Determining Proteins in Milk Using Dumas Analysis

The protein content of milk plays a central role in its quality and flavor, as well as the characteristics of its consequent by-products. As a result, the amount of protein in milk and other dairy products is a key variable for quality control. There are three broadly utilized approaches for measuring the protein content of dairy products: Dumas, Kjeldahl, and Near Infrared Reflectance spectroscopy (NIR).

The Dumas methodology requires the sample to be burned in a high-temperature oven (usually exceeding 900 °C) and then in a reduction reactor. This converts all the nitrogen in the sample to N2, which subsequently undergoes measurement. The Kjeldahl methodology digests the sample in a strong, heated acid for 90 minutes. This converts all the nitrogen in the sample to ammonia, which subsequently undergoes distillation and titration. Consequently, both the Dumas and Kjeldahl methodologies are, in effect, total nitrogen determinations.

The protein content is quantified via multiplication by an appropriate factor. The NIR methodology requires determining the wavelengths of light that a sample absorbs, and the subsequent relation of that information to protein content. This approach performs a direct measurement of protein content, but necessitates sample-specific calibrations with a separate methodology.

Of the three approaches, the Kjeldahl methodology is the most labor intensive, the slowest and the most expensive to run. The rapid, reliable and automated analysis by Dumas is a sensible option for any dairy product sample, either as a primary analyzer or for calibration of an NIR system.

Instrumentation

Boasting more than 110 years of experience manufacturing elemental analyzers and over 50 years of experience manufacturing customized Dumas N/Protein analyzers, Elementar has recently launched the rapid MAX N exceed analyzer. This instrument incorporates ease of operation and high throughput with the dependable determination of nitrogen, even among complex samples and at low concentrations.

The 90-position autosampler uses stainless steel crucibles that can sustain up to 5 g of solid or 5 mL of liquid. Each position of the random-access autosampler is permanently available which, when coupled with the instrument’s user-friendly software, allows time-critical samples to be easily upgraded in measurement priority.

The crucibles are inserted into the combustion furnace by a gripper arm with an integrated oxygen inlet. As the oxygen is dosed directly at the sample, less oxygen is required to achieve complete combustion, which is an important feature of Elementar’s unparalleled low price-per-sample.

Moreover, Elementar’s proprietary EAS REGAINER® and EAS REDUCTOR® technology enables additional savings. This system adopts a non-toxic, metal-free methodology which binds excess oxygen and regenerates the metals, thus reducing the nitrogen oxides from combustion to nitrogen gas for reliable detection. As a result, the reduction tube filling can examine more than 1000 samples before the need for replacement. This significantly reduces one of the main cost drivers for Dumas analysis without compromising analytical performance.

Further savings can be achieved by utilizing argon, rather than the usual helium, as a carrier gas. Due to its capacity to measure up to 1 g of organic material, the rapid MAX N exceed can still generate precise, reproducible results, even among reasonably heterogeneous samples, such as milkshakes or yogurt with fruit.

Bolstered by a sturdy three-stage gas drying system, the instrument can also efficiently undertake routine measurement of several grams of aqueous solutions, such as milk. Furthermore, because the same upright crucibles can be utilized for solids or liquids, relaying between solid and liquid samples necessitates no extra chemicals or materials, such as absorbers or sample liners.

A Case Study

Immergut, a company which has been processing milk and milk-related goods for more than 100 years, now offers a product portfolio of around 300 products across multiple brands, encompassing a broad spectrum of milk- and fruit-based beverages. Following the consequential increased demand on their quality control lab, Immergut required a quicker option to replace their existing Kjeldahl protein determination.

In the fall of 2016, a rapid MAX N exceed was established in their lab. Personnel completed training in its utilization and maintenance in only four hours. Over the next fourteen working days, almost 800 samples were examined with the rapid MAX N exceed alongside comparison measurements that were undertaken using the existing Kjeldahl systems (see Table 1).

The Dumas measurements were completed in triplicate, while the Kjeldahl values were completed in duplicate (except where no repeatability is provided, in which case only one determination was undertaken). The Dumas and Kjeldahl quantifications were all performed onsite by Immergut’s personnel. A subset of the samples was also dispatched to an external lab implementing Kjeldahl analysis.

Table 1. A selection of sample types and the Kjeldahl values for %N and %protein.

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

Comparison with DIN EN ISO 14891

The precision requirements for repetitions with the same method within labs (repeatability, r95) and between labs (reproducibility, R95) are provided in DIN EN ISO 14891 “Milk and milk products – Determination of nitrogen content – Routine method using combustion according to the Dumas principle”. The precision requirement states that two independent quantifications should manifest an absolute difference in mass percent nitrogen less than the matrix-dependent accepted value.

As a conservative estimate for the heterogeneity of samples in this study, the requirement for reduced fat milk (r95 of 0.080 %N and R95 of 0.093 %N) was applied to all samples. Figure 1 demonstrates the difference of the two Kjeldahl measurements and the maximum difference of the three Dumas measurements for each sample. Each method surpassed the repeatability requirement by a factor of two or more for each 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).

While this case study encompasses different methods, the reproducibility requirements of the standard provide an appropriate benchmark for establishing their equivalence. Figure 2 demonstrates the greatest difference between a Kjeldahl value and Dumas value for each sample. By way of comparison, the greatest difference between the Kjeldahl results from the external lab and Immergut is also exhibited.

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.

Each comparison surpassed the reproducibility requirement by a factor of two or more for each sample. It is worth noting that neither the Dumas nor external lab values exhibited systematic variation from the Immergut Kjeldahl values.

Summary

For determining protein in milk and milk by-products, both the Dumas and Kjeldahl analysis approaches exhibit top quality analytical performance. In the case of an example set of thirty nine products deriving from Immergut, the average repeatability of a newly installed rapid MAX N exceed was 0.007 %N absolute, compared to the 0.0011 %N absolute displayed by the Kjeldahl technique.

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

While the analytical performance of each methodology comfortably satisfies international standards criteria, the performance of the rapid MAX N surpassed that of Kjeldahl analysis in both comparisons. Furthermore, as well as superior analytical performance, Immergut enjoyed improved throughput, enhanced ease of use and reduced operating costs (0.50€ per sample) by selecting the rapid MAX N exceed over Kjeldahl analysis.

Boasting an average analysis time of approximately six minutes, calibrations for NIR systems can now be performed efficiently and effectively, guaranteeing top-quality results in all aspects of production. These advantages and enhancements render the rapid MAX N exceed an ideal solution, enabling any producer of milk and other dairy products to minimize costs, maximize productivity and achieve higher quality results.

This information has been sourced, reviewed and adapted from materials provided by Elementar Analysensysteme GmbH.

For more information on this source, please visit Elementar Analysensysteme GmbH.

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