Separation and Identification of Chiral N-acylcalix[4]arene Amino Acid Derivatives by Use of Reversed-Phase HPLC S. J. Thibodeaux / M. Safichez Pefia / Y. Zhang / S. A. Shamsi / I. M. Warner* Department of Chemistry, Louisiana State University, Baton Rouge, LA 70803, USA Key Words Column liquid chromatography p-tert butyl calix[4]arene derivatives Summary This work focuses on the separation and identification of p-tert butyl calix[4]arene derivatives. An isocratic mixture of 65 % (v/v) acetonitrile and 35 % (v/v) water with 0.1% (v/v) phosphoric acid was used as the mobile phase. A Bio-Rad Bio-Sil ODS-5S (250 mmx 4 mm) column was used as the stationary phase with UV detec- tion of the analytes at 274 nm. The reversed-phase high performance liquid chromatographic method which was developed provided baseline separation of five p-tert- butyl calix[4]arene derivatives in twenty minutes. Introduction Cali• (CX) chemistry is a rapidly developing area of supramolecular chemistry. This is due in part to the simple synthesis of CXs which can be achieved from the condensation of p-tert-butyl phenol and formaldehyde [1, 2]. Many researchers have found that the complexa- tion and solubility properties of CXs can be modified through reactions to the lower and upper rims [1-4]. These modifications have resulted in widespread appli- cations of CX such as ionophores for ion-selective mem- branes and electrodes [5], and for stationary phases in chromatography [6, 7]. One of the more recent areas of CX research involves the development of chiral cali• arenes [8-11]. Shinkai et al. synthesized chiral CXs by attaching chiral aliphatic chains [8] and amino acid moieties [10] to the lower and upper rim, respectively. In addition, Bayard has modified the upper rim of CX by the introduction of two chiral units of 2-(methoxyme- tyl) pyrrolidine [9]. Our work has centered on develop- ing new chiral calixarene compounds in which the amino acids are attached to the lower rim. These modifi- cations have resulted in a water-soluble derivative of CX that was used as a pseudo- stationary phase for elec- trokinetic capillary chromatography (EKC) [12, 13]. It is well established that reversed-phase-HPLC (RP-HPLC) is an excellent technique for the separation of non-volatile compounds with high molecular weight. Both gradient [14] and isocratic [15, 16] RP-HPLC methods have been reported for the separation of a va- riety of CX derivatives. However, the mobile phase con- ditions for RP-HPLC was modified for the work pre- sented here. This manuscript describes the development of a RP-HPLC method for the baseline separation of six p-tert-butyl-calix[4]arene derivatives (Figure 1). Experimental Reagents HPLC grade acetonitrile (ACN) and ACS grade phos- phoric acid were purchased from EM Science (Gibb- stown, N J) and used as received. All mobile phases were prepared with triply distilled water (18.6 Mf~). The filter system was purchased from U.S. Filter (Lowell, MA). The p-tert-butyl calix[4]arene derivatives were synthe- sized using the procedure reported by Safichez Pefia, et al. [12]. HPLC Procedures The chromatographic system used in this study con- sisted of a model AS-100T automatic sampler, model 2700 solvent delivery system, Bio-Sil ODS-5S column (250 mm by 4 mm), and a Bio- Dimension UV/VIS de- tector; all were purchased from the Bio-Rad (Hercules, CA). An analytical guard column with C18 packing was purchased from Alltech (Deerfield, IL) to protect the Bio-Sil ODS column from hydrolysis. The mobile phase was prepared by using appropriate % (v/v) of ACN, wa- ter, and phosphoric acid. The mobile phase was soni- cared for five minutes, and then filtered using 0.45 [am membrane filters purchased from Gelman Science (Ann Arbor, MI). The analytical column was equili- brated with the new mobile phase for at least 30 rain. The analytes were prepared in 100 % ACN at 50 ppm. Spiking was done with 100 ppm standards. Twenty mi- 142 Chromatographia Vol. 49, No. 3/4, February 1999 0009-5893/99/02 142-05 $ 03.00/0 9 1999 Friedr. Vieweg & Sohn Verlagsgesellschaft mbH Original