (0.04 %N) in the range of
1.0 – 1.1 grams.
RSD %
Run
1
Table 3 shows the Nitrogen / Protein data of a starch sample of approximately
400 ppm N weighed in a range of 1000 – 1100 mg.
The protein content was calculated automatically by the Eager Xperience dedicated
software using 6.25 as protein factor.
N%
is of their differing nitrogen content.
ect in the determination of nitrogen,
ples.
N%
Table 5 shows the statistic
calculated automatically b
protein factor.
No memory effect was ob
the nitrogen.
TABLE 4. Random sequ
TABLE 2. Nitrogen data of starch (0.25 %N) in the range of 700 mg to 2 grams.
Sample weight (mg)
Table 4 shows the sequen
randomly to evaluate mem
content.
RSD % 2
B
C
5.0535
4 Nitrogen/Protein Determination in Starch by Flash Combustion using Large Sample Weight as an Alternative to the Kjeldahl Method
D
E
F
G
N
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
1 gram.
Average N %
RSD %
Table 4 shows the sequence of analysis of starch samples in trace level analyzed
randomly to evaluate memory effect when changing the sample type and nitrogen
content.
Table 6 shows the N/Prot
liquid sample was adsorb
the tin capsule and the sa
Table 5 shows the statistical data of the relative samples. The protein content was
calculated automatically by the Eager Xperience dedicated software using 6.25 as
protein factor.
No memory effect was observed indicating complete combustion and conversion of
the nitrogen.
TABLE 6. Nitrogen / Pro
N%
0.0355
TABLE 4. Random sequence of starch samples at trace level.
0.2527
1.0520
he range of 700 mg to 2 grams.
Average N %
0.2556
RSD %
1.4550
ch sample of approximately
.
by the Eager Xperience dedicated
Protein %
0.0366
Run
Starch ID
Weight (mg)
N%
Protein %
1
A
1000.4
0.0177
0.1107
2
B
1002.5
0.0404
0.2528
3
C
1051.5
0.0162
0.1015
4
D
1050.4
0.0380
0.2378
5
B
1099.6
0.0404
0.2525
6
A
1100.9
0.0179
0.1120
7
E
1000.9
0.0356
0.2228
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
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
RSD % 2
Starch ID
B
0.2947
C
0.2756
0.2736
0.2640
0.2636
5.0535
D
0.3022
0.2685
E
0.2841
0.2667
0.2619
0.0380
0.0350
0.0354
An overlay of chromatogr
the FLASH 4000 analyzin
chromatogram is obtaine
173015 uV/sec while the
glucose at 1 gram giving
FIGURE 3. Overlay of ch
TABLE 7. Nitrogen / Pro
TABLE 5. Statistical data of starch samples at trace level.
A
%N) in the range of
0.0352
F
G
N%
0.0177
RSD %
0.0179
0.5618
0.0404
0.1430
0.0178
0.0404
0.0403
0.0162
0.0167
0.0162
0.0380
0.0382
0.0387
0.0356
0.0354
0.0365
0.0143
0.0150
0.0191
0.0188
1.7638
0.9414
1.6352
3.3787
1.1194
Protein %
0.1107
Sample
0.1120
0.5886
0.2525
0.1816
0.1115
0.2528
0.2519
0.1015
0.1046
0.1014
0.2378
0.2387
0.2419
0.2228
0.2212
0.2280
0.0914
0.0935
0.1196
0.1174
N
RSD %
1.7750
0.8998
1.5872
3.3285
1.3128
1
0.2
3
0.0
2
0.0
Conclusion
The FLASH 4000 analyze
determination due to:.

Excellent reproduc

No memory effect

Nitrogen determina
effect.

Combustion metho
ISO, etc).
AOAC is a trademark of The Ass
Association of Cereal Chemists.
trademark of The American Soci
Fish Oil Organization. ISO is a tr
of IMERYS MINERALS CALIFOR
its subsidiaries
This information is not intended t
intellectual property rights of othe
Thermo Scientific Poster Note • PN42212_PITTCON 2014_E_02/14S 5
amples in trace level analyzed
g the sample type and nitrogen
Table 6 shows the N/Protein data obtained of a slurry starch sample analysis. The
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
dicated software using 6.25 as
TABLE 6. Nitrogen / Protein data of slurry starch.
N%
e combustion and conversion of
RSD %
0.0355
at trace level.
N%
Protein %
0.0177
0.1107
0.0404
0.2528
0.0162
0.1015
0.0380
0.2378
0.0404
0.2525
0.0179
0.1120
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
0.2200
0.2186
0.0354
0.2214
An overlay of chromatograms is shown in Figure 3 to demonstrate the performance of
the FLASH 4000 analyzing samples at about 100 ppm nitrogen at 1 gram. The black
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
glucose at 1 gram giving a Nitrogen Area of 34223 uV/sec.
FIGURE 3. Overlay of chromatograms
TABLE 7. Nitrogen / Protein data comparison
RSD %
0.5886
0.2525
0.1816
0.1115
0.2528
0.2519
0.1015
0.1046
0.1014
0.2378
0.2387
0.2419
0.2228
0.2212
0.2280
0.0914
0.0935
0.1196
0.1174
1.7750
0.8998
1.5872
3.3285
1.3128
3.1968
0.2373
0.0350
Sample
0.1120
0.2291
3.1947
0.0380
ace level.
0.1107
RSD %
0.2216
0.0352
0.0366
Protein %
Protein %
FLASH 4000
Kjeldahl Method
N%
Protein %
N%
Protein %
1
0.2527
1.5794
0.2504
1.5650
3
0.0358
0.2237
0.0360
0.2250
2
0.0404
0.2525
0.0399
0.2494
Conclusion
The FLASH 4000 analyzer demonstrates the best solution for Nitrogen / Protein
determination due to:.

Excellent reproducibility and accuracy.

No memory effect when changing the sample and content of nitrogen.

Nitrogen determination in a wide range from trace to high content without matrix
effect.

Combustion method is approved by official organizations (AOAC, AACC,AOCS,
ISO, etc).
AOAC is a trademark of The Association of Official Analytical Chemists. AACC is a trademark of The American
Association of Cereal Chemists. AOCS is a trademark of The American Oil Chemists' Society. ASBC is a
trademark of The American Society of Brewing Chemists. IFFO is a trademark of The International Fishmeal and
Fish Oil Organization. ISO is a trademark of The International Standards Organization. Chromosorb is a trademark
of IMERYS MINERALS CALIFORNIA, INC. All other trademarks are the property of Thermo Fisher Scientific and
its subsidiaries
This information is not intended to encourage use of these products in any manners that might infringe the
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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trademark of The Association of Official Analytical Chemists. AACC is a trademark of The American Association of Cereal Chemists.
AOCS is a trademark of The American Oil Chemists’ Society. ASBC is a trademark of The American Society of Brewing Chemists.
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