At this point if optimum separation is not achieved the mobile phase composition can be reduced or increased incrementally in 5-10%B steps. If the desired resolution is still not achieved the mobile phase solvents used or the column chemistry may need to be reconsidered, or gradient elution employed. If a gradient separation has been recommended (i.e. Δt |

t The final mobile phase composition for the gradient method (%B |

t Using the calculated values of %B |

Figure 3: Chromatogram produced using optimized %B |

It should be noted that although optimizing initial and final %B in this way can give the desired separation the gradient elution profile can be further optimized. In the chromatogram in Figure 3 there is a critical peak pair (1) which would indicate that this separation needs further optimization. The gradient profile can be further optimized by calculating an appropriate gradient slope (t |

S = shape selectivity factor (for small molecules use 5 or calculate Equation 8) |

Δφ = change in %B expressed as a decimal V |

Using the same values of initial and final %B and the new gradient time the chromatogram in Figure 4 was obtained. |

Figure 4: Figure 2: Chromatogram produced using optimized %B_{inital(gradient)} and %B_{final(gradient)} and gradient time (t_{g}). |

If there are still any critical peak pairs the separation may require some further optimization, this may require the use of segmented gradient profiles. The re-equilibration time of a gradient method is a parameter that should be considered and optimized. |

V V |

F = flow rate (mL/min.) For this separation the optimum re-equilibration time would be 7.2 minutes. |