The optimization of peak capacity in g
radient elutionRPLC is essential fo
r the sepa
ration of multicomponentsamples such as those encounte
red in p
roteomic
resea
rch. In this wo
rk, we study the effect of g
radient time(
tG), flow
rate (
F), tempe
ratu
re (
T), and final eluentst
rength (
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final) on the peak capacity of sepa
rations ofpeptides that a
re
rep
resentative of the
range in peptidesfound in a t
ryptic digest. We find that the
re a
re ve
ry st
ronginte
ractions between the individual va
riables (e.g., flow
rate and g
radient time) which make the optimization quitecomplicated. On a given column, one should fi
rst set theg
radient time to the longest tole
rable and then set thetempe
ratu
re to the highest achievable with the inst
rument.Next, the flow
rate should be optimized using a
reasonablebut a
rbit
ra
ry value of
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final. Last, the final eluent st
rengthshould be adjusted so that the last solute elutes as closeas possible to the g
radient time. We also develop an easilyimplemented, highly efficient, and effective Monte Ca
rlosea
rch st
rategy to simultaneously optimize all the va
riables. We find that g
radient steepness is an impo
rtantpa
ramete
r that influences peak capacity and an optimum
range of g
radient steepness exists in which the peakcapacity is maximized.