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The key design factors in hydrodynamic L-LC are sun & planet radii, beta values (even if compensation of changing rotation speed is made), coil winding technique, tubing bore etc. For hydrostatic L-LC the key design parameters are chamber shape/design/insertions, chamber volume, sun radii etc. For both L-LC principles as more factors are changed, the percentage success of Process Scale-up and cross compatibility between different L-LC fundamentally different design modes will reduce.

Confusion 4. Solvent selection in L-LC is not scientific. Incorrect, it is governed by same principles as S-LC

From discussions, non L-LC chromatographers have repeatedly said that they see L-LC biphasic solvent choice in published methods, as a variety of abstract solvent mixes or quote, “Witches Brews”. At best, they see the Arizona etc methods as “semi-defined scales of set ratios, in supposed sequences, for which L-LC researchers chose convenient, unrealistic standards to argue their validity”.

Our biphasic solvent selection research (presently being prepared for publication) shows this does not need to be so. Biphasic solvent selection in L-LC is governed by exactly the same principles as S-LC, with target & matrix solubility, functionalities, polarities, molecular weights, dipole moments, shape configurations, complex and micelle formation, pH etc all taken into account and utilised in comparison to known solvent properties, as defined by the Synder Triangle and solvent polarity series.

Confusion 5. At Big Prep 5 it was confirmed, a Company has over one million bioactive compounds in its library, and all only required S- LC. One could ask do we need L-LC? The answer is definitely yes. To assist this statement, we would add that a Quattro CCC was recently custom designed for a USA Pharmaceutical Company for High Throughput Preparation application. This custom build shows that not only is L-LC’s maintenance of compound integrity applicable in searching for unknown bio-actives/taste/colour/nutriceuticals etc in natural products, but it may possibly one day have a place in mainstream Pharmaceuticals as well (see Results & Discussion).

Why is the answer above yes? The answer above is yes in part because the above initial statement is self-fulfilling prophecy. If research only ever used S-LC to define a library, by definition any compound that would be absorbed by the phase, or would have been degraded by the phase would not be in the library. Perhaps it would be well for us all to remember that silica is used as a catalyst for certain hydrolysis reactions. The question that should be asked is how many bioactive targets or cytotoxic contaminants may be missed owing to use of only S-LC? (Ref Results & Discussion). The polarity limit of RP-C18 / NP-silica etc in HPLC etc is alarmingly small, compared to L-LC. With stop rotation-wash-off or elution-extrusion each L-LC run can go from

infinitely polar, to infinitely non-polar (or vice versa). If using a switching valve, Head to Tail may be reversed during a run, and even change from a reverse phase to normal phase run (or vice versa) at any time of the users choosing.

Confusion 6. L-LC always has low plate counts. Why bother with L-LC? The reply is, L-LC has massively higher stationary phase retention than S-LC, thus achieves resolution with low plate count, plus L-LC can achieve selectivity through its vast stationary phase options. L-LC can use half to less than a tenth of solvent to prepare the same mass of target in same matrix when cross-compared to SLC. L-LC has a very high loading capacity (5 to 15% of coil volume), plus only requires low cost, low-pressure liquid pumps. All these factors make further consideration of L-LC important as cheaper more “Green” techniques are researched.

Regarding low plate per metre (p/m) counts, it should be noted that the percentage of stationary phase is fundamental to full resolution equation, though this factor is often deleted in HPLC texts. The reason HPLC etc requires high plate counts is in part that it has a very low percentage of stationary phase relative to the total content of the containing vessel.

In Flash and HPLC the numbers of stationary phases are extremely limited compared to L-LC. Therefore resolution by massive changes in selectivity is limited in S-LC.

L-LC traditionally uses 70 to 98% stationary phase. In a reverse phase C18 column, the C18 might be 10 to 21% of the stationary phase, which is itself a small percentage of total container void volume/void mass. In L-LC logical biphasic solvent selection, with options of isocratic (1,2), linear or step gradients (3), ionic liquids, pH based frontal chromatography (called pH Zone refining in CCC), micelles, reverse micelles, aqueous/aqueous, aqueous/organic, organic/organic, stop rotation-wash-off, elution- extrusion are all usable using the same L-LC system. All these possibilities are created only by choice of different biphasic or even triphasic solvents. Inorganic cations (inclusive precious metals, transition metals, radioactive isotopes) and anions, plus organic compounds, even certain shape orientations/chiral compounds, can be resolved with a single L-LC instrument.

Confusion 7. L-LC is mainly a stand-alone chromatography technique. Incorrect, Sequential L-LC and HPLC has been for the last 10 years our preferred operation mode in Contract Preparations of targets.

Almost every difficult application at the AECS- QuikPrep Ltd laboratory uses L-LC to polarity fractionate highly complex matrices (that would poison an HPLC column with one injection), into narrow polarity bands. We would stress the mutual benefit of Sequential L-LC and HPLC, and never view L-LC and HPLC as mutually exclusive. The L-LC fractions are so extremely restricted in polarity, that they only require isocratic HPLC to complete our standard Sequential L-LC to HPLC runs. 95 to 99+% pure

target can be obtained after a single Generic Gradient L-LC run and Sequential HPLC Prep column (see below). This Sequential L-LC and HPLC principle can be repeatedly successful, even when starting from unknown, and totally uncharacterised, raw natural product or crude synthesis materials. Sequential L-LC to L-LC, utilising different biphasic solvents for the second L-LC run, is only used if targets irreversibly adsorb or degrade on 5um, end capped C18 etc HPLC phases.


Figure 1

Figure 2

Fig 1 & 2 show the chassis and coil/volume options of the Quattro CCC™ model range. (“J” Type Planetary Centrifuge, open, constant id tubing, wound on a planetary bobbin, with no rotating seals). The bobbins (planetary rotating body, holds the coiled columns) can have tubing with different material choice. Options include PTFE, Stainless Steel or Titanium. Tubing bore for id can vary from 0.5 to 12.5 mm, and volumes from 7 to 3000 ml for a single rotor assembly. A single bobbin can have two coils. All models except the entry IntroPrep™ have two dynamically balanced bobbins, with up to 4 coils as an option. Each coil can be used independently for same or different preparations, or used in any combination, in series with any coil or multiple of coils of the same id. Uniquely for hydrodynamic L-LC model ranges, all models share the same key L-LC design parameters, inclusive of the same sun & planet radii, speed ranges, beta values, winding techniques and only tubing bore is varied. This model range is also the only one that allows even the largest bore to be tested on a laboratory based unit, prior to introduction to process based preparation. Hybrid coil winding, that is multiple id.’s in the same instrument or bobbin, can be manufactured produced. Multiple bobbin sets for a single chassis are available. In this way the major

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