Characterization of triacontyl (C-30) liquid chromatographic columns
Introduction
It has been estimated that over 90 % of all small molecule chromatographic separations are performed with alkyl modified silica columns operated in the reversed-phase mode, and most of these separations utilize octadecyl (C-18) stationary phases [1]. It is widely recognized that the properties of C-18 columns vary significantly among columns from different manufacturers and even among different lots of a column from the same manufacturer [2], [3], [4]. Dissimilarities among commercial columns can be attributed to variations in the physical and chemical properties of the microparticulate silica sorbents and the synthetic approaches used in the preparation of these materials. Distinctions in column properties are often intentionally created to address specific separation needs and to broaden choices in column selection for unique applications (e.g., endcapped and nonendcapped stationary phases). As such, the availability of columns with dissimilar properties provide opportunities for the development and optimization of new chromatographic methods.
The synthesis and characterization of triacontyl (C-30) stationary phases for liquid chromatography was first described by Sander et al. [5] This early study addressed the need for improved separations of polar and nonpolar carotenoid isomers and was an outgrowth of research on shape selectivity for other classes of compounds with constrained molecular shapes [6,7]. Changes in column selectivity were observed for C-18 stationary phases prepared with polymeric surface modification chemistry (“polymeric phases”) compared with stationary phases prepared with monomeric surface modification chemistry (“monomeric phases”) [6,8]. Polymeric syntheses utilize trifunctional silanes in combination with water in solution to form silane oligomers that then react with the silica. Monomeric syntheses are usually carried out under anhydrous conditions with monofunctional silanes to result in surface modification without the formation of oligomeric silane percursors. Changes in selectivity were also noted for different length alkyl stationary phases [9]. The initial C-30 column developed by Sander et al. utilized a polymeric surface modification scheme of silica with trichlorotriacontylsilane, in a manner analogous to the preparation of polymeric C-18 columns [8].
Both polymeric C-18 and polymeric C-30 columns exhibit enhanced separations of constrained-shape solutes compared with corresponding columns prepared with monomeric synthesis schemes [10]. Polymeric C-18 columns have proven to be especially useful for the analysis of polycyclic aromatic hydrocarbon (PAH) isomers [11,12], whereas polymeric C-30 columns are well suited to the analysis of carotenoid isomers and certain vitamins [13,14]. The selectivity differences among monomeric and polymeric C-18 and C-30 columns have been attributed to changes in stationary phase order that result from differences in the surface modification chemistry employed, bonding density, alkyl phase length, and column temperature [9,[15], [16], [17], [18]].
The recent availability of C-30 columns from commercial sources has made possible an examination of variations in performance that are characteristic of this class of columns. In previous studies, SRM 869b was used to provide a metric of shape selectivity for monomeric and polymeric C-18 columns, particularly for application to separations of PAHs [19]. This test is now utilized for C-30 columns with correlation to separations of carotenoids to characterize shape selectivity. Representative columns were further studied to evaluate flow-related kinetic performance for correlation with stationary phase properties.
Section snippets
Reagents
All solvents were HPLC grade obtained from commercial sources. SRM 869b Column Selectivity Test Mixture for Liquid Chromatography was obtained from the National Institute of Standards and Technology (NIST) Office of Reference Materials. Carotenoid reference standards were obtained from commercial sources: lutein (CAS 127-40-2), β-cryptoxanthin (CAS 472-70-8), trans-α-carotene (CAS 7488-99-5), and trans-β-carotene (CAS 7235-40-7) were obtained from Sigma-Aldrich (St. Louis, MO); zeaxanthin (CAS
Results and discussion
To assess the scope of characteristics exhibited among commercial C-30 columns, representative examples were acquired and studied. For comparison, monomeric and polymeric C-18 columns were included to illustrate the influence of chain length and bonding chemistry. Differences in column selectivity towards carotenoid isomers were examined under various environmental and mobile phase conditions, and aspects of column kinetic performance related to flow were evaluated for the van Deemter model.
Conclusions
A broad spectrum of performance characteristics are exhibited by the C-30 columns included in this study. For these columns, the elution order and retention behavior of polar and nonpolar carotenoid isomers is well correlated with the shape separation factor (αTBN/BaP) as determined with SRM 869b. C-30 column selectivity spans the range of properties exhibited by monomeric-like and polymeric-like C-18 columns; however, unique separations are possible with C-30 columns that are not achieved with
Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgments
The authors thank David Duewer for assistance with numerical methods used to determine van Deemter coefficients and David Duewer and David Sheen for helpful discussions on principal component analysis. This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.
References (24)
- et al.
High molecular-shape-selective stationary phases for reversed-phase liquid chromatography: a review
TrAC-Trends Anal. Chem.
(2018) - et al.
C-30 Stationary phases for the analysis of food by liquid chromatography
J. Chromatogr. A
(2000) - et al.
Comparison of different analytical techniques for the analysis of carotenoids in tamarillo (Solanum betaceum Cay.)
Arch. Biochem. Biophys.
(2018) - et al.
Study on the elution order of carotenoids on endcapped C-18 and C-30 reverse silica stationary phases. A review of the database
J. Food Composit. Anal.
(2016) - et al.
Influence of synthetic routes on the conformational order and mobility of C-18 and C-30 stationary phases
J. Chromatogr. A
(2006) HPLC Columns, Theory, Technology, and Practice
(1997)- et al.
A Simple and Interactive Column Classification for Reversed-Phase Liquid Chromatography: The Carotenoid Test, Part I: Studied Properties, Probes, and Fundamentals
LC GC N. Am.
(2016) - et al.
A simple and interactive column classification for reversed-phase liquid chromatography: the carotenoid test, part II: additional studies and practical use of the classification map
LC GC North America
(2017) - et al.
Chromatographic classification of commercially available reverse-phase HPLC columns
Chromatographia
(1997) - et al.
Development of engineered stationary phases for the separation of carotenoid isomers
Anal. Chem
(1994)
Determination of column selectivity toward polycyclic aromatic hydrocarbons
J. High Resolut. Chromatogr. Chromatogr. Commun
Influence of stationary phase chemistry on shape recognition in liquid chromatography
Anal. Chem.
Cited by (11)
Integrated analytical approaches for the characterization of Spirulina and Chlorella microalgae
2022, Journal of Pharmaceutical and Biomedical AnalysisCitation Excerpt :The mobile phase (A) consisted of methanol-acetonitrile-water (79:14:7 v/v/v) as described before, whereas the mobile phase B consisted of acetone (100%), instead of chloroform (100%) [18,19]. The use of acetone as the new mobile phase B, whose compatibility with the column was assessed in a previous study [21] resulted in a stable efficiency during the chromatographic analyses, allowing a better separation of the selected analytes. Furthermore, it preserved the components of the HPLC, such as gaskets and O-rings, without the risk of instrumental damages due to the use of chloroform.
Method development for PPB culture screening, pigment analysis with UPLC-UV-HRMS vs. spectrophotometric methods, and spectral decomposition-based analysis
2022, TalantaCitation Excerpt :Yet, in nature carotenoids exist in different isomer forms (the main difference with synthetic products), which results in different bioavailability and effects when ingested with the diet [16,17]. A non-endcapped reverse phase (RP)-HPLC column with triacontyl (C30) can be used to maximise chromatographic resolution and selectivity of carotenoids and their isomers [18,19]. High-performance and ultra-performance liquid chromatography coupled with mass spectrometry (HPLC-MS, UPLC-MS) and using suitable analytical standards are the most effective tools for the detection and quantification of PPB carotenoids [20,21].
Comprehensive profiling of conjugated fatty acid isomers and their lipid oxidation products by two-dimensional chiral RP×RP liquid chromatography hyphenated to UV- and SWATH-MS-detection
2022, Analytica Chimica ActaCitation Excerpt :Only the unconjugated C18 PUFAs were sufficiently separated, based on the number of double bonds yet without resolution of α- and γ-linolenic acid (see Fig. 1d, red trace). It is well known from the literature that C30-phases exhibit enhanced shape selectivities which has led to unique selectivity for carotenoid isomers and other E/Z isomer mixtures [34,35]. In fact, the use of a shape-selective C30-RP-stationary phase led to a general improvement of the separations, especially for the conjugated dienes (three out of four isomers were separated; Fig. 1c, blue trace).
Reversed-phase chromatographic separation and downstream precipitation of lupane- and oleanane-type triterpenoids: Experiments and modeling based on the method of moments
2021, Separation and Purification TechnologyCitation Excerpt :Moreover, they are known to provide higher sample loadings and more reproducible retention behavior than C18 phases when operated in highly aqueous solvent environments [44,46]. Recently, a comparison between multiple monomeric and polymeric C30 and C18 stationary phases was accomplished by Sander et al. [47] and better separations of carotenoid isomers were obtained with C30 columns than with C18 columns. It is known that C30 stationary phases, particularly the Acclaim C30 ones, provide good selectivities for triterpenic acids fractionation [48], but for the specific betulinic and oleanolic acids separation very limited work is available related to mobile phase selection.
Enhanced separation of bioactive triterpenic acids with a triacontylsilyl silica gel adsorbent: From impulse and breakthrough experiments to the design of a simulated moving bed unit
2020, Separation and Purification TechnologyCitation Excerpt :The retention behavior of stationary phases is temperature-dependent, since at lower temperatures adsorption is higher and the concentration wave velocity decreases (this is very clear for linear isotherms). With relation to selectivity, very distinct trends may result: (i) Sander et al. [77] found that selectivity of monomeric-like C30 adsorbent towards tetrabenzonaphthalene and benzo[α]pyrene increases with increasing temperature from 5 to 50 °C; (ii) the same authors reported modest changes in selectivity over 10 – 40 °C in the case of carotenoid isomers using polymeric-like C30 column; (iii) Sánchez-Ávila et al. [78] reported better separation results at low temperature (optimum selectivity at 5 °C from the studied range of 5 – 35 °C) for triterpenic acids and dialcohols over a C18 stationary phase. Regarding our experimental selectivities plotted in Fig. 2, two distinct effects appear, namely, the addition of water to methanol improves the separation while the addition of acetonitrile has the opposite effect.