High-performance liquid chromatographic determination of major organic acids in apple juices and

:D 1987 Fri~dr. VJu'weg& Sohn V,:rldgsgesellscha~:mbH
rapid spectral detector and an IBM data station were used for HPLC analysis. The HPLC columns were 250 x 4 m m i.d. LiChrosorb RP18 (C18) and RP-8 (C8) 5-/zm particle size. Processed apple juice and cider samples were purchased from local markets. Before injection all samples were filtered through 0.45/1m Millipore filters, and the carbonated ciders were first degassed for 5min in an ultrasonic bath. When acid concentrations were too high, the samples were diluted to 2 - 5 ~ Brix.
Introduction
Among the substances under analysis in apple, apple juice and ciders, organic acids are becoming increasingly important because of their role in maintaining quality and nutritive value in addition to other roles still unknown. These acids directly and indirectly affect the quality of apples and their industrial drinking derivatives. Directly, because they influence the flavour and stability of juices (and may be added to prevent settling of solids or darkening of the juices, or to modify the flavour) and ciders. For example, it is well known that natural malolactic fermentation brings about several consequences: firstly, the acidity of the cider diminishes and secondly, it leads to the replacement of malic acid by lactic acid which has a less marked taste. As a result, a net gustative improvement of ciders is achieved, as the cider becomes lighter. On the other hand, some of the acids, especially malic, are indirectly important because they can be used as indicators of apple maturity. Moreover the determination of the organic acids makes it possible to prove the adulteration of apple beverages, e.g. high citric Chromatograoh~a Vol. 24, 1987 0009 5893/87 0347 04 ~ 03 00/0
Experimental
All reagents were of analytical-reagent grade and were used without further purification. Malic, succinic, citric, lactic and ascorbic acids were purchased from Merck (Darmstadt, FRG) and Sigma (St. Louis, MO, USA}. Buffer solutions consisted of 10-2M potassium dihydrogen ,phosphate (KH2PO4), adjusted to different pHs with phosphoric acid (H3PO4). The solvents used for the mobile phase were of HPLC grade (Merck) and were employed as supplied. A LKB (Bromma, Sweden) liquid chromatograph equipped with a Model 2150 pump, a Rheodyne Model 7125 injection valve equipped with a 20-#1 loop, a LKB Model 2140 347
Summary
A reversed-phase high-pressure liquid chromatographic method is presented for the simultaneous separation and determination of malic, citric, lactic, succinic and ascorbic acids in apple juices and ciders. After filtration and/or degasification, the organic acids in the sample are separated on a LiChrosorb/C18 column and quantified by using a rapid diode array detector. The method is considered to be a suitable choice for the accurate and precise determination (C.V. 5%) of these compoesent, an o p t i m u m pH = 2.25 was selected for further studies. Lactic and ascorbic acids were coeluted at all pHs tested. This did not present any problem in the analysis of real samples because firstly, lactic acid is a fermentation product and should not be present in apple juices, and secondly, ascorbic acid is not usually a common additive in natural ciders (where lactic acid should be controlled). In any case, if the acids were all present in the same sample, the diode array detector w o u l d minimize the problem because it would allow the acquisition of spectral information within a defined wavelength range during elution, generating a data matrix (Abs,),, t: spectrochromatogram or ;k, t: isogram) for subsequent processing. As can be seen in the t w o dimensional contour plots (Fig. 2), the overlapping of ascorbic and lactic acids was at a minimum at 206nm. Ascorbic acid can be determined free of interferences at 270nm, while the other acids, in the absence of ascorbic acid, could be determined with more sensitivity (the shorter the wavelength used the more sensitive the corresponding determination, as shown by Fig. 3 at 206nm). The effect of the ionic strength of the eluent on the retention time of carboxilic acids seems to be slight, but it can be observed that when the ionic strength decreases, the w i d t h of the peaks increases and the chromatograms lose resolution. Investigations on the effects of increasing temperature and mobile phase f l o w rate show a decrease in analysis time for both, but a parallel decrease in resolution. This fact is in contradiction w i t h the results reported by other authors, who recommended an o p t i m u m temperature of 45~ [9].