Effect of mobile phase pH on the retention of nucleotides on different stationary phases for high-performance liquid chr
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ORIGINAL PAPER
Effect of mobile phase pH on the retention of nucleotides on different stationary phases for high-performance liquid chromatography S. Studzińska & B. Buszewski
Received: 4 October 2012 / Revised: 15 November 2012 / Accepted: 16 November 2012 / Published online: 14 December 2012 # The Author(s) 2012. This article is published with open access at Springerlink.com
Abstract The main aim of the present investigation was to study the retention and separation of eight nucleotides with the use of seven stationary phases in RP HPLC mode. Two octadecyl columns were used; however, aminopropyl, alkylamide, cholesterol, alkyl-phosphate, and phenyl were also studied. Special attention was paid to the mobile phase buffer pH, since it appears that this factor is very influential in the case of chromatographic separation of nucleotides. The retention of nucleotides was greater for mobile phase pH4.0 in comparison with pH7.0 (except for octadecyl and phenyl packing). This is a consequence of protonization of polar groups present on the stationary phase surface. It was proved that aminopropyl, alkyl phosphate, alkylamide packing materials are not suitable for the resolution of nucleotides. On the other hand, cholesterol and phenyl stationary phases are alternatives for conventional octadecyl phases. Application of cholesterol packing allows separation of small, polar nucleotides, which is impossible to achieve in the case of octadecyl phase. Moreover, a phenyl support allows separation of nucleotides in a shorter time in comparison with octadecyl packing. Keywords High-performance liquid chromatography . Nucleotides . Stationary phases . Separation . Retention mechanism Abbreviations A Adenosine 5′-monophosphate disodium salt C Cytidine 5′-monophosphate
S. Studzińska : B. Buszewski (*) Environmental Chemistry and Bioanalytics, Faculty of Chemistry, Nicolaus Copernicus University, 7 Gagarin St., 87-100 Toruń, Poland e-mail: [email protected] S. Studzińska e-mail: [email protected]
DA DC DG DT G SG-AP SG-C18 SG-CHOL SG-NH2 SG-PC10 SG-Ph U
2′-Deoxyadenosine 5′-monophosphate disodium salt 2′-Deoxycytidine 5′-monophosphate sodium salt 2′-Deoxyguanosine 5′-monophosphate sodium salt hydrate Thymidine 5′-monophosphate disodium salt hydrate Guanosine 5′-monophosphate disodium salt hydrate Alkylamide stationary phase Octadecyl stationary phase Cholesterol stationary phase Aminopropyl stationary phase Phosphoalkyl stationary phase Phenyl stationary phase Uridine 5′-monophosphate disodium salt
Introduction Nucleotides are essential for living organisms and are the building blocks of nucleic acids [1, 2]. Moreover, these compounds are driving forces for cell growth and control changes of energy. Nucleotides take part in many cellular and intercellular processes, playing important regulatory functions [1–3]. Their determination is of great importance in the fields of chemistry, biochemistry, medicine, genetics, and environment. Determination of nucleotide content in the cell provides information for understanding cellular energy
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