How to determine peak purity in agilent chemstation

Here are two examples to illustrate the compounding effect of less than 100% efficiency in the synthesis of a 100nt vs the synthesis of a 50nt. This measure is especially important for long oligos, because as the length of an oligo increases, small differences in coupling efficiency have dramatic effects on the yield of the full-length product. Coupling efficiency in oligonucleotide manufacturing refers to the success rate of a synthesizer adding a new base to a growing nucleic acid chain. Protection of 2вЂІ-OH and deprotection at the end of the synthesis may not be accomplished at 100% yield, so the overall efficiency of RNA synthesis is compromised compared to DNA. The synthesis as well as the purification of oligoribonucleotides are intrinsically less efficient compared to the synthesis and purification of their deoxy counterparts, primarily due to the presence of the 2вЂІ-OH that needs to be protected during synthesis 11, 12, 13, 14, 15, 16. Most chromatography suppliers claim applications for oligonucleotide analysis, but in reality good analytical methods exist for up to 25nt 9, 10 and become less optimal as the length increases 5, 6. HPLC remains the most widely used analytical technology especially for purity determination and batch-to-batch comparison of compounds poised for pharmaceutical use 8. Since then a steady improvement in instrumentation and column packings have yielded methods for analysis and purification of both synthetic materials as well as compounds from biological fluids 4, 5, 6, 7. HPLCвЂ™s first application was the resolution of nucleic acids exploiting IEX normal mode 3, even though this type of chromatography was later abandoned. As a pump forces the MP through the column, the components of the mixture interact with the stationary phase - the particleвЂ™s surface - to different degrees and are separated in the process. In HPLC, a liquid mobile phase (MP) carries a mixture of compounds through a column packed with particles. A major revolutionary step in chromatography was the advent of high-performance liquid chromatography (HPLC) instruments invented 50 years ago 1, 2. separation of a mixture into its components, approximately 120 years ago is credited to Mikhail Tsvet, a Russian-Italian botanist. Unless well resolving HPLC methods are used for batch-to-batch evaluation of man-made RNAs, process development will remain shortsighted, and observed off-target effects in-vitro or in-vivo may be partially related to low purity and the presence of shorter sequences. We demonstrate the relative stability of RNA in the denaturing/basic IEX mobile phase, lay out a protocol to determine the on-the-column stability of any RNA, and establish the applicability of this method for quality testing of sgRNA, tRNA, and mRNA. Here we report that IP-RP is less suitable compared to the conventional ion-exchange (IEX) for analysis of 100nt RNAs.

In an attempt to exploit a single HPLC method and obtain both identity as well as purity, ion-pair reversed-phase chromatography (IP-RP) at high temperature in the presence of an organic cosolvent is the current analytical strategy. Analytical methods for RNAs up to the 60 nucleotides (nt) have been reported, but they fall short for purity evaluation of 100nt long, used as single guide RNA (sgRNA) in CRISPR technology, and promoted as pharmaceuticals. Synthetic RNA oligos exhibit purity decreasing as a function of length, because the efficiency of the total synthesis is the numerical product of the individual step efficiencies, typically below 98%.

How to determine peak purity in agilent chemstation