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Troubleshooting Poor Yields, Impurities and Methods

Method development is a critical part of achieving consistent performance due to variability in reagents, phosphoramidites, and handling procedures.

 

Important: Always follow phosphoramidite vendor recommendations before applying the troubleshooting suggestions below.

Common Problems and Solutions

🔹 Problem: High n–1 Impurities

Possible Causes & Solutions:

  • Cause: Bad detritylation yields:

  • Solutions:

    • Double the deblock times

    • Modify pulse times

    • Increase number of repetitions

For K&A Systems : Sometimes the initial column receives less deblock than the remaining columns.  This can be because of an air bubble in the manifold that leads to initial column receiving less deblock volume.  This can be mediated by removing purges between repetitions or adding a deblock "prime" to ensure the manifold is filled with deblock.  

WARNING! At the moment the K&A does not have a native prime option so a deblock prime is generally recommended at the end of the cycle before the initial deblock in a cycle.  The very first deblock section goes to the trityl monitors so having a prime at the beginning of the cycle might give you faulty trityl histograms data.


  • Cause: Bad coupling and capping:

  • Solutions:

    • Double coupling and capping times or repetitions

  • Cause: Bad oxidation yields:

    • Increase oxidation time

    • Try reversing Ox/Cap steps or reorder as Cap/Ox/Cap or Cap/Ox/Ox/Cap

NOTE: When incorporating thiolation reagent (Sulfur) it is generally recommended you go Sulf/CAP as some capping reagents interfere with the thiolation process.  

  • Old or wet reagents (especially phosphoramidites and activator):

    • Replace with freshly prepared reagents in dry ACN

    • Use drying traps on:

      • All phosphoramidites

      • ACN bottle

      • Activator bottle

      • Note: Drying traps take ~24 hours to remove ~80% of water

    • In humid environments this is even more crucial

  • Slow reaction rates on first cycle:

    • Double detritylation and coupling times for the first cycle only

🔹 Problem: High 5’-Failure Sequences (n–1, n–2, n–3…)

Possible Causes & Solutions:

  • Poor coupling:

    • Double coupling times or repetitions

  • Poor oxidation (rare):

    • Increase oxidation time

    • Increase I₂ concentration above 20 mM (unless using dmf-dG phosphoramidite)

🔹 Problem: 5’-Failures With Phosphate + 3’-Deletions With Phosphate (or Phosphate + 98)

Cause: Excessive depurination
Solution:

  • Limit deblock exposure to ≤60 seconds (especially when using TCA)

  • Test alternative deblocks (different acid or concentration), then adjust time

🔹 Problem: High n–1, Very Few 5’-Failures

Cause: Poor capping
Solution:

  • Replace capping solutions

  • Increase capping times

  • Test different capping formulations

🔹 Problem: n+1 Impurities

Cause: Acidic activator removes DMT → forms dimer → reacts with 5’-OH
Solution:

  • Switch to a less acidic activator (e.g., DCI or tetrazole)

🔹 Problem: n+x (x = 2, 3, 4...) Impurities After FLP Peak

Cause: Exocyclic amine protecting groups lost pre-synthesis → oligo branching
Solution:

  • Replace phosphoramidites

🔹 Problem: High m+275 or m+261 with Universal Linker

Cause: Variable linker degradation depending on Me/Ph group and succinimide ring
Solution:

  • Apply longer or harsher cleavage/deprotection conditions

🔹 Problem: m+366 and/or m+286 (Impurities elute long after FLP)

Cause: Trityl adds to unoxidized P(III)
Solution:

  • Increase oxidation time

  • Raise I₂ concentration >20 mM (unless using dmf-dG phosphoramidite)

🔹 Problem: High m+41 Impurity

Cause: Incomplete capping
Solution:

  • Adjust capping time to reduce m+41 without increasing n–1

  • Try alternative capping solutions

🔹 Problem: Excessive m+53 Peak (Just After FLP)

Cause: Incomplete DEA reaction
Solution:

  • Increase DEA reaction time (post-synthesis, pre-cleavage)

  • For first few additions of DEA (or hindered amine), pass through columns in <10 seconds to quickly rinse acrylonitrile from oligo

Mass Analysis Tips

Before LC-MS analysis of your own oligos, generate and reference the following:

  • All 5’-deletions (e.g., for 5’-CATGT-3’:

    • 5’-ATGT-3’, 5’-TGT-3’, 5’-GT-3’)

  • All 3’-deletions (e.g.,

    • 5’-CATG-3’, 5’-CAT-3’, 5’-CA-3’)

  • All 5’-failures with phosphate

    • e.g., 5’-ATGT–○P–3’, 5’-TGT–○P–3’

  • All 3’-failures with phosphate

    • e.g., ○P–CATG-3’, ○P–CAT-3’, ○P–CA-3’

Note: “5’-failures” can originate from 3’-end deletions, and “3’-failures” from 5’-end deletions—these terms refer to the reactive site (5’-OH vs. 3’-OH).

  • Include other common impurities in your mass list

  • Use exact masses, not average masses

  • Generating the mass list can be streamlined using an Excel template with your sequence