Liquid Chromatography Retention Research Articles

Retention of nucleic acids in ion-pair reversed-phase high-performance liquid chromatography depends not only on base composition but also on base sequence.

The study on nucleic acid retention in ion-pair reversed-phase high-performance liquid chromatography mainly focuses on size-dependence, however, other factors influencing retention behaviors have not been comprehensively clarified up to date. In this present work, the retention behaviors of oligonucleotides and double-stranded DNAs were investigated on silica-based C18 stationary phase by ion-pair reversed-phase high-performance liquid chromatography. It is found that the retention of oligonucleotides was influenced by base composition and base sequence as well as size, and oligonucleotides prone to self-dimerization have weaker retention than those not prone to self-dimerization but with the same base composition. However, homo-oligonucleotides are suitable for the size-dependent separation as a special case of oligonucleotides. For double-stranded DNAs, the retention is also influenced by base composition and base sequence, as well as size. This may be attributed to the interaction of exposed bases in major or minor grooves with the hydrophobic alky chains of stationary phase. In addition, no specific influence of guanine and cytosine content was confirmed on retention of double-stranded DNAs. Notably, the space effect resulted from the stereostructure of nucleic acids also influences the retention behavior in ion-pair reversed-phase high-performance liquid chromatography.

Study of the slope of the linear relationship between retention and mobile phase composition (Snyder-Soczewiñski model) in normal phase liquid chromatography with bonded and charge-transfer phases

The Snyder model and the Soczewiñski model are compared on classic NPLC bonded phases using literature data, and on the charge transfer 2, 4-dinitroanilinopropyl (DNAP) column using experimentally collected data. Overall, the Snyder model slightly better predicts the n-slope than the Soczewiñski model. However, both models give comparable uncertainty in predicting n-slope for a given compound. The number of aromatic double bonds was the most suitable descriptor for estimating the relative n-slope of PAHs, as it correlated with behavior better than the number of aromatic rings and is simpler to calculate than the solute adsorption area. On the DNAP phase, a modified Soczewiñski model is suggested to allow for the significant contribution of the aromatic rings to the n-slope. For classic NPLC bonded phases and DNAP columns, the contribution of polar group to the n-slope parallels the adsorption energy of each polar group.

Separation and Purification of Ombuoside from Gynostemma Pentaphyllum by Microwave-Assisted Extraction Coupled with High-Speed Counter-current Chromatography.

A rapid and efficient method for the separation and purification of ombuoside from Gynostemma pentaphyllum by microwave-assisted extraction coupled with high-speed counter-current chromatography (HSCCC) was successfully developed. Using an orthogonal array design L9 (34), the extraction conditions, including microwave power, irradiation time, solid-to-liquid ratio and extraction times, were optimized. Ombuoside was isolated and purified from the crude extraction byHSCCC with two-phase solvent system composed of n-hexane:ethyl acetate:ethanol:water (5:6:5:5, v/v) in a single run. A 210 mg quantity of the crude extract containing 2.16% ombuoside was loaded, yielding 3.9 mg of ombuoside at 96.7% purity. The chemical structure of ombuoside was determined by comparison with the high-performance liquid chromatography retention time of standard substance as well as UV, FT-IR, ESI-MS, 1H NMR and 13C NMR spectra. The purified ombuoside had strong 1,1-diphenyl-2-picrylhydrazyl and hydroxyl free radical scavenging activities.

Identification and Characterization of Metabolites of Irinotecan in-vivo and in-vitro matrixes by HPLC/LC-MS

In the present study metabolite identification and characterization has done by using HPLC and LC-MS. During method development various mobile phases have tried for identification of metabolites. The matrixes selected for in- vivo study were urine because nearly all the metabolites of irinotecan were obtained in it. The extraction mixtures have selected to retain maximum amount of analyte with less effort. During experiment four extraction solvents were used in six different concentrations out of which TBME suit our method. In-vitro study done by Human Liver microsomes by using Phosphate buffer (pH 7.4) and NADPH as co-factors for initiation of enzymatic reaction. Irinotecan is a prodrug that is converted in the liver to an active metabolite, SN-38. It is eliminate in Bile and Faeces and thus its dose reduced in Hepatic Failure. Irinotecan act by inhibiting Topoisomerase-1.It is the enzyme which nicks, introduces negative supercoils and reseals the DNA strand. Conventionally, drug metabolite identification in the past has usually been based on the comparison of ultraviolet (UV) spectral data and high-performance liquid chromatography (HPLC) retention times of isolated ‘unknown’ metabolites with those of synthesised standards. Such a method of detecting and characterising drug metabolites is an uncertain, time-consuming and expensive process, as well as affording very limited structural information. Furthermore, Phase I metabolism of a drug candidate often results in only minor structural modification of the parent compound; these minor changes can make it particularly difficult to determine suitable chromatographic conditions to effect HPLC separation of metabolites. This study describes contemporary approach to identification and characterization of xenobiotic metabolites in complex biological fluids derived from drug metabolism studies.

Optimal Hydrophobicity in Ring-Opening Metathesis Polymerization-Based Protein Mimics Required for siRNA Internalization.

Exploring the role of polymer structure for the internalization of biologically relevant cargo, specifically siRNA, is of critical importance to the development of improved delivery reagents. Herein, we report guanidinium-rich protein transduction domain mimics (PTDMs) based on a ring-opening metathesis polymerization scaffold containing tunable hydrophobic moieties that promote siRNA internalization. Structure-activity relationships using Jurkat T cells and HeLa cells were explored to determine how the length of the hydrophobic block and the hydrophobic side chain compositions of these PTDMs impacted siRNA internalization. To explore the hydrophobic block length, two different series of diblock copolymers were synthesized: one series with symmetric block lengths and one with asymmetric block lengths. At similar cationic block lengths, asymmetric and symmetric PTDMs promoted siRNA internalization in the same percentages of the cell population regardless of the hydrophobic block length; however, with 20 repeat units of cationic charge, the asymmetric block length had greater siRNA internalization, highlighting the nontrivial relationships between hydrophobicity and overall cationic charge. To further probe how the hydrophobic side chains impacted siRNA internalization, an additional series of asymmetric PTDMs was synthesized that featured a fixed hydrophobic block length of five repeat units that contained either dimethyl (dMe), methyl phenyl (MePh), or diphenyl (dPh) side chains and varied cationic block lengths. This series was further expanded to incorporate hydrophobic blocks consisting of diethyl (dEt), diisobutyl (diBu), and dicyclohexyl (dCy) based repeat units to better define the hydrophobic window for which our PTDMs had optimal activity. High-performance liquid chromatography retention times quantified the relative hydrophobicities of the noncationic building blocks. PTDMs containing the MePh, diBu, and dPh hydrophobic blocks were shown to have superior siRNA internalization capabilities compared to their more and less hydrophobic counterparts, demonstrating a critical window of relative hydrophobicity for optimal internalization. This better understanding of how hydrophobicity impacts PTDM-induced internalization efficiencies will help guide the development of future delivery reagents.

Application of solvation model to prediction of the solute retention in liquid chromatography over a wide range of mobile-phase compositions

Possibilities have been studied of using a solvation model to predict the retention behavior of solutes in liquid chromatography mobile phases of water-acetonitrile and water-methanol with the organic solvent content varying from 1 to 100 vol%. Twenty-one test solutes, both aliphatic and aromatic compounds, have been selected on the basis of two-level factorial designs. Using the multiple linear regression analysis, regression coefficients, which are characteristics of the stationary and mobile-phase system, were calculated for different mobile phases in the solvation model. Regression coefficients have been used for the prediction of the retention behavior. Unbiased results have been obtained by using two sets, one training and the other the testing set. The predicted retention has been compared with the experimental data. The methanol-water system provided good results at low and medium methanol concentrations; the retention prediction was unsatisfactory for mobile phases containing more than 90% of methanol. The acetonitrile-water system yielded similar results, but the retention prediction ceased to be at acetonitrile concentrations greater than 80%. The retention has primarily been determined by cohesive and acid-base interactions. The dependences of the regression coefficients on the mobile-phase composition were similar for the acetonitrile-water and methanol-water systems.

Influence of carboxylic ion-pairing reagents on retention of peptides in thin-layer chromatography systems with C18 silica-based adsorbents

One of the main problems related to chromatography of peptides concerns adverse interactions of their strong basic groups with free silanol groups of the silica based stationary phase. Influence of type and concentration of ion-pairing regents on peptide retention in reversed-phase high-performance liquid chromatography (RP-HPLC) systems has been discussed before. Here we present influence of these mobile phase additives on retention of some peptide standards in high-performance thin-layer chromatography (HPTLC) systems with C18 silica-based adsorbents. We prove, that due to different characteristic of adsorbents used in both techniques (RP HPLC and HPTLC), influence of ion-pairing reagents on retention of basic and/or amphoteric compounds also may be quite different. C18 silica-based HPTLC adsorbents provide more complex mechanism of retention and should be rather considered as mixed-mode adsorbents.

Evaluation of the dual retention properties of stationary phases based on silica hydride: Perfluorinated bonded material.

The synthesis of a new perfluorinated stationary phase based on silica hydride using a hydrosilation reaction was investigated. The material was characterized by elemental analysis, diffuse reflectance infrared Fourier transform spectroscopy and (13) C cross-polarization magic-angle spinning NMR spectroscopy. The retention properties of this new material were tested in the reversed-phase and normal-phase mode. Variable buffer strength experiments at two pH conditions for selected polar compounds were used to compare the new phase to hydrophilic interaction liquid chromatography retention. These results and previous data reported in the literature were used to postulate differences in the retention mechanism between hydrophilic interaction liquid chromatography and silica hydride-based stationary phases.

RePLiCal: A QconCAT Protein for Retention Time Standardization in Proteomics Studies.

This study introduces a new reversed-phase liquid chromatography retention time (RT) standard, RePLiCal (Reversed-phase liquid chromatography calibrant), produced using QconCAT technology. The synthetic protein contains 27 lysine-terminating calibrant peptides, meaning that the same complement of standards can be generated using either Lys-C or trypsin-based digestion protocols. RePLiCal was designed such that each constituent peptide is unique with respect to all eukaryotic proteomes, thereby enabling integration into a wide range of proteomic analyses. RePLiCal has been benchmarked against three commercially available peptide RT standard kits and outperforms all in terms of LC gradient coverage. RePLiCal also provides a higher number of calibrant points for chromatographic retention time standardization and normalization. The standard provides stable RTs over long analysis times and can be readily transferred between different LC gradients and nUHPLC instruments. Moreover, RePLiCal can be used to predict RTs for other peptides in a timely manner. Furthermore, it is shown that RePLiCal can be used effectively to evaluate trapping column performance for nUHPLC instruments using trap-elute configurations, to optimize gradients to maximize peptide and protein identification rates, and to recalibrate the m/z scale of mass spectrometry data post-acquisition.

Dependence of reversed-phase liquid chromatographic retention for aripiprazole and some related chemical impurities on the main elution parameters

ABSTRACTThe present study focuses on the chromatographic behavior of a novel atypical antipsychotic drug, aripiprazole, and its five chemical related impurities. The experiments were performed on a Zorbax SB-C18 column (150 mm × 4.6 mm, 5 µm particle size), whereas the detection was provided by an UV-Vis DAD detector set at 254 nm. The influences of the organic modifier, column temperature, and the pH of the mobile phase on the retention of the six compounds were studied. From these dependences some molecular descriptors and thermodynamic parameters that are important in drug characterization were evaluated. Also, the effect of two different acids added to the mobile phase (phosphoric acid and trifluoroacetic acid) on the retention behavior for chosen compounds was studied.

Microwave-Assisted Extraction and Purification of Arctiin and Arctigenin from Fructus Arctii by High-Speed Countercurrent Chromatography.

An efficient method for the rapid extraction, separation and purification of bioactive lignans, arctiin and arctigenin, from Fructus arctii by microwave-assisted extraction coupled with high-speed countercurrent chromatography was developed. The optimal extraction conditions of arctiin and arctigenin were evaluated by orthogonal array. Arctigenin could be converted from arctiin by hydrochloric acid hydrolysis. The separations were performed at a preparative scale with two-phase solvents composed of ethyl acetate-ethanol-water (5 : 1 : 5, v/v/v) for arctiin, and n-hexane-ethyl acetate-ethanol-water (4 : 4 : 3 : 4, v/v/v/v) for arctigenin. From 500 mg of crude extract sample, 122.3 mg of arctiin and 45.7 mg of arctigenin were obtained with the purity of 98.46 and 96.57%, and the recovery of 94.3 and 81.6%, respectively. Their structures were determined by comparison with the high-performance liquid chromatography retention time of standard substance as well as UV, FT-IR, electrospray ion source (ESI)-MS, (1)H-NMR and (13)C-NMR spectrum. According to the antioxidant activity assay, arctigenin had stronger 1,1-diphenyl-2-picrylhydrazyl free radicals scavenging activity.

Applications of Poly(Ethylene)Glycol (PEG) in Separation Science

The wide range of applications of poly(ethylene)glycol (PEG) in primarily chromatography and other closely related analytical methods has been reviewed. PEG has been used as mobile phase modifier in capillary electrophoresis (CE) as well as ion exchange, size exclusion, and hydrophobic interaction liquid chromatography (LC) methods. Generally in the presence of PEG, LC retention of macromolecules is altered and stability of their structure is maintained. PEG was effective in CE as a permanent coating for fused silica capillaries to shield free silanol groups that can cause protein adsorption to the wall resulting in band broadening and low recovery of the separated proteins. In gas chromatography, PEG-based stationary phases were applied for separation of polar analytes. PEG could also serve as an extraction medium in solid phase microextraction and aqueous two phase systems. Selected analytical applications, primarily LC and CE, involving PEG to facilitate the determination of either small molecules or macromolecules such as proteins in their native form are discussed and representative figures provided.

Gradient liquid chromatographic retention time prediction for suspect screening applications: A critical assessment of a generalised artificial neural network-based approach across 10 multi-residue reversed-phase analytical methods

For the first time, the performance of a generalised artificial neural network (ANN) approach for the prediction of 2492 chromatographic retention times (tR) is presented for a total of 1117 chemically diverse compounds present in a range of complex matrices and across 10 gradient reversed-phase liquid chromatography-(high resolution) mass spectrometry methods. Probabilistic, generalised regression, radial basis function as well as 2- and 3-layer multilayer perceptron-type neural networks were investigated to determine the most robust and accurate model for this purpose. Multi-layer perceptrons most frequently yielded the best correlations in 8 out of 10 methods. Averaged correlations of predicted versus measured tR across all methods were R2=0.918, 0.924 and 0.898 for the training, verification and test sets respectively. Predictions of blind test compounds (n=8–84 cases) resulted in an average absolute accuracy of 1.02±0.54min for all methods. Within this variation, absolute accuracy was observed to marginally improve for shorter runtimes, but was found to be relatively consistent with respect to analyte retention ranges (~5%). Finally, optimised and replicated network dependency on molecular descriptor data is presented and critically discussed across all methods. Overall, ANNs were considered especially suitable for suspects screening applications and could potentially be utilised in bracketed-type analyses in combination with high resolution mass spectrometry.

Retention projection enables accurate calculation of liquid chromatographic retention times across labs and methods

Identification of small molecules by liquid chromatography–mass spectrometry (LC–MS) can be greatly improved if the chromatographic retention information is used along with mass spectral information to narrow down the lists of candidates. Linear retention indexing remains the standard for sharing retention data across labs, but it is unreliable because it cannot properly account for differences in the experimental conditions used by various labs, even when the differences are relatively small and unintentional. On the other hand, an approach called “retention projection” properly accounts for many intentional differences in experimental conditions, and when combined with a “back-calculation” methodology described recently, it also accounts for unintentional differences. In this study, the accuracy of this methodology is compared with linear retention indexing across eight different labs. When each lab ran a test mixture under a range of multi-segment gradients and flow rates they selected independently, retention projections averaged 22-fold more accurate for uncharged compounds because they properly accounted for these intentional differences, which were more pronounced in steep gradients. When each lab ran the test mixture under nominally the same conditions, which is the ideal situation to reproduce linear retention indices, retention projections still averaged 2-fold more accurate because they properly accounted for many unintentional differences between the LC systems. To the best of our knowledge, this is the most successful study to date aiming to calculate (or even just to reproduce) LC gradient retention across labs, and it is the only study in which retention was reliably calculated under various multi-segment gradients and flow rates chosen independently by labs.

Calculation of retention time tolerance windows with absolute confidence from shared liquid chromatographic retention data

Compound identification by liquid chromatography–mass spectrometry (LC–MS) is a tedious process, mainly because authentic standards must be run on a user's system to be able to confidently reject a potential identity from its retention time and mass spectral properties. Instead, it would be preferable to use shared retention time/index data to narrow down the identity, but shared data cannot be used to reject candidates with an absolute level of confidence because the data are strongly affected by differences between HPLC systems and experimental conditions. However, a technique called “retention projection” was recently shown to account for many of the differences. In this manuscript, we discuss an approach to calculate appropriate retention time tolerance windows for projected retention times, potentially making it possible to exclude candidates with an absolute level of confidence, without needing to have authentic standards of each candidate on hand. In a range of multi-segment gradients and flow rates run among seven different labs, the new approach calculated tolerance windows that were significantly more appropriate for each retention projection than global tolerance windows calculated for retention projections or linear retention indices. Though there were still some small differences between the labs that evidently were not taken into account, the calculated tolerance windows only needed to be relaxed by 50% to make them appropriate for all labs. Even then, 42% of the tolerance windows calculated in this study without standards were narrower than those required by WADA for positive identification, where standards must be run contemporaneously.

Abstract 4619: DNA adductome analyses at multiple sites of human gastric mucosa, resected for gastric cancer

Abstract DNA adducts are recognized as an initiative step of mutagenesis, but the measurement of them is still a technical challenge. So-called adductome approach, based on LC-MS/MS, is a popular procedure to identify multiple DNA adducts in various tissues at a single experiments. Recently we reported 7 lipid peroxidation-induced DNA adducts in human gastric mucosa resected for gastric cancer in two countries for the first time, and showed the profile of these DNA adducts could indicate geo-pathological background of gastric mucosa (Matsuda 2013). Though DNA adductome approach posesses the high throghput potential in terms of finding numerous specis of DNA adducts having different molecular weights, there are still challenges to overcome. First of all, the numbers of the standards of DNA adducts molecules available are less than the species with identified molecular weight. This implies there are much more DNA modifications in human tissues than previously characterized. The other challaenge is that the absolute quantifications of these adducts requires isotopic standard chemicals. In this study, we strengthened DNA adductome method by including as many as known adducts in the assay system. Mass/Charge value and liquid chromatography retention time of known 254 DNA addcuts were collected and incoporated into LC-MS/MS machine (4000-QTRAP; Thermo). By this procedure we could measure the 254 DNA adducts at one time in MRM mode. Using this system, we measured 254 DNA adducts in the following human gastric mucosa. Written informed consents were obtained from the patients who took gastrectomy for gastric cancer. Under the hypothesis of field cancerization of gastric mucosa, in order to analyze the spatial influence of the exposure of the potential carcinogens toward cancer site, muocsal tissue at multiple sites with different distance to cancer, (10cm and more distant from the tumor site in the stomach to the site adjacent to the tumor) were taken. Three to 10 sites/case were investigated for DNA adducts measurements. Totally 11 cases and 77 sites are analyzed. Considerable numbers of known DNA adducts were present in human gastric mucosa in different quantities. Among them, tobacco-related and lipid-peroxidation-induced DNA adducts were noted. We also did the same measurements in proximal, middle, and distal gastric mucosa from autospy cases without gastric cancer and compared them to those of the cancer cases. Among them, etheno-dA was able to be quantitatively measured using isotopic standard. The mean level of etheno dA in the gastric mucosa of autopsy cases without gastric cancer was 8.02/107 nucleotides, while that of gastric cancer patients mucosae was 4.80/107 nucleotides. These comprehensive identifications in adducts in human gastric mucosa will provide the basic information for dosimetry and risk assessment of environmental chemicals toward gastric cancer. Citation Format: Nobuya Kurabe, Ippei Ohnishi, Masako Suzuki, Yusuke Inoue, Tomoaki Kahyo, Moriya Iwaizumi, Yoshitaka Matsushima, Yukari Totsuka, Hitoshi Nakagama, Masako Kasami, Hideto Ochiai, Keigo Matsumoto, Shioto Suzuki, Fumihiko Tanioka, Haruhiko Sugimura. DNA adductome analyses at multiple sites of human gastric mucosa, resected for gastric cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4619. doi:10.1158/1538-7445.AM2015-4619

Investigation on Selectivity of Mercaptopropylsilica and Silver Nanoparticles- Functionalised Mercaptopropylsilica

Mercaptopropylsilica and silver nanoparticle (AgNP)-coated mercaptopropylsilica (Nucleosil 300, particle size 5 m and pore size 10 nm) were prepared and their suitability as stationary phases for reversed-phase high-performance liquid chromatography (RP-HPLC) was evaluated. The retention mechanism was investigated for both ionogenic and non-ionogenic compounds using 150 x 4.6 mm i.d. columns, with emphasis on non-specific hydrophobic interactions and the possibility of specific hydrogen-bonding and donor-acceptor interactions between solutes and stationary phases. The retention of more than 40 substances including pyridines, anilines, phenols, benzoic acids and others was investigated. A different selectivity was obtained in the case of a series of anilines and pyridines. However, both phases showed a non-orthogonal retention mechanism, the latter being related to electron donor-acceptor interactions. The presence of hydrophilic interaction liquid chromatography (HILIC) retention behavior was demonstrated for mercaptopropylsilica phase. Keywords: Coordination, hydrogen bonding, hydrophilic interaction liquid chromatography, mercaptopropylsilica, reversedphase high performance liquid chromatography, silver nanoparticles, stationary phase.

Determination of the solubilising character of 2-methoxyethyl-(dimethyl)ethylammonium tris(pentafluoroethyl)trifluorophosphate based on the Abraham solvation parameter model

Gas–liquid chromatographic retention factors have been measured for 42 different organic probes on a 2-methoxyethyl(dimethyl)ethylammonium tris(pentafluoroethyl)trifluorophosphate, ([MeoeM2EAm]+[FAP]–), stationary phase capillary column at 323 and 353 K. The measured retention factors were combined with published gas-to-liquid partition coefficient data for solutes dissolved in ([MeoeM2EAm]+[FAP]–) and with published gas-to-water partition coefficient data to yield 107 gas-to-anhydrous ionic liquid and 105 water-to-anhydrous ionic liquid partition coefficients. Abraham model correlations for describing solute transfer into ([MeoeM2EAm]+[FAP]–) were derived from the three sets of experimental partition coefficient data. The derived correlations back-calculated the experimental gas-to-([MeoeM2EAm]+[FAP]–) and water-to-([MeoeM2EAm]+[FAP]–) partition coefficient data to within 0.13 and 0.15 log units, respectively.

Hydrophilic interaction liquid chromatography for the separation of acidic agricultural compounds.

Organic acids with very low pKa require extremely low pH conditions to achieve adequate retention in reversed-phase liquid chromatography, but an extremely low pH mobile phase can cause instrument reliability problems and limit the choice of columns. Hydrophilic interaction chromatography is a potential alternative to reversed-phase liquid chromatography for the separation of organic acids using more moderate conditions. However, the hydrophilic interaction chromatography separation mechanism is known to be very complex and involves multiple competing mechanisms. In the present study, a hydrophilic interaction chromatography column packed with bare silica core-shell particles was used as the separation column and six agricultural organic acids were used as model analytes to evaluate the effects of buffer concentration, buffer pH, and temperature on sample loading capacity, selectivity, retention, and repeatability. It was found that using a higher concentration of buffer can lead to a significant improvement in the overall performance and reproducibility of the separation. Investigation of column equilibration time revealed that a very long equilibration time is needed when changing mobile phase conditions in between runs. This limitation needs to be acknowledged in hydrophilic interaction chromatography method development and sufficient equilibration time needs to be allowed in method scouting.

Tuning retention and selectivity in reversed-phase liquid chromatography by using functionalized multi-walled carbon nanotubes

Aim of this work was to explore the possibility of retention and selectivity tuning in reversed-phase liquid chromatography by means of chemically modified multi-walled carbon nanotubes (MWCNTs). These were synthesized by derivatizing pristine MWCNTs with amino-terminated alkyl chains containing polar embedded groups. A novel hybrid material based on functionalized MWCNTs (MWCNTs-R-NH2) was prepared, characterized and tested. The idea was to design a mixed-mode separation medium basing its sorption properties on the peculiar characteristics of MWCNTs combined with the chemical interactions provided by the functional chains introduced on the nanotube skeleton. MWCNTs-R-NH2 were easily grafted to silica microspheres by gamma radiation (using a 60Co source) in the presence of polybutadiene as the linking agent. The composite was characterized by scanning electron microscopy (SEM) and Brunauer, Emmett and Teller (BET) analysis in terms of structural morphology, surface area and porosity. The MWCNTs-R-NH2 sorbent was tested as stationary phase. The reversed-phase behaviour was first proved by analysis of alkylbenzenes, while the key role of CNT derivatization in addressing the selectivity/affinity towards the solutes was evidenced by testing three classes of analytes, viz. barbiturates, steroid hormones and alkaloids. These compounds, with different molecular structure and polarity, were here analysed for the first time on CNT-based LC stationary phases. The behaviour of the novel sorbent was compared in terms of retention capability and resolution with that observed using unmodified MWCNTs, pointing out the mixed-mode characteristics of the MWCNTs-R-NH2 material. The same test mixtures were analysed also on a conventional mono-modal separation sorbent (C18) to highlight the particular behaviour of the (derivatized)MWCNTs-based stationary phases. The novel material showed better performance in separation of polar compounds, i.e. barbiturates and alkaloids, than the unmodified MWCNTs and than the C18 column. Results showed that MWCNT functionalization is powerful to modulate retention/selectivity in reversed-phase liquid chromatography.