21 Apple Juice Analysis
The chromatogram in Figure 2 results from the injection of 20 µL of apple juice which was diluted with ultra-pure water. Depending on the sample, it is advisable to filtrate the sample in addition, to avoid introducing particles to the column and the detector. The alkaline eluent (100 mmol/L sodium hydroxide / 10 mmol/L sodium acetate) ensures that the sugars are present in dissociated form (as anions) and can therefore be separated in the column by the ion exchanger.
Figure 2: Determination of glucose, fructose, and sucrose in apple juice. Except for simple dilution, no sample preparation is required. Column: Metrosep Carb 2 - 150/4.0; eluent 100 mmol/L sodium hydroxide / 10 mmol/L sodium acetate; column temperature: 30°C; sample volume: 20 µL; flow rate: 0.5 mL/min; amperometric detector: PAD mode; working electrode (WE): Au, reference electrode (RE): Pd, working potential: 50 mV; dilution 1:1000.
Because carbohydrates are electrochemically active, they can be detected amperometrically. During amperometric detection, the analytes are oxidised to a working electrode by applying a potential to the latter. This results in an electrical current that reveals the concentration. Over time, however, carbohydrates form deposits on the working electrode when a continuous potential is applied. The amperometric detector is therefore operated in PAD (pulsed amperometric detection) mode. Here, a three-stage cyclic potential ensures that after measuring the current, i.e., after the determination stage, the electrode is cleaned from the adsorbed molecules and eventually conditioned [4].
Residual Lactose in ‘Lactose-Free’ Products
Figure 3: Determination of lactose traces in milk declared lactose-free, diluted 1:100 and spiked with 100 mg/L lactose, after Inline Dialysis. Column: Metrosep Carb 2 - 150/4.0; eluent: 5 mmol/L sodium hydroxide / 2 mmol/L sodium acetate; column temperature 40°C; sample volume 20 µL; flow 0.8 mL/min, amperometric detector: PAD mode; WE: Au; RE: Pd; working potential: 50 mV.
Figure 4: Lactose is composed of the monosaccharides galactose and glucose. The hydrolysis of lactose illustrated here is catalysed by the enzyme lactase.
A key part of the quality control of products declared lactose-free is the determination of residual lactose. The ion chromatogram in Figure 3 illustrates the determination of lactose in ‘lactose-free’ milk to which 100 mg/L lactose was added. Again, the separation takes place under strongly alkaline conditions (eluent of 5 mmol sodium hydroxide / 2 mmol sodium acetate) and the analyte is detected by pulsed amperometry. The high concentrations of galactose and glucose illustrated in the chromatogram are a result of the enzymatic breakdown of lactose into these very monosaccharide constituents (Figure 4). Because of its fat- and protein-rich matrix, milk must undergo dialysis before being analysed. With the Metrohm Inline Sample Preparation, which includes an Inline Dialysis option, this is a fully automated process, and therefore does not involve any additional effort.
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