HPLC Method for Adenine on Primesep 100 by SIELC Technologies

High Performance Liquid Chromatography (HPLC) Method for Analysis of Adenine

Adenine is a chemical compound with the molecular formula C₅H₅. It is a building block for DNA and RNA as one of the four essential nucleotide bases. It is vital as a part of the main energy-carrying molecule in cells. When in DNA, it pairs with thymine, while in RNA, it pairs with uracil.

Adenine can be retained and analyzed using the Primesep 100 stationary phase column. The analysis utilizes an isocratic method with a simple mobile phase consisting of water and acetonitrile (MeCN) with a sulfuric acid buffer. Detection is performed using UV.

HPLC Method for Adenine on Primesep 100 by SIELC Technologies

High Performance Liquid Chromatography (HPLC) Method for Analysis of Adenine

Adenine is a chemical compound with the molecular formula C₅H₅N₅. It is a building block for DNA and RNA as one of the four essential nucleotide bases. It is vital as a part of the main energy-carrying molecule in cells. When in DNA, it pairs with thymine, while in RNA, it pairs with uracil.

Adenine can be retained and analyzed using the Primesep 100 stationary phase column. The analysis utilizes an isocratic method with a simple mobile phase consisting of water and acetonitrile (MeCN) with a sulfuric acid buffer. Detection is performed using UV.

HPLC Method for Separation of Adenine, Deoxyadenosine and Adenosine on BIST B+ by SIELC Technologies.

Separation type: Bridge Ion Separation Technology, or BIST™ by SIELC Technologies

Description

HPLC Method for Uracil, Thymine, Adenine, Cytosine, Guanine on Newcrom AH by SIELC Technologies

High Performance Liquid Chromatography (HPLC) Method for Analysis of Uracil, Thymine, Adenine, Cytosine, Guanine.

Uracil, Thymine, Guanine, Cytosine and Adenine are the nucleobases found in RNA and DNA.

Uracil, also noted as U and Ura, has C₄H₄N₂O₂ chemical formula. It is a derivative of pyrimidine that is rarely found in DNA, working more often in RNA in transcription by binding to adenine through hydrogen bonds. You can find detailed UV spectra of Uracil and information about its various lambda maxima by visiting the following link.

Thymine, also noted as T and Thy, has the chemical formula C₅H₆N₂O₂. It is a a pyrimidine nucleobase that may be produced through methylation of uracil. In DNA, it creates a double hydrogen bond to Adenine to hold together the structure of DNA.

Guanine, also noted as G and Gua, has the chemical formula C₅H₅N₅O. By forming three hydrogen bonds with the Cytosine, it creates a base pair. It’s name comes from the Spanish term “guano”, meaning bird or bat dropping, as that is said to have been how it was first discovered. Outside of DNA, Guanine that is harvested from fish scales, is occasionally used in cosmetics for it’s luster.

Cytosine, also noted as C and Cyt, has the chemical formula C₄H₅N₃O. In DNA, it pairs with Guanine to create a base pair. In RNA, it is synonymous with Uracil, being an interchangeable third base. Not only that, due to it’s instability, it can change into Uracil through spontaneous deamination.

Adenine, also noted as A and Ade, has the chemical formula C₅H₅N₅. Besides DNA and RNA, Adenine can also be found in Adenosine triphosphate (ATP), which is a nucleotide triphosphate that provides energy in cells required for bodily functions. In DNA, it partners with Thymine via two hydrogen bonds, while in RNA it bonds to Uracil for protein synthesis.

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.

HPLC Method for Adenosine, Cordycepin, Adenine on Newcrom AH by SIELC Technologies

High Performance Liquid Chromatography (HPLC) Method for Analysis of Adenosine, Cordycepin, Adenine.

Adenosine is a key building block of energy-carrying molecules with the chemical formula C₁₀H₁₃N₅O₄. It has a variety of other uses, including being a inhibitory neurotransmitter which helps with sleep and acting as a blood flow regulator. Medicinally, it is used as treatment for supraventricular tachycardia (SVT). You can find detailed UV spectra of Adenosine, Cordycepin, Adenine and information about its various lambda maxima by visiting the following link.

Cordycepin is an adenosine analogue with the chemical formula C₁₀H₁₃N₅O₃. It is reported to prevent cell reproduction in various cancer cells. It also might possess antioxidant and anti-inflammatory properties, when considered in addition with it’s ability to cross the blood-brain barrier, it may become widely used in pharmaceuticals.

Due to cordycepin having a very similar structure to adenosine, it has shown to have inhibitive properties on the COVID-19 coronavirus. However, due to their similar structures, the separation of the two sugars can be challenging. Both sugars can be separated isocratically in about six minutes on the Newcrom AH mixed-mode column, which has both hydrophobic and cationic exchange properties. The mobile phase consists of acetonitrile (ACN, MeCN) and water with ammonium formate as a buffer which makes it mass-spec (MS) compatible. It can also be UV detected at 260nm.

Application Notes: Nucleosides are glycosylamines consisting of a nucleobase linked to a ribose or a deoxyribose sugar. Nucleoside are building blocks for DNA and RNA. These compounds are very polar in nature and contain groups available for hydrogen bonding interactions. Method for separation of adenine and adenosine were developed using a hydrogen-bonding method. There is a strong correlation between retention time for adenine/adenosine and the mobile phase composition, which consists of acetonitrile and methanol. Order of elution for compounds depends on the amount of acetonitrile and methanol.  Furthermore, ellution of adenine and adenosine can be reversed based on the composition of the mobile phase. Our method is compatible with LC/MS and preparative chromatography.

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.

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.