Optimal Drying of HPLC fractions

by in Application Notes, BPS News

Part 3

Controlling the pressure

Another crucial parameter to consider when drying water /ACN HPLC fractions using a centrifugal evaporator is pressure control.

Figure 3: Boiling point of water/ACN mixes at different pressures

Figure 3: Boiling point of water/ACN mixes at different pressures

In the past, evaporators dried all solvents by applying full vacuum. However it is now understood that at pressures below 6 mbar, the boiling point of water drops below 0°C – its freezing point – and thus any water in samples freezes before it is evaporated. This makes for very slow drying. A minimum pressure of 6 mbar or more when evaporating water is therefore essential.

With mixtures of ACN and water the problem is more complex ( Figure 3 ). Running at a steady 8 mbar may not freeze pure water, but at this pressure ACN boils at –25°C and this can freeze the water that is mixed with it in a typical HPLC fraction. The secret is therefore to choose a pressure at which ACN boils above 0°C, and use this pressure until the ACN is all gone, then switch to a suitable pressure for water (e.g. 8 mbar). To use this approach you need a system that can switch pressures at a predetermined part of the run.

Overcoming solvent interactions

Another issue frequently facing evaporation of Water / ACN HPLC fractions is the 5% or so of samples that take significantly longer to dry than the rest. The usual cause of this is an interaction between the compounds involved and the solvent (water) which results in the solvent boiling point being significantly elevated.

To overcome this problem the secret to drying these ‘ stubborn fractions’ is to follow the main run with a period at full vacuum. This will not cause freezing because by now the remaining water has a modified boiling point curve and cannot be made to boil as low as 0°C. Again, this requires a system that can automatically change pressure at a pre-set time during the run.

Continuous operation and condenser capacities

Figure4: The design of Genevac's continuous condenser minimises downtime

Figure 4: The design of Genevac’s continuous condenser minimises downtime

Finally, consider the job the system is doing, and look at the condenser (trap) capacity. If your system is running continuously then bear in mind that at some point most condensers need to be thawed out and drained (perhaps after every 1 or 2 runs, depending on total solvent load and trap capacity). Evidently this will have an impact on your overall throughput of HPLC fractions.

The “continuous” condenser design (Figure 4) (two condensers in one, which alternate between working and defrosting) operating on the Genevac Model HT-4 offers a novel solution to this problem.

Conclusion

Centrifugal evaporation represents an elegant technique to overcome the sample drying bottleneck in evaporating HPLC fractions in High Throughput Medicinal Chemistry and Product Purification laboratories.

When drying HPLC fractions that often can be mixtures of Water/Acetonitrile.

•Ensure precautions are taken to prevent bumping

•Protect your samples from overheating

• Don’t confuse capacity with throughput – compare actual run times

•Use a multi-stage pressure approach to prevent freezing and optimise runtimes

•Don’t forget to factor in condenser defrost times into your planning.

Learn more about evaporation>>

The HT Series evaporator>>

Genevac’s other centrifugal evaporators>>

Download this entire article as a pdf>>

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