kjeldahl; sample preparation; digestion; milk

Chapter 40

The detectives shine a light on Kjeldahl – Part 1: Sample preparation & Digestion

📂 Workshop overview: Having returned from the successful dioxin conference where the detectives got to grips with the determination of dioxins in fish; Shallot Holmes wonders if the detectives can also decode the mysteries of nitrogen and protein determination. He challenges the food detectives to provide their clients with an in-depth investigation into an age-old laboratory process – the Kjeldahl method. In this first installment, find out all about the importance of sample preparation and the important digestion step.

It was approaching the end of the year, and the detectives had no case to sink their teeth into, so they sat discussing the dioxins conference they had been lucky enough to attend. Like all detectives, they were fascinated with poisonous substances and could talk about them for days. Shallot Holmes reflected on the year and thought about his clients and the importance of detecting protein and nitrogen. The detectives had solved several cases that involved the determination of protein in Milk, Beer, and Plant-based meat. Shallot Holmes began to formulate a plan that would keep the detectives busy and serve as an end of year gift to their loyal clients.

Shallot Holmes rose to his feet. “Right, detectives, your attention please” The detectives sat up and looked over to Shallot Holmes, pacing back and forward excitedly. “Over this past year, a particular method has repeatedly cropped up in our cases. Can anyone guess what the process is?”

The detectives racked their brains until Nancy Drew spoke up, “Rotary Evaporation?”

“Good thought, but no, that’s not it. In fact, by the end of this year, laboratories will have been using this process for 140 years,” declared Shallot Holmes.

The detectives looked puzzled until Shallot Holmes put their minds at rest. “The process I am speaking of is the Kjeldahl method, used for nitrogen and protein determination; Johan Kjeldahl published his method in 1883, the same year the electric lamp was patented. So, detectives, it is time for us to shine a light on the Kjeldahl method”.
Shallot went on to explain his idea. He said that although they had compared Kjeldahl to NIR and described how to speed up the digestion process; they had never provided a detailed overview so clients could determine their own methods. “It is a capital mistake to theorize before one has data. Insensibly one begins to twist facts to suit theories, instead of theories to suit facts,” declared Holmes.

He tasked each of the detectives with investigating a step in the Kjeldahl process, and they would reconvene to share their findings and pass the knowledge on to their clients. As it’s a lot of information to get through, Shallot Holmes divided the workload into two workshops. “In the first workshop, I shall give a brief introduction to sample preparation,” said Shallot Holmes. “And Eggcule Poirot shall investigate the 1st step of the Kjeldahl process, which is the digestion stage”.

“For our second workshop, Miss Mapple will explain the separation step, and Nancy Beef will do the final step, which involves titration and the volumetric determination of the amount of ammonia”. Shallot Holmes noted that he wanted an overview of the process as well as advice for things that their clients should be aware of.

“What about Lieutenant Cornlumbo? You haven’t given him anything to investigate at all!” shouted Nancy Beef, thinking Cornlumbo had got off lightly. Shallot explained that he had a special task for Lieutenant Cornlumbo that would result in a special prize for one of the detectives. The detectives looked puzzled yet excited and set off to work on their respective tasks.

A week later, the detectives reconvened for the first workshop. Shallot Holmes began by explaining the importance of sample preparation. He explained that the Kjeldahl method allows for calculating protein content based on the determined nitrogen. The first consideration was the preparation of the sample being tested. Sample size explained Holmes had a significant implication on the subsequent steps, and a general rule was that particle size should not be larger than 1mm. There were two critical points:

– Sample amount
– Homogeneity

“If the sample size is too low, titration volume will be too low and, therefore, inaccurate. If the sample size is too high, there will not be enough boric acid to trap all the ammonia, and the result may appear lower than expected. Homogeneity is essential to ensure reproducibility. If samples are not homogenous, there will be high standard deviations in repeated determinations”. Shallot Holmes described a useful measure for inhomogeneity that could be used by their clients, which was the relative standard deviation (RSD) expressed as a % of the mean value. A 6 – 30 % protein content of homogenous samples shows RSD values < 1 %. RSD values > 1 % are a strong indication of insufficient homogeneity and will not result in high-precision measurements. The optimal sample amount depended on the expected nitrogen content, which has implications on the choice of titrant concentration and size of sample tube required.

Next up was Eggcule Poirot to talk about digestion. This involved carbonization, where organically bonded nitrogen is converted into ammonium sulfate. “For digestion, two critical factors our clients must be aware of are oxidization and temperature,” explained Eggcule. “Organic material is oxidized with sulfuric acid, and this process is enhanced with oxidizing agents called catalysts. Historically, catalysts used included selenium, mercury, copper, and titanium”. Eggcule, who was obsessed with safety – as the story of Humpty Dumpty had haunted him as a child – was quick to point out that mercury and selenium were highly toxic and should be avoided.

“As for temperature, the desired sample temperature is between 350°C and 380°C and should not exceed 390°C as nitrogen losses occur due to the transformation of ammonia into elemental nitrogen gas N2. The reaction rate doubles with every 10°C increase; however, you cannot simply apply more heat. Increasing the boiling point of sulfuric acid (approx. 335°C) requires inert salts. Nowadays, catalyst tablets are available that have been optimized to add the right amount of catalyst and create the ideal ratio of salt to sulfuric acid for specific processes”.

Eggcule described how the temperature of the sample changed throughout the digestion process and how important it was to control. Increasing the temperature would speed up the process, but higher temperatures decompose the carbonized material to carbon dioxide, which is reflected by foam formation – expanding black sample material in the sample tube. “Foam formation is something our clients must contend with, and it is mainly an issue with large samples. Certain catalysts will, however, reduce foaming, as can additives such as silicone or stearic acid. Slowly increasing the temperature can also help prevent foaming. Optimal digestion conditions are achieved when the condensation zone remains 5 cm below the constriction of the sample tube. Towards the end of the digestion stage, foam decomposes, and a clear liquid indicates the completion of the chemical reaction. Once the liquid is clear, a further 30-minute digestion period is recommended to allow complete mineralization”.

“Great work, Eggcule,” declared Shallot Holmes. “Understanding the importance of sample preparation and the factors that influence the digestion stage is sure to help our clients avoid several common mistakes when undertaking Kjeldahl determinations”. The rest of the detectives seemed very impressed. The pressure was now on for Miss Mapple and Nancy Drew, who were due to give their presentations at the next workshop. Meanwhile, Lieutenant Cornlumbo was smirking in the corner, only adding to the intrigue of the other detectives. Just what had he been tasked to do? And what on earth was the special prize Holmes mentioned? Be sure to check out the next workshop to find out.