The
considerable effectiveness of an appropriate linear gradient can be see
by comparing the complexity of the gradient hplc bioactive natural product
extract, Slide 23 to the extremely
high purity of the Quattro CCC purified bioactive ingredient shown it
Slide 24. This slide also shows that
95% of this active ingredient was contained in a single 4 ml fraction
which from the UV trace of the CCC run Slide
22 appeared to be identical to the previous fraction, which in
fact contained only a minor trace of this bioactive component.
We should like to stress that online monitoring, especially by UV can
be very misleading in CCC, and it is best to fraction collect and do offline
tlc, hplc, hplc-ms, nmr or bioactivity assessment of these collected fractions
in order to achieve a true picture of the resolving power of Quattro CCC's.
In hplc it is relatively easy to extrapolate an isocratic condition from a gradient because only the mobile phase is changing.
In HSCCC if one makes an isocratic mix based on the elution profile, then that isocratic mix will have a different stationary phase solvent ratio from that in the column. This occurs as both phases in HSCCC are interactive and inside a dynamically changing coil is a very different equilibrium environment from a static unchanging test tube.
This will happen for both linear and step gradients, but the constancy of the step isocratic portion, allows easier optimization than in the linear gradient situation.
In addition, with step gradients, the additional wash-off of stationary phase once stepped initiated rapidly equilibrates and stabilizes. In Slide 20 a typical complex wash-off profile of a linear gradient is shown. These complex wash-off profiles also cause considerable extra complication when trying to optimize an isocratic separation from a linear gradient.
When method developing a novel HSCCC separation where test tube partitioning prescreening has proven unsuccessful, it is recommended to first try a reverse phase gradient and fractionate the complex matrix into predictable polarity bands. It is possible that component(s) of interest may still be in the presence of other components ( sometimes a single CCC run can yield individual component purities of 98% + ).
The gradient composition of the fraction of interest is noted.
The HSCCC chromatographer can then utilize the different selectivity of reverse and normal phase gradients, see Slide 21 and run a new normal phase gradient, which brackets the organic modifier elution concentration for the collected fraction of interest, which requires addition purification.