Using an Automated Kjeldahl Analyzer: Practical Notes

The fully automated Kjeldahl nitrogen analyzer is the instrument of choice for protein determination and is widely used to measure protein content in food. It offers high sensitivity, fast analysis, a wide application range and small sample requirements, and the equipment and its operation are comparatively simple — which is why it is the most widely used protein-determination instrument today.

1.1 Before switching on

Make sure every solution is sufficient for the planned determinations: check the deionized water, the alkali solution and the receiving solution, and confirm that the waste in the waste tank has been emptied in time. If the deionized water, alkali solution or receiving solution falls below its level sensor — or the waste level rises above its sensor — the instrument will alarm and the running test will be stopped, interrupting normal determination work.

1.2 Open the cooling-water valve first

Before switching on, make sure the cooling-water valve is open. If it is closed, the instrument can still pass its self-check — but during distillation the temperature of the distillation unit runs too high, the instrument alarms and prompts you to open the cooling-water valve. Once it is opened, the instrument continues to work, but the test has already been stopped; and running without cooling-water protection can damage the distillation unit.

1.3 Start-up: purge titrator bubbles

With 1.1 and 1.2 confirmed, switch the instrument on. It runs a self-check and, once passed, waits for program selection. First enter manual mode and purge any bubbles from the titrator. Bubbles affect the determination result: once a bubble enters, it occupies volume that should belong to the standard titrant acid, so the delivered acid volume falls short and the measured result comes out low.

1.4 Blank determination

For blank determination, measure the water blank first — the water blank checks the stability of the instrument. The instrument is stable when two consecutive blank readings differ by less than 0.05 and the water-blank value is below 0.2. Then measure the reagent blank and enter the blank value into the instrument. If you skip the water blank and measure the reagent blank directly — that is, while the instrument is still unstable — you will get a falsely high reagent-blank value and the determination results will be biased low.

1.5 Sample determination

1.5.1 Weighing

Mix liquid samples thoroughly before sampling; drawing directly from the upper layer without prior mixing affects the result. Mix solid samples well, weigh them on weighing paper, and use tweezers to deliver the sample to the bottom of the digestion tube. Sample that improper handling leaves on the tube wall should be rinsed down to the bottom of the tube with the sulfuric acid. If it remains on the wall and is not rinsed down, the determination result will be biased low.

1.5.2 Digestion

When adding acid, take into account how the sample's composition affects acid consumption. With too little acid, the sample is digested incompletely and results run low. With too much acid, the excess acid remains after neutralization with alkali on the instrument, the free ammonia cannot be distilled over, and the determination result is seriously affected.

During digestion, pre-digest at 220 °C for 1 hour first, then raise the temperature to 420 °C. The purpose of pre-digestion is to let the sample and the sulfuric acid react slowly at first. Without pre-digestion, concentrated sulfuric acid reacts violently with the sample at high temperature and causes charring — the determination result runs low, and the charred particles can also block the tubing during waste discharge so that the instrument cannot work normally.

1.6 Determining the recovery rate

Recovery is determined with ammonium sulfate and with urea respectively. Using ammonium sulfate alone is simple and fast and requires no digestion — but it can only verify the instrument's measuring part; it cannot provide quality assurance for the digestion pre-treatment. Urea must be digested before determination, so it provides quality assurance for the entire process — pre-treatment and instrument measurement alike.