Thymine

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.

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.