Determination of ammonia in beers by pervaporation flow injection analysis and spectrophotometric detection
Introduction
Beer is a complex mixture of compounds and its composition is determined by the materials and technology used in its production. The concentration of nitrogenous compounds is relatively high and this group includes protein, peptides, amino acids, volatile and non-volatile amines, and ammonia. Protein predominates with levels up to 6 g l−1 [1], [2], [3] whereas assimilable amino acids derived from protein may be as high as 0.6 g l−1 [3]. In comparison, the concentrations of biogenic amines [1], [4], small volatile amines [5], [6], [7] and ammonia [8], [9] tend to be in the low mg l−1 range.
Ammonium salts together with amino acids are important nitrogenous sources in yeast fermentation processes in beer brewing [10], [11]. Ammonium salts provide amino groups in transamination reactions and amino acids deficiency during the fermentation process results in the formation of by-products which may change the taste of beer [11]. For these reasons, analysis of ammonia in beer is important.
The determination of ammonia in beer is difficult because of the presence of other nitrogen containing compounds. The ammonia ion-selective electrode has been recommended previously [8], [9] using standard addition instead of the calibration method and it was claimed that there was some interference from ethanol and small volatile amines. In our laboratories (see later), it has been difficult to obtain reliable measurements for some beers using an ammonia probe.
High concentrations of protein, peptides and amino acids have been reported to interfere in the determination of ammonia using spectrophotometric procedures based on the Berthelot reaction (phenol/hypochlorite) or the modified Berthelot reaction [12], [13], [14]. Searle found that protein gives a negative interference arising from a decrease in available hypochlorite [12]. Individual amino acids interfered strongly at 20 μM in the modified Berthelot method but not the phenol-based Berthelot method, although peptone (from casein) interfered in both methods [14]. Therefore, batch spectrophotometric procedures or direct flow injection (FIA) methods using either of the Berthelot reactions are unsuitable for the determination of ammonia in beer because of interferences from the likely high concentrations of protein and amino acids.
Yokoi et al. have demonstrated that beers contain proteins of different hydrophobicities and that hydrophobic proteins play an important role in foam formation in beers [15]. Therefore, gas diffusion flow injection analysis would be unsuitable for the determination of ammonia in beers because the high level of hydrophobic protein would lead to blockage of the pores in the hydrophobic membrane used in this technique.
Pervaporation flow injection (PFI) systems are frequently used for the determination of volatile analytes in complex samples (e.g. emulsions, suspensions, corrosive compounds, etc.) [16], [17], [18]. A major advantage of PFI over other separation methods is that it requires minimal sample preparation. In addition, deterioration in the permeability of the pervaporation membranes can be avoided [16], [17], [18], since the donor stream (which carries the injected sample) does not contact the membrane.
The work in this paper focuses on developing a PFI method utilising the modified Berthelot reaction for determination of ammonia in beers which requires minimal sample preparation and is able to suppress interference from protein, amino acids and small volatile amines. This method is compared with the existing PFI mixed indicator method [19] and the ammonia probe method [8], [9].
Section snippets
Solutions preparation
Ammonium chloride (BDH, Australia) and diethylamine hydrochloride (Fluka, USA) were used to prepare standard solutions. All solutions were prepared using NANOpure water (Barnstead, Dubuque, IA, 17 MΩ cm) and were degassed using a sonicator for 15 min before use.
The composition of donor and acceptor streams was selected based on previous published reports [20], [21], [22] on the modified Berthelot method. These reagents were NaOH (BDH), sodium dichloroisocyanurate (Ajax, Australia), sodium
Ion-selective electrode measurements
Previous investigators recommended the use of the ammonia probe to determine ammonia in beers [8], [9]. In both cases, samples were diluted about 4-fold to overcome interference from alcohol and it was claimed that more reliable results were achieved in about 10 min using single-point standard addition rather than a calibration procedure. For fermentation samples, it was recommended that samples be centrifuged for 20 min before analysis [9].
In the present study, preliminary measurements
Conclusions
On the basis of the results obtained, it is concluded that the combination of PFI with detection using the modified Berthelot reaction is a more reliable method for the determination of ammonia in samples containing proteins, amino acids and volatile small amines. A sample throughput rate of 11 h−1 was achieved for 4.0 mg l−1 ammonia samples. This method offers a wide linear detection range from 0.1 to 40.0 mg l−1 and a detection limit of 0.05 mg l−1. It was successfully applied for the
Acknowledgements
We are grateful to the Australian Research Council for financial support.
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