Preparative HPLC (Purification)

When we have a crude mixture (not a purified compound), most of the time we need to isolate a single compound out of it. HPLC can be used for this purpose because its chromatography column can separate the mixture into components.

Purification steps

1. Method Selection

  1. First we take an aliquot (a small sample) from the mixture and inject it into the LCMS instrument.

  2. We run a QC (analysis) of the mixture and identify when the component-of-interest elutes. Say it was 1.2-1.25 min.

It’s possible though that our first separation method won’t give us good resolution, so we may need to try multiple method - that’s our Method Selection.

2. Purification

  1. Now we can inject the whole mixture (or split it into smaller parts if there’s a risk of column overload), but now instead of passing it to the detector we direct the analytes to a vial around 1.2-1.25 min.

  2. Now we have a vial with our analyte together with the HPLC solvent(s).

  3. (Optional) We can run another QC - inject an aliquot of the resulting mixture to check the purification step.

The part of the mixture that we send to the vial is called a Fraction, and we use a Fraction Collector to gather the fraction from the injections.

3. Drydown and reconstitution

  1. Now it’s time to dry out the HPLC solvent. In order for the analyte to turn into a salt, we could add either an acid or a base depending on its pKa.

  2. After we got the powder, we weigh it and calculate the yield of the reaction. Note, that because it’s a salt and not a pure compound, the weighing doesn’t translate into the number of moles unless we somehow incorporate the salt count (the number of counter-ions to our analyte). Because we don’t always know the salt count, the amount of the substance is often approximated.

  3. Optionally, we could dissolve it back - now in the solvent that we need and the concentration that we need. This will also allow us to run a final QC - to check that the whole procedure succeeded.

QC & Prep columns

During Method Selection we inject just a small aliquot. This means we can run it on a different, smaller column than what we need for the actual prep step.

For this to work we first need to run a study/calculations to compare the 2 columns - so that we could translate the Retention Time of one to the other.

Focused Gradients

The actual Purification is usually run to isolate a single analyte out of mixture. So if the whole QC run takes 5 minutes, and we actually need only 1.2-1.4 min where the target analyte is eluted, the rest of the time is spent for nothing.

Since gradient elution is a common technique in such experiments, we can use it to speed things up. E.g. if QC shows that the compound is eluted when running at 50% of Solvent A, then during the Prep phase we can ramp up to 50% quickly to get our compound of interest. After the fraction is collected, we ramp up Solvent B to flush the rest of the compounds out of the column.

For this we need to map the regions of the QC chromatogram to different HPLC methods programmed to specific gradients. See more details on how you can do this with Peaksel.

Peaksel capabilities

Peaksel is an analysis software, so it can’t run the instruments directly. But in batch-level visualizations you can generate sample lists based on the QC data.

Using the calculated columns you can do Method Selection, export a CSV report and open it in Excel or acquisition software for fine-tuning.

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