Feeling the Heat: Troubleshooting Hydrolysis for Accurate Hot Extraction
In the heart of the Black Forest in Germany, The Label Lab provides comprehensive analytical services to ensure accurate labeling of fat content, nutritional values, and other key parameters. Recently, they invested in a state-of-the-art UniversalExtractor for faster, more efficient hot extractions. The goal? Streamline operations and boost profits while maintaining a high standard of precision. However, in the lab, things are not quite going to plan!
Wilhelm Wackler and Heinz Heißluft are trying to get to grips with the sample preparation for the new instrument. The day had started well enough: the sausage samples were carefully weighed and homogenized. But now, during the hydrolysis, foam rapidly forms inside the vessel. Wilhelm’s heart skips a beat as he fumbles with the controls, trying to lower the heat, but the foam overflows, spilling onto the lab bench. Heinz quickly yanks Wilhelm’s wobbly hands away just in time as the hot, acidic froth hisses dangerously close to the top of the vessel.
More hot air rises from Heinz as he begins to panic. The foam swells, and they watch helplessly as it overflows, spilling onto the lab bench. The acid hisses and sputters on the surface, sending Wilhelm scrambling for paper towels and gloves, but the mess spreads faster than they can control. They finally manage to reduce the heat and calm the foaming disaster. They finished the hydrolysis, but they are now behind schedule. In a panic, Wilhelm rushes to put the sample in the microwave oven, his hands trembling uncontrollably.
Rushing to make up for lost time, Wilhelm puts the sample in the microwave on high heat. But when they open the door, a wave of hot air rises. The sample has darkened, and the fat has oxidized, leaving burnt residue stuck to the glass. But anyway, they placed the samples in the extraction unit and performed a hot extraction. The results? Not even close to the declared values. Concerned about The Label Lab’s reputation, Wilhelm and Heinz call in the food detectives.
Meanwhile, in the detective’s office, the detectives enjoy the fondue Cornlumbo made with the cheese he acquired from their last client. “Thank you, Cornlumbo, this is delicious,” says Miss Mapple. “It’s so moreish, I can’t get enough,” says Eggcule as he tears another piece of bread to dip in the cheese. As Shallot Holmes leans over, he drops his bread into the fondue pot. “Aha, you must now pay a penalty; you must kiss the person sitting to your left,” shouts Cornlumbo. “I have to kiss Miss Mapple because I dropped my bread,” says Holmes. “Anyone who drops anything into the fondue pot must pay a penalty,” says Cornlumbo. Miss Mapple’s cheeks turn red as Holmes leans over and gives her a peck on the cheek. Cornlumbo looks to his left at Nancy Beef, reaches forward with some bread, and drops it into the fondue pot just as the phone rings. “Ooh, saved by the bell,” shouts Nancy as she leaps from her chair to answer the phone.
Wilhelm Wackler and Heinz Heißluft explain the problems they’ve been having, and Nancy, in need of a quick escape, volunteers to help and bolts out the door.
Why is acid hydrolysis necessary when analyzing fat content?
Nancy arrives at The Label Lab and gathers evidence to build her case. She sees that they are analyzing the fat content of sausages and asks if they performed acid hydrolysis. In sausages, the fat is often bound to proteins, carbohydrates, or within the structure of the muscle tissue. Acid hydrolysis breaks down proteins and other structures that encapsulate the fat, releasing it to be effectively extracted.
Luckily for Nancy, Wilhelm and Heinz wrote down each step of their process so that they could effortlessly repeat the steps and optimize the method over time. Nancy investigates their methodology for clues and takes notes as she reads.
What are the steps for Fat determination by hot extraction?
- Sample homogenization.
- Hydrolysis of the sample.
- Drying of the hydrolyzed sample.
- Hot extraction of the fat.
- Drying of the extract.
- Weighing of the extract.
- Calculation of fat content.
What are the steps for Acid hydrolysis?
Preparation of the glass sample tubes:
- 1. Add 20 g of quartz sand and 2 g Celite® 545 to the glass sample tube and compact the sand by gently tapping the glass sample tube onto the table.
Hydrolyzing the sample matrix:
- 2. Place 2 g Celite® 545 in the hydrolysis vessel.
- 3. Add up to 10 g homogeneous sample to the hydrolysis vessel and note the weight of the sample.
- 4. Add 50 mL hydrochloric acid (4 M) and form a suspension by gently swirling the vessel.
- 5. Add another 50 mL hydrochloric acid (4 M) making sure to rinse any remaining sample off the glass wall.
- 6. Preheat the hydrolysis instrument for 10 min.
- 7. Insert the samples into the unit and lower the vessels.
- 8. Connect the aspiration tubes and start the vacuum pump.
- 9. Reduce the heat to level 2.5 when one position is boiling.
Hydrolyze the sample for 30 min after constant boiling is observed in each position
- 10. At the end of the hydrolysis time, add 50 mL of warm (50 °C) deionized water to each vessel.
- 11. Switch off the heating and lift the hydrolysis vessels to the top position to filter the hydrolysate.
- 12. Wash each vessel by gradually adding at least 400 mL of warm deionized water until it reaches a neutral pH.
- 13. Check the pH with a pH paper on the bottom of the frit.
- 14. Stir the Celite® layers with a spatula to loosen the pulp.
- 15. Wipe off the spatula with a piece of tissue and add it to the top of the sample.
- 16. Dry the glass sample tubes in a microwave oven.
- 17. Allow the glass sample tubes to cool to room temperature in a desiccator.
What are Nancy’s Top Tips for Hot Extraction?
“Can you tell me how you performed step 1?” asks Nancy. “We did it just how it says; we added the quartz sand and Celite® to the glass sample tube,” says Wilhelm. “Did you mix them?” asks Nancy. “Wilhelm Wackler tends to shake up most things he touches,” says Heinz. “Then I suggest that this step is a job for you, Heinz, as the sand and the Celite® layer should not be mixed. Otherwise, the Celite phase may break through the frit and affect the results by increasing the recovery or blocking the frit,” Nancy explains. “Also, in step 14, when you stir the Celite® layers, you must avoid touching the sand layer.”
“In step 3, you say, ‘add up to 10g of sample; how do you choose the sample amount?” asks Nancy. “Well, we don’t want to overload the system, so we thought no more than 10 g would be fine,” says Wilhelm. “You’re right, but the approximate fat content of the sample should dictate the sample size. You can use the following as a guide,” says Nancy.
- <10 %: 7–10 g
- 10–20 %: 3.5–7 g
- 20–50 %: 1.5–3.5 g
- 50–80 %: 1–1.5 g
- 80–100 %: 0.7–1 g
“You mentioned you had a problem with violent foaming. Did you reduce the heat when one of the solutions started to boil?” Nancy asked. Heinz glanced at Wilhelm, who looked embarrassed and began stammering, “We… um… at the beginning… when we started…”
“There was a phone call, and we didn’t notice when it started boiling,” Heinz blurted out. “By the time we came back, there was already a lot of foam, and acid was sputtering everywhere. We eventually reduced the heat, but it was too late.”
Nancy stared at them both. “The beginning is critical for hydrolysis. You need to keep a close eye on it. Once it’s boiling without foam, you can leave it, but not before.”
“You also said you managed to burn the sample in the microwave. I’d suggest starting at a high heat initially, for example, 15 min at 640 W, then reducing the temperature to 480 W for 9 minutes. You may need to adapt this depending on your microwave. Fat may decompose above 105 ºC, and oxidized fat can result in an excessive recovery,” says Nancy.
“Finally, I’d suggest one final step: 18. Add another layer of quartz sand (20 g) to prevent the Celite® from being re-suspended in the condensed solvent. If you follow these guidelines, I’m sure you’ll solve all the problems you’ve been having,” says Nancy. “Oh, thank you so much, Nancy; I’m so happy I could kiss you!” shouts Heinz. “Umm, that’s ok, your thanks are enough,” says Nancy as she bolts out the door.