Uracil

HPLC Method for Analysis of Uracil, Thymine, Hypoxanthine, Guanine on Newcrom AH Column by SIELC Technologies

Separation type: Liquid Chromatography Mixed-mode

Uracil, Thymine, Hypoxanthine, and Guanine are all nitrogenous bases, each playing distinct roles in the biochemistry of nucleic acids.

Uracil (U):

• Found in: RNA
• Pairs with: Adenine (A)
• Structure: Pyrimidine
• Role: Uracil replaces thymine in RNA. In DNA, adenine pairs with thymine, but in RNA, adenine pairs with uracil.

Thymine (T):

• Found in: DNA
• Pairs with: Adenine (A)
• Structure: Pyrimidine
• Role: Thymine is specific to DNA, distinguishing it from RNA. It’s the base that pairs with adenine through two hydrogen bonds.

Hypoxanthine:

• A naturally occurring purine derivative. It’s not directly a base in standard DNA or RNA, but it plays a significant role in the metabolism of purines.
• It is the base form of the nucleoside inosine, which can be found in certain tRNAs and plays a role in wobble base pairing.
• Hypoxanthine is also an intermediate in the purine degradation pathway, leading to the production of uric acid.

Guanine (G):

• Found in: Both DNA and RNA
• Pairs with: Cytosine (C)
• Structure: Purine
• Role: Guanine is one of the four main nucleobases in the nucleic acids DNA and RNA. It forms three hydrogen bonds with cytosine, contributing to the stability of the nucleic acid structures.

These nitrogenous bases are crucial for the structure, replication, and function of nucleic acids. Understanding their properties and interactions is fundamental to molecular biology and genetics.

Nucleotides can be retained and analyzed on a mixed-mode Newcrom AH column with a mobile phase consisting of water, Acetonitrile (MeCN), and ammonium formate. This analytical method can detect compounds with high resolution and peak symmetry using UV detection at 260 nm

High Performance Liquid Chromatography (HPLC) Method for Analyses of Uracil, Thymine, Hypoxanthine and Guanine

Condition

Description

Uracil, Thymine, Guanine, Cytosine and Adenine are the nucleobases found in RNA and DNA.  The nucleobases are difficult to separate on reverse-phase columns due to their polar, hydrophilic and ionic nature.  Using the Newcrom AH mixed-mode column, the nucleobases can be easily separated isocratically using low organic mobile phase (5% acetonitrile) or pure water, if organic mobile phase is undesirable, with ammonium formate buffer, making the method both UV and Mass Spec compatible.

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.

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.

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.

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.

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.

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 HPLC 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. This HPLC method can utilize UV, ELSD, and LC/MS detection.

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.

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.