doi:10.1016/j.chroma.2006.01.090 
Copyright © 2006 Elsevier B.V. All rights reserved.
The effect of chaotropic mobile phase additives on the separation of selected alkaloids in reversed-phase high-performance liquid chromatography
J. Fliegera, 
aDepartment of Inorganic and Analytical Chemistry, Medical University of Lublin, 20-081 Lublin, Staszica 6, Poland
Received 15 November 2005;
revised 16 January 2006;
accepted 19 January 2006.
Available online 20 March 2006.
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Abstract
The retention behavior of selected alkaloids from different classes was studied. The effect of chaotropic salts additives to the mobile phase on chromatographic parameters of protonated basic analytes was investigated on Zorbax Extend-C18 column. The influence of the type of salts and their concentration on retention, efficiency, peak symmetry and separation selectivity of investigated alkaloids was established. Buffered acetonitrile–water mobile phase was chosen because of significant retention of added liophilic ions due to strong dispersive π–π interactions. These conditions are responsible for great contribution of electrostatic forces in the retention of protonated bases. The addition of salt, such as hexafluorophosphate, perchlorate, trifluoroacetate leads to the increase in retention, efficiency and separation selectivity of examined analytes. The influence of added salts on increase in retention parameters could be expressed as follows: H2PO4− < CF3COO− < ClO4− < PF6−. This order is in agreement with ability of salts to “salting-in” effect according to Hofmeister series. Obtained chromatograms of alkaloids mixture illustrate suitability of chaotropic effect to improve their separation selectivity.
Keywords: Reversed-phase HPLC; Alkaloids; Salt effect; Chaotropic counteranions
Fig. 1. Experimental retention factor for investigated alkaloids vs. perchlorate concentration in mobile phase: 30% ACN/10 mM phosphate buffer (pH 2.7).
Fig. 2. Experimental retention factor for investigated alkaloids vs. trifluoroacetate concentration in mobile phase: 30% ACN/10 mM phosphate buffer (pH 2.7).
Fig. 3. Experimental retention factor for investigated alkaloids vs. hexafluorophosphate concentration in mobile phase: 30% ACN/10 mM phosphate buffer (pH 2.7).
Fig. 4. Effect of anionic additive type on the retention of investigated alkaloids. (*) For emetine and berberine the strongest retention was observed when hexafluorophosphate salt was added to the mobile phase. Their retention factors were higher than 25.
Fig. 5. Chromatograms of a mixtures of alkaloids (A—caffenine, B—laudanozine, C—colchicine, D—boldine, E—strychnine, F—cinchonine, G—quinine) with different organic anions in the mobile phase.
Fig. 6. The effect of different anionic additives on retention, peak symmetry and efficiency of narcotine.
Table 1.
k-Retention parameter, As-tailing factor and N-theoretical plate number values for investigated alkaloids obtained in eluent systems studied
(–) Criterion for asymmetry or theoretical plates not satisfied. (*) Very strong retention. All calculations were performed using HSM program. The following equation is used to calculate the number of theoretical plates according to USP standards: N = 16(RT/W)2, where RT = the actual full retention time of the appropriate peak, W = the peak width obtained by drawing tangents to each side of the peak and calculating the distance between the two points where the tangents meet the baseline. The HSM program uses the following equation to calculate asymmetry: As = 1/2(1 + B/A), where A and B are evaluated at a 5% peak height of an appropriate peak. The capacity factor (k) is calculated as follows: k = tR/t0 − 1, where tR = the actual retention time of the individual peak, t0 = the elution time of the unretained sample (thiourea).
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