Primesep 200 separates with baseline resolution nucleic bases (uracil, thymine, cytosine, guanine, and adenine) by a combination of cation exchange and reversed phase. Uracil typically does not retain on reversed-phase column and is often used as an unretained void volume marker for C18 and C8 columns. Primesep 200 has an embedded anionic functional group which helps retain polar compounds polar and ion-exchange mechanisms. Excellent peak shape results with a mass spec compatible mobile phase of water, acetonitrile (MeCN, ACN) and trifluoracetic acid (TFA) with UV detection at 270 nm.
HPLC Application for Separation of Nucleotide Bases
Nucleotide bases are parts of DNA and RNA. Adenine and guanine are purine-bases; uracil, thymine and cytosine are pyrimidine-bases. In the view of chromatography these compounds are very polar and similar in properties. It is hard to obtain base line separation on traditional C18 as peaks of nucleotide bases co-elute even at low organic concentration. In this application nucleobases are well retained and separated on Primesep 200 mixed-mode column. Compounds are retained by weak reverse phase and weak ion-exchange mechanisms. Method can utilize UV, ELSD, and LC/MS detection.
HPLC Separation of 8 Generic Compounds on Primesep Columns
Mixed-mode HPLC columns allow to analyze compounds with drastically different properties in one run. Acidic, basic, and neutral compounds can be separated in one run using either isocratic or gradient conditions. In this application, neutral hydrophilic (uracil, phenol and hydroquinone), neutral hydrophobic (toluene), hydrophilic acidic (benzoic acid), hydrophilic basic (lutidine) and hydrophobic basic (amitriptyline) are separated using gradient of ACN. Neutral compounds are retained by reversed-phase mechanism, hydrophilic acidic compound become more hydrophobic at lower pH and retain by reversed-phase mechanism too. Basic compounds are retained by cation exchange mechanism, and hydrophobic basic compounds are retained by reversed-phase and cation-exchange mechanisms. All compounds are resolved within 17 minutes on a short column. Method can be applied to various polar and hydrophobic compounds, which can be separated on one column and in one run. Mixed-mode columns can operate in single or combination of several modes: reversed-phase, ion-exchange, ion-exclusion and HILIC. This mixed-mode HPLC column can be used as a general column for separation of wide range of compounds.
HPLC Separation of Acidic, Basic, and Neutral Compounds
Primesep 100 and Primesep 200 columns can be used as a universal column for analysis of wide range of compounds. These mixed-mode reversed-phase ion-exchange HPLC columns can provide a valuable alternative to traditional reversed-phase column. Amines, amino acids, quaternary amines, and various zwitter-ions can be analyzed along with hydrophobic compounds and organic and inorganic counter-ions. In this application, 8 compounds with different hydrophobic, hydrophilic, basic and acidic properties are separated based on their properties. Primesep 100 column is a mixed-mode HPLC column with a C12 carbon chain and carboxylic acid on the surface with pKa of 1. Primesep 200 column is a mixed-mode HPLC column with a C12 carbon chain and carboxylic acid on the surface with pKa of 2. These columns can be used with 100% organic (ACN) and 100% aqueous mobile phases. This HPLC method can be adopted as a generic and robust approach for analysis of acidic, basic and neutral compounds within the same run.
Uracil is a naturally occurring pyrimidine derivative. Uracil is used for drug delivery and as a pharmaceutical. Uridine is a product of uracil and sugar ribose. Uridine is one of components of ribonucleic acid. Uracil and uridine are slightly basic in nature. Both compounds are hydrophilic and uracil is very often is used as a void marker in reversed-phase chromatography. Uracil and uridine are separated based on their polar properties on a Primesep N HILIC column. Uracil and uridine are monitored by UV.
HPLC Separation of Nucleic Bases at pH 4 and 5 on Obelisc N
Nucleic bases are biological compounds found in genetic molecules (DNA, RNA). They can be separated on an Obelisc N column, which offers very polar characteristics and can be used with positively or negatively charged groups. Closely-eluted adenosine and uridine can be further separated by simply adjusting the pH of the mobile phase. Mobile phase is water and acetonitrile (MeCN, ACN) with Ammonium Acetate as buffer. UV detection at 250nm.
HILIC Retention of Uridine and Uracil on Sielc's HILIC Columns
Uridine and uracil are separated on three HILIC columns from SIELC. Primesep N, Primesep S, and Obelisc N columns all have different polarity and different embedded acidic groups on the surface of silica gel: Primesep N is a normal-phase HPLC column with embedded acidic groups with a pKa of about 5. Primesep S is a normal-phase HPLC column with embedded acidic groups with a pKa of about 3. The Primesep S stationary phase retains basic compounds by cation-exchange at pH > 3.
In traditional HILIC mode, a charged or neutral polar analyte interacts with a water layer on the polar stationary phase surface. On Obelisc N the charges are greatly separated and independently accessible, resulting in different selectivity compared to traditional HILIC and silica columns. Mobile phase composition changes the conformation of the long hydrophilic chain. All three columns can be used to analyze uridine and uracil by HPLC with UV, LC/MS and ELSD detection.
Primesep S, Primesep N, Obelisc N, 4.6x150 mm, 5 µm, 100A
HPLC Separation of Uracil and Uridine in HILIC Mode on Primesep S2 Column
Uracil and uridine are separated based on their polarity. The mechanism of retention is pure HILIC mode. Retention time is controlled by varying the amount of ACN. Buffer concentration and pH will affect ionization and polarity of the stationary phase, and thus indirectly affect retention of neutral compounds. Column is compatible with all general detection techniques.