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Protecting groups like Fmoc and coupling both steps are essential to monitoring the Fmoc SPPS (Solid Phase Peptide Synthesis) reaction completion. Reliable methods are used to detect the unreacted number of amino groups for monitoring these two essential reaction steps of coupling and cleavage. The ability to detect the complete coupling, incomplete coupling or failure of coupling we use many colour tests in the laboratory and based on this the Fmoc peptide chemistry allows the control of the completion of the Fmoc cleavage. The most important test used is the Kaiser test and highly recommended to monitor the coupling and cleavage steps. If the result of colour tests is positive after coupling, then the second coupling should be performed. Then again use the colour test to detect the level of coupling. If the result is still slightly positive, repeat coupling with the smaller modification of reagents such as used PyBOP instead of HOBT AND HOAT. These colour tests help in revealing the presence of unreacted amino-functional groups. Thus, we need to block these free N-terminal of amino- acids which help in avoiding the making of deletion of sequence.
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