Ion-suppression techniques

Similarly to the methods used to characterize natural chlorophylls, RP-HPLC has been chosen by several authors to identify the individual components in Cn chlorophyllin preparations and in foods. The same ODS columns, mobile phase and ion pairing or ion suppressing techniques coupled to online photodiode UV-Vis and/or fluorescence detectors have been used. ° ... 

Ion suppression is not often applied to strong acids or strong bases because of the extremes in pH that would be required for retention. Suppression by the addition of buffers is restricted to the range pH 3-8, because silica-based stationary phases are unstable in solutions of either high or low pH (i.e., above pH 8 or below pH 2). These restrictions do not apply to polymeric supports, and polymer-based stationary phases can be used in the separation of a wider range of solutes using the ion-suppression technique. 

FIGURE 5-19. Separation of phenolic acids using the ion suppression technique. Mobile phase 5% acetic acid in water. (Chromatogram reprinted from L. W. Wulf and C. W. Nagel, J. Chromatography, 116, 271 (1976) with permission.)... 

A number of HPLC methods are available for the analysis of LSD. Two are presented here, namely (i) an ion-suppression technique, and (ii) an ion-paired method. The majority of techniques that are used exploit the same physicochemical characteristics of the systems as those described here. 

Stockton and Irgolic separated As(III), As(V), arsenobetaine and arsenocholine by high performance liquid chromatography using a reversed-phase ion suppression technique. This method seemed applicable to the separation of these arsenicals with better resolution than the conventional ion-exchange chromatography using resins. 

Fig. 5-54. Separation of some long-chain fatty acids by means of ion suppression technique. - Separator column IonPac NS1 (10 pm) eluent (A) 3 10 5 mol/L HC1 / acetonitrile / methanol (70 24 6 v/v/v), (B) 3 10-5 mol/L HC1 / acetonitrile / methanol (16 60 24 v/v/ v) gradient linear, 100% A in 15 min to 100% B flow rate 1 mL/min detection suppressed conductivity injection volume 50 pL solute concentrations 100 ppm butyric acid (1), 100 ppm caproic acid (2), 200 ppm ca-prylic acid (3), 200 ppm capric acid (4), 300 ppm lauric acid (5), 300 ppm myristic acid (6), and 400 ppm palmitic acid (7).

The ion suppression technique can be used to great effect for the analysis of weak acids or bases. For the analysis of acidic compounds the technique consists of the addition of a small amount of acetic or phosphoric acid to the mobile phase. By reducing the eluant pH dissociation of the sample molecules is suppressed. They thus have decreased affinity for the eluant and are retained to a greater extent by the ODS phase. The range of BPC is considerably extended using techniques such as ionic suppression and this mode of LC using ODS bonded phases finds wide application. 

The utility of ion suppression techniques in the analysis of ionisable molecules by reverse phase chromatography is limited to samples of weakly basic or acidic compounds. The analysis of stronger acids (pATj < 3) or stronger bases (pA j, > 8) would require eluant of pH, <2 or >8, respectively. Reverse phase chromatography with chemically bonded stationary phases is, however, restricted to eluant pH >2 and <8 for reasons previously discussed. 

Thus the resolution achieved in a chromatogram is largely dependent upon values of a and k, both of which are strongly dependent on the nature of the eluant in terms of solvent strength, organic solvent, mobile phase pH (ion suppression techniques) and eluant additives (IPRs). 

Figure 657 Separation of long-chain fatty acids utilizing an ion-suppression technique. Separator column lonPac NS1,10pm eluent (A) 3.10" mol/L HCI/MeCN/MeOH (70 24 6 v/v/v) and (B) 3 10"= mol/L HCI/MeCN/MeOH (16 60 24 v/v/v) gradient linear, 100% A in 15 min to 100% B flow rate 1 mL/min ...

As can be seen from Figure 6.63, very small and symmetric peaks result when a salt gradient is applied. Separations of this kind are not possible with either the ion-suppression technique or with ion-pair chromatography. This is especially true for aromatic polycarboxylic acids, which elute from a mixed-mode phase such as OmniPac PAX-500 according to their valency (Figure 6.64b). When separating on chemically bonded silica under ion-suppression conditions (Figure 6.64a), a shorter analysis time is observed but not all components of the test mixture are separated. Moreover, penta- and... 

The separation of anti-inflammatory drugs (see Table 6-4) on OmniPac PAX-500 shown in Fig. 6-67 is also based on the combination of anion exchange- and reversed-phase interactions. It represents an interesting alternative to the ion-suppression technique, because a number of these compounds bear carboxyl groups and are strongly hydrophobic due to their aromatic character. The structural versatility of compounds, which can be separated within 20 minutes with a combined sodium carbonate/acetonitrile gradient, is characteristic for such a separation. In principle, this separation could also be carried out with a chloride salt in the mobile phase, but the concentration required for elution would be higher than that required for carbonate. 

In the area of vitamins. Fig. 9-193 shows the gradient elution of water-soluble vitamins using Spherisorb ODS 2, at which the most important compounds of this kind can be analyzed in less than 15 minutes. Under these conditions, ascorbic acid elutes close to the system void, which hampers its determination in real-world samples. Among the numerous HPLC methods published in hterature for determining ascorbic add, those that operate under addic conditions are preferable because ascorbic add oxidizes under alkaline conditions. Such methods include ion-ejodusion chromatography [299] and ion-suppression techniques [300]. An optimal separation of ascorbic acid and isoascorbic acid (which interferes with the determination of ascorbic add in plasma) is obtained on macro-porous polymeric stationary phases with a purely aqueous sodium ddiydro-genphosphate eluant of high concentration [301]. Maximal selectivity between the two compounds is obtained at pH 2.14. To avoid ascorbic acid oxidation. 

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