The Quest for the Quickest Kjeldahl: How to Automate Protein Determination

Chapter 61
📂 Case overview: Shallot Holmes puts the team to the test by finding out which detective can speed up the Kjeldahl process the most without hindering the accuracy of the results. Who will come out on top in the quest for the quickest Kjeldahl?

How can you Prevent Heartburn?

It’s a sunny day, and the detectives are in the office waiting for Shallot Holmes, who is seeing a client about a potential case. It’s been a few hours, and the detectives are hungry, especially the resident gannet Cornlumbo. Miss Mapple heats up some homemade tomato soup she bought and offers some to the detectives. Cornlumbo, however, says he cannot eat tomato soup as tomatoes are too acidic and give him heartburn. Miss Mapple tells Cornlumbo that she also suffers from heartburn but that he should try her soup as she has a trick for toning down the acidity of the tomatoes. The detectives are all intrigued by Miss Mapple’s trick and ask how she neutralizes the acidity. “Well, I add a pinch of baking soda to the tomatoes, which has a double benefit. It helps neutralize the acid by increasing the pH, and it also helps break down the cell walls of the tomatoes, softening them more quickly,” she says. Cornlumbo tries a bowl of soup, and Miss Mapple is right – no heartburn!
 

Adding a pinch of baking soda to tomatoes helps neutralize the acid by increasing the pH; it also helps break down the cell walls of the tomatoes.

How can IR Heating Technology Accelerate Kjeldahl?

Shallot Holmes enters the office with the details of the latest case. The client uses the Kjeldahl method for protein determination, and they are looking for a way to speed up the process. Shallot asks the detectives if they have any ideas for how they can speed up the process. Eggcule Poirot kicks things off by suggesting that the client use a digester with IR heating technology. “Traditional heating methods rely on conduction, but Infrared heating is a more efficient way to transfer energy. Digesters that use IR offer faster heat-up times, and IR radiation can penetrate the sample directly, achieving desired temperatures quicker and reducing the digestion time. Another advantage is the even distribution of heat that may lead to a more consistent digestion process,” says Eggcule. Holmes is suitably impressed and asks the other detectives if they have any suggestions.
 

Digesters that use IR offer faster heat-up times; IR radiation can penetrate the sample directly, achieving desired temperatures quicker and reducing the digestion time.

How can Automation Speed up Protein Determination?

Nancy Beef suggests using automatic titration. “By adding an automatic titrator to the distillation unit, as soon as the distillation is complete, the titrator can start the titration process without manual intervention. This reduces the time lag between distillation and titration, making the process more efficient; it also reduces the chance of human error,” says Nancy. Cornlumbo agrees that automation is the key to speeding up modern laboratory processes and that it could be taken a step further. “The client could perform online titration whereby the titration starts while the distillation is still in progress. This can save several minutes as you don’t have to wait for the distillation to be complete before starting the titration,” says Cornlumbo.

By adding an automatic titrator to the distillation unit, as soon as the distillation is complete, the titrator can start the titration process without manual intervention.

“The introduction of samples can also be automated with modern instruments that feature an autosampler,” says Nancy. She explains that automated samplers can be programmed to handle multiple samples in a sequence. They can load the sample, start the digestion, and even initiate the titration process. Such a system can be set up to run overnight, allowing for the continuous processing of samples. Also, just like the automated titrator, the autosampler benefits from no human interference; each sample is handled in a consistent manner, reducing variability and improving reproducibility. Holmes tells the detectives that he can think of one more way the client could speed up their process and wonders if any of them can guess.

Automated samplers can be programmed to handle multiple samples in a sequence. They can load the sample, start the digestion, and even initiate the titration process.

Do Catalysts Speed up the Digestion Process?

The detectives all try their best to think of another method, but it isn’t until Miss Mapple remembers her trick with the soup. Adding the touch of bicarbonate of soda helped speed up the softening of the tomatoes, acting a bit like a catalyst. Miss Mapple explains to the group that a titanium catalyst combined with hydrogen peroxide can accelerate the digestion process. Kjeldahl catalysts are typically tablets composed of around 97% salts that increase the boiling temperature of the sulfuric acid, and the remaining 1-3% is the catalyst that increases the speed and efficiency of the digestion process. Titanium helps break down compounds, while hydrogen peroxide serves as an oxidizing agent that facilitates the breakdown of organic matter. Hydrogen peroxide also works as an anti-foaming agent, which is particularly advantageous if the sample contains fat or carbohydrates.

Using a titanium catalyst combined with hydrogen peroxide can accelerate the digestion process.

“Excellent work, detectives,” says Holmes. “The automation suggestions you made will definitely speed up the client’s processes without affecting their methods; however, the addition of catalysts or oxidizing agents will have to be verified,” says Holmes, who explains the importance of validating any new process or modification to ensure the results are accurate and comparable to the traditional method. Holmes suggests a little competition between the detectives to decipher the best method to suggest to the client – a race! Eggcule is to perform a standard Kjeldahl digestion the way the client currently does, Miss Mapple will perform an accelerated digestion with H2O2, and Nancy will perform an accelerated digestion with H2O2 and a Kjeldahl Titanium tablet. It is down to Cornlumbo to prepare the samples, time the detectives, and write up the results.

The detectives head to the lab and perform their protein analysis one by one, while Cornlumbo takes notes and compares the results to reference samples. At the end of the day, the detectives reconvene, and Cornlumbo shows the detectives a breakdown of the time taken by each method, the results compared to a reference sample showing the Relative Standard Deviation (RSD), and finally, a comparison chart showing the advantages and disadvantages of each method.

Standard Kjeldahl Digestion.

Accelerated Digestion with H2O2.

Accelerated Digestion with H2O2 and Titanium Catalyst.

Results of the Nitrogen and Protein Determination Compared to a Reference Sample (declared protein content 14.5 g/ 100 g).

Comparison of the Digestion Methods

Shallot is very impressed with the work of the detectives and is keen to share their findings with the client, who he is sure will be very happy with the optimizations achieved by the detectives. To celebrate their success, the detectives enjoy a meal together, and as soon as they finish their dessert, Eggcule suggests they all enjoy a nice glass of Chartreuse but only “To speed up the digestion process! – Cheers!”