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Protein Analysis

In October 2013, Lab Manager, along with BUCHI Corporation, hosted a Product Spotlight webinar to discuss the various techniques that are in use for protein analysis.

by Tanuja Koppal, PhD
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Comparing Kjeldahl, Dumas, and Near-IR

William Ickes, product specialist for Kjeldahl and Dumas at BUCHI Corporation, explained the workings of Kjeldahl, Dumas, and near-infrared (NIR) spectroscopy for protein analysis for various applications and outlined the strengths and weaknesses of each technique. Tonya Schoenfuss PhD, assistant professor in the Department of Food Science and Nutrition at the University of Minnesota, discussed the use of NIR spectroscopy for analyzing proteins in dairy products. Their presentations were followed by a question-and-answer session in which attendees asked questions and received feedback from the two experts. The live webinar was attended by a global audience, with varying levels of expertise and representatives from diverse industries. This event provided them with a unique opportunity to interact with the experts in real time and to seek their guidance and advice on various issues related to the analysis of proteins at different stages of the food process chain. The event was moderated by Tanuja Koppal, PhD, contributing editor for Lab Manager. Here are some key topics that were addressed.

What are the analytical challenges that need to be overcome for protein analysis?

Ickes: There are many analytical challenges for measuring protein content in food, feed, and forage in today’s modern industries. These challenges include the reliability of the analytical method, compliance with official methods, speed of analysis, sample throughput, unattended operation, variation in sample types, investment, and running costs. The three most important methods for protein determination—Kjeldahl, Dumas, and NIR spectroscopy—can meet the challenges of protein analysis, but the strengths and weaknesses of each technique should be considered according to the demands of the instrument’s place in the product life cycle.

What are the pros and cons of some of the existing methodologies available for protein analysis, namely Kjeldahl, Dumas, and NIR spectroscopy? (See chart below comparing the principles and features of the three techniques.)

Chart 1Ickes: Each technique, whether it be Kjeldahl, Dumas, or NIR spectroscopy, has its pros and cons. The pros of Kjeldahl include a robust technique that has the ability to cope with varying sample matrices, compliance with official methods, a high level of automation, and lower initial costs. The cons include the speed of analysis and use of chemicals for analysis. Dumas has fast analysis time, high throughput, a high level of automation, no use of harmful chemicals, and the ability to quickly reevaluate suspect samples. On the other hand, Dumas has limited ability to cope with sample matrices and to comply with official methods for some sample types, and it has higher initial costs. The advantages of NIR spectroscopy include fast speed of analysis, no use of chemicals, and multicomponent analysis. The disadvantages are its [issues of] compliance with official methods and initial start-up costs.

What are some of the key criteria that determine the best technique that should be used for protein analysis? (See chart below listing applicability of each technique.)

Chart 2Ickes: Some of the key criteria that will determine the best technique for protein analysis are determined by where in the product life cycle the analysis will take place and what the data generated will be used for. If the data is used for label claims, then a method such as Kjeldahl may be appropriate because of its compliance with official methods. If the data is to be used to monitor the manufacturing process or to screen incoming goods, then a faster analytical technique such as Dumas or NIR spectroscopy should be considered.

What factors need to be considered when optimizing the technique or method?

Ickes: Some of the factors that should be considered when optimizing a technique or method for protein analysis are the range of protein content in the sample, the homogeneity of the sample matrix, the speed required for analysis to ensure that there are no bottlenecks in the production process, and the analytical accuracy and precision needed for the results.

How would you analyze unknown proteins in your food sample?

Ickes: To analyze unknown protein content in a food sample, Kjeldahl would be the most appropriate method initially because it directly measures the nitrogen content of the amino acids. Two things to consider when analyzing a sample for unknown protein amount are the correction factor for the specific sample type, which is determined by empirical data from amino acid analysis, and any contribution from nonprotein nitrogen, which can be determined by Kjeldahl after the precipitation of the protein with trichloroacetic acid followed by filtration.

What are some of the trends and improvisations taking place in protein analysis?

Ickes: The trend for Kjeldahl analysis of protein is that the technique is still widely used due to its robustness, ability to cope with virtually any sample matrix, and compliance with official methods. There is also a trend toward increasing the use of alternative techniques such as Dumas and NIR spectroscopy, which use less chemicals and have a faster analysis time. With the evolution of the hardware and software of NIR spectroscopy, it continues to be a growing trend for the analysis of protein in food.

What are the challenges associated with sample preparation for NIR spectroscopy?

Schoenfuss: NIR spectroscopy is actually a very flexible system—you don’t have to have liquids or have to dilute a sample. The challenge is that you need the samples for NIR spectroscopy to be consistent. You don’t want the temperature or particle size to vary. You don’t want the thickness of the material in a petri dish or how much you have compressed a paste into the dish to vary. So developing a standard operating procedure for how the sample will be prepared, before you start your calibration work, is very important. You also have to decide on the presentation method, because this can’t change either.

What is the effect of particle size and sample color when using NIR spectroscopy for protein analysis?

Schoenfuss: In our work, we did not find particle size differences due to the fat in fluid milk to be an issue for quantification of components, which is contrary to what is seen in mid-IR analysis. But for dry ingredients such as flour and grains, it can be very important. Calibrations can even be developed to estimate the particle size of powders using NIR spectroscopy.

Can we determine amino acid profiles or protein ratings using NIR spectroscopy?

Schoenfuss: NIR spectroscopy has been used to identify and quantify amino acids, and it is also possible to determine protein ratings.

How important is it to use the right standards and calibration methods for protein analysis?

Schoenfuss: The quality of a calibration is directly dependent on the quality of your wet chemistry data for protein analysis. So if that data is inaccurate, the calibration will be inaccurate as well.

For more information on protein determination, visit or  

William H. Ickes is currently the product specialist for Kjeldahl and Dumas at BUCHI Corporation in the USA. He is a graduate of Millersville University and has a Bachelor of Science in chemistry and biology. He formerly worked as a pharmaceutical raw materials chemist at Lancaster Laboratories as well as a technical support specialist and service specialist at BUCHI Corporation.

Dr. Tonya Schoenfuss is a professor in the Department of Food Science and Nutrition at the University of Minnesota and conducts research on dairy products and ingredients; she also teaches food quality, product development, and dairy product technology classes. She has a BS in dairy science from Cal Poly San Luis Obispo, an MS in food science from Virginia Tech, and a PhD in dairy science from Louisiana State University.