Determining Proteins in Milk Using Dumas Analysis

Milk contains proteins that play a major role in its flavor, quality, and the properties of its resultant by-products. Therefore, the amount of protein present in all dairy products, including milk, is an important variable for quality control.

Three widely used methods are available for quantifying the protein content of dairy products. These include Near Infrared Reflectance spectroscopy (NIR), Kjeldahl, and Dumas methodologies.

In the Dumas methodology, the sample needs to be burned in a high-temperature oven (often surpassing 900 °C) and subsequently in a reduction reactor. This process changes all the nitrogen present in the sample to N2, which then undergoes measurement.

In the Kjeldahl methodology, the sample is digested in a strong, heated acid for a period of 90 minutes. This process changes all the nitrogen present in the sample to ammonia, which then undergoes distillation and titration. Hence, both the Kjeldahl and Dumas methodologies are effectively total nitrogen determinations.

The protein content is measured through multiplication by a suitable factor. In the NIR methodology, the wavelengths of light absorbed by a sample need to be determined and the ensuing relation of that data to protein content also needs to be determined. While this method directly quantifies the protein content, it requires sample-specific calibrations with an individual method.

Among the three techniques, the Kjeldahl methodology is the most costly to run, the most labor-intensive, and the slowest. The fast, consistent, and automated analysis by the Dumas methodology offers a practical option for all types of dairy product samples, either for calibration of a NIR system or as a primary analyzer.

Image Credit: Elementar Analysensysteme GmbH.

Image Credit: Elementar Analysensysteme GmbH.

Instrumentation

Elementar has over 110 years of experience in developing elemental analyzers and more than five decades of experience in developing customized Dumas N/Protein analyzers. The company has recently introduced the fast MAX N exceed analyzer. This instrument integrates high throughput and ease of operation with a reliable determination of nitrogen, even at low concentrations and among challenging samples.

Stainless steel crucibles used by the 90-position autosampler can sustain up to 5 mL of liquid or 5 g of solid. Every position of the random-access autosampler is eternally available which, when combined with the user-friendly software of the instrument, enables easy scale-up of time-critical samples in measurement priority.

The crucibles are integrated into the combustion furnace through a gripper arm with a built-in oxygen inlet. Since oxygen is directly dosed at the sample, less amount of oxygen is needed to realize complete combustion.            

In addition, the proprietary EAS REGAINER® and EAS REDUCTOR® technology from Elementar allow more savings. This system uses a metal-free, non-toxic methodology that binds surplus amounts of oxygen and regenerates the metals, decreasing the nitrogen oxides from combustion to nitrogen gas for consistent detection.

Consequently, the reduction of tube filling can assess over 1000 samples before there is a need to replace this tube. This considerably decreases one of the key cost drivers for Dumas analysis without affecting the analytical performance.

Additional savings can be realized by using argon as a carrier gas, instead of the usual helium. The rapid MAX N exceed has a capacity to quantify up to 1 g of organic material and can produce accurate and reproducible results, even among rationally heterogeneous samples such as yogurt or milkshakes with fruit.

The instrument, which is supported by a rugged three-stage gas drying system, can efficiently perform regular measurement of several grams of aqueous solutions such as milk.

Since the same upright crucibles can also be used for liquids or solids, relaying between liquid and solid samples does not require any additional materials or chemicals, including sample liners or absorbers.

A Case Study

Immergut is a company that has been processing milk and milk-related goods for over a century. It currently provides a product range of about 300 products across many brands, covering a wide range of fruit-based and milk-based beverages.

Subsequent to the consequential increased demand for its quality control laboratory, Immergut needed a faster option to substitute its current Kjeldahl protein determination. Soon afterward, a rapid MAX N exceed was established in Immergut’s laboratory in the fall of 2016.

The company’s personnel were able to complete training in the system’s utilization and maintenance in a span of just four hours. Over the next 14 working days, the rapid MAX N exceed was used to analyze nearly 800 samples and simultaneously comparison measurements were made using the current Kjeldahl systems (refer to Table 1).

Table 1. A selection of sample types and the Kjeldahl values for %N and %protein. Source: Elementar Analysensysteme GmbH.

  SAMPLE N [%] PROTEIN [%]
1 Yogurt Drink Raspberry 0.307 1.96
2 Cream 0.309 1.97
3 Milkshake Cafe 0.342 2.18
4 Milkshake Vanilla 0.393 2.51
5 Milkshake Chocolate 0.434 2.77
6 Milkshake Strawberry 0.470 3.00
7 Lactose-free Milk 0.495 3.16
8 Healing Whey 0.496 3.17
9 Whole Milk 0.519 3.31
10 Yogurt Drink Strawberry-Banana 0.521 3.32
11 Goat milk 0.522 3.33
12 Soy Drink Chocolate 0.534 3.41
13 Low-fat Milk 0.561 3.58
14 Diet Drink Yogurt Strawberry 0.562 3.58
15 Soy Drink Nature 0.568 3.62
16 Yogurt Drink Passion Peach 0.603 3.85
17 Cappuccino Milk 0.603 3.85
18 Protein Drink/Nutrition Suppl. Van 0.628 4.01
19 Sports Drink Chocolate 0.628 4.01
20 Sports Drink Mocha 0.634 4.04
21 Yogurt Drink Banana 0.646 4.12
22 Yogurt Drink Red Fruits 0.693 4.42
23 Sports Drink Chocolate 0.873 5.57
24 Sports Drink Strawberry 0.876 5.59
25 Protein Drink Vanilla 0.908 5.79
26 Sports Drink Vanilla 0.917 5.85
27 Sports Drink Chocolate 0.972 6.20
28 Protein Drink Vanilla 0.988 6.30
29 Coconut Water Pineapple 0.993 6.34
30 Coconut Water Natural 1.016 6.48
31 Coconut Water Pure 1.039 6.63
32 Protein Drink Vanilla 1.119 7.14
33 Protein Shake Cafe 1.129 7.20
34 Protein Drink Chocolate 1.154 7.36
35 Protein Water Passion Fruit 1.161 7.41
36 Protein Shake Strawberry 1.165 7.44
37 Protein Drink Chocolate 1.290 8.23
38 Protein Drink Strawberry 1.317 8.40
39 Protein Shake Vanilla 1.495 9.54

 

The Dumas measurements were made in triplicate, whereas the Kjeldahl values were made in duplicate (but in cases where there was no repeatability, only a single determination was undertaken). Immergut’s personnel carried out all Kjeldahl and Dumas quantifications onsite. In addition, a subset of the samples was also sent to an external laboratory that implemented Kjeldahl analysis.

Image Credit: Elementar Analysensysteme GmbH.

Image Credit: Elementar Analysensysteme GmbH.

Comparison with DIN EN ISO 14891

DIN EN ISO 14891 “Milk and milk products—Determination of nitrogen content—Routine method using combustion according to the Dumas principle” includes the precision requirements for repetitions with the same technique between laboratories (reproducibility, R95) and within laboratories (repeatability, r95).

According to the precision requirement, two separate quantifications should manifest a complete variation in mass percent nitrogen that is below the matrix-dependent accepted value.

As a conservative estimate for sample heterogeneity in this analysis, the need for reduced fat milk (R95 of 0.093%N and r95 of 0.080%N) was used on all samples.

Figure 1 shows the variation of the two Kjeldahl measurements and also demonstrates the highest difference of the three Dumas measurements for every sample. It was observed that each technique exceeded the repeatability prerequisite by a factor of two or more, for every sample.

A comparison between the difference of two Kjeldahl analyses of each sample (red squares) and the maximum difference of three Dumas analyses of each sample (blue diamonds).

Figure 1. A comparison between the difference of two Kjeldahl analyses of each sample (red squares) and the maximum difference of three Dumas analyses of each sample (blue diamonds). Image Credit: Elementar Analysensysteme GmbH.

While this case study covers different methods, the standard’s reproducibility requirements offer a suitable benchmark for ascertaining their equivalence. The greatest variance between a Dumas value and Kjeldahl value for an individual sample is shown in Figure 2. For comparison purposes, the highest variance between the Kjeldahl results from Immergut and the external laboratory is also shown.

A comparison of the largest difference between a Kjeldahl and a Dumas value for each sample analyzed by Immergut (green triangles) and the largest difference between the Kjeldahl results from the external lab and Immergut (purple circles). For both series, values are positive or negative reflecting measurements above or below the Immergut Kjeldahl value, respectively.

Figure 2. A comparison of the largest difference between a Kjeldahl and a Dumas value for each sample analyzed by Immergut (green triangles) and the largest difference between the Kjeldahl results from the external lab and Immergut (purple circles). For both series, values are positive or negative reflecting measurements above or below the Immergut Kjeldahl value, respectively. Image Credit: Elementar Analysensysteme GmbH.

Each comparison exceeded the reproducibility need by a factor of two or more for every sample. It must be noted that neither the external laboratory nor the Dumas values showed a systematic change from the Immergut Kjeldahl values.

Summary

To determine protein content in milk and milk by-products, both the Kjeldahl and Dumas analysis methods display excellent quality analytical performance. With regard to an example set of 39 products obtained from Immergut, the average repeatability of a recently installed rapid MAX N exceed was 0.007 %N absolute, as opposed to the 0.0011 %N absolute exhibited by the Kjeldahl approach.

The average reproducibility of the measurements between the rapid MAX N exceed and the Kjeldahl systems at Immergut was 0.022, and the average reproducibility of the measurements between Immergut and an external Kjeldahl analysis was 0.026 %N absolute.

Although the analytical performance of each technique easily fulfills international standards criteria, the performance of the rapid MAX N exceeded that of Kjeldahl analysis in both comparisons. In addition to excellent analytical performance, Immergut enjoyed enhanced throughput, reduced operating costs (0.50€ for each sample), and improved ease of use by choosing the rapid MAX N exceed over Kjeldahl analysis.

With an average analysis time of about six minutes, calibrations for NIR systems can currently be carried out effectively and efficiently, ensuring excellent quality outcomes in all facets of production.

Such improvements and benefits make the rapid MAX N exceed a perfect solution, allowing any producer of milk and other dairy products to maximize productivity, reduce costs, and realize higher quality results.

About Elementar Analysensysteme GmbH

Elementar is the world’s leading German manufacturer of analyzers for high performance analysis of organic and inorganic elements. Arisen from the Analytical Instrumentation Department of the Heraeus technology group, we have developed and manufactured elemental analyzers for more than 110 years in the Rhine Main area near Frankfurt, Germany. We help our customers to support the quality of life whenever water, nutrition, education, health, environment, climate, energy or product quality are involved. Through our premier brand Elementar we offer an unmatched combination of innovative technologies, tailor-made solutions and comprehensive support.

Today’s product portfolio includes dedicated analyzers for CHNOS elemental analysis of micro and macro samples, water analysis (TOC/TNb), determination of N/Protein, analysis of metals and other inorganic materials, and instruments for stable isotope analysis (IRMS). Our longtime experience and continuous innovation ensure our products continue to advance science across agriculture, chemical, environmental, energy, materials and forensics markets in more than 80 countries.


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Last updated: Mar 2, 2020 at 8:38 AM

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