# HPLC Purification of Peptides

Peptides are usually purified by preparative or semi-preparative HPLC. The factors such as gradient, flow rate, and such are determined by the size of the column, the sequence of the peptide and the resolution of the desired peptide from impurities. In order to achieve an efficient preparative scale purification, a development process is required in which optimizes gradient and flow rate for efficient separation.

## Solvents

The solvents typically utilized in HPLC of peptides are water (Solvent A) and acetonitrile (ACN) (Solvent B). These can be easily lyophilized to yield the isolated peptide. 0.1% trifluoroacetic acid (TFA) is added as an ion pairing reagent to improve peak width and peak symmetry of basic peptides and to maximize retention of acidic peptides.

Peptides typically are eluted from the HPLC column utilizing a linear gradient starting at a low percentage of organic solvent and ending with a higher percentage. Peptide gradients are usually shallow, with a rate of increase of 1% to 4% per minute in a 20 minute time span and a flow rate of 1.0 ml/min.

## Purification Process Development

Before scaling up to a preparative or semi-preparative column, the separation must be optimized on an analytical column with the same packing material as the larger column. The first step is to determine the solvent composition required to elute the peptide. This can be accomplished by injecting a sample of the peptide on the analytical column, eluting it with a gradient of 20% ACN to 90% ACN and observing the retention time and void volume time.

- Inject a sample of the peptide and elute it with a gradient of 20%B to 90%B in 20 minutes at a flow rate of 1.0 ml/min. Including a small air bubble in the sample will facilitate determining the void volume time.

- Measure the retention time (Tr) of the peptide and void volume time (Tv). The void volume time is the time needed for compound not retained by the column, such as a tiny air bubble, to travel through the system.

- Calculate the percent change rate of the gradient. If the gradient was 20%B to 90%B in twenty minutes, this would be (90% – 20%)/20 minutes, or 70%/20 minutes which is equal to 3.5%/minute.

- Calculate the solvent composition that elutes the peptide. Solvent composition = %B at gradient start +(Tr – Tv)*3.5%/min.

If the peptide elutes at the void volume, try a gradient of 10%B to 80%B or 5%B to 75%B.

Inject another sample and elute it with a 20 minute linear gradient starting with the starting solvent composition and ending with the solvent composition calculated above. The peptide should elute between 3 and 6 column-volumes.

## Transferring the Process to the Preparative or Semi-preparative Column

The first step in transferring the process to the larger column is to determine the void volume of the preparative HPLC system. This can be achieved by setting the flow rate of the prep system to 1.0 ml/min and injecting a blank sample containing an air bubble. The time it takes to elute the bubble corresponds to the void volume in milliliters (Void volume = elution time x flow rate [1 ml/min]).

The peptide’s retention time on the larger scale column can be estimated utilizing this formula:

Rt(prep) =(Vprep/Vanal)*Rt(anal)

Where Rt(prep) = Retention time of the peptide on the preparative column

Rt(anal) = Retion time of the peptide on the analytical column

Vprep = Void volume of the prep column

Vanal = Void volume of the analytical column

Alternatively, the flow rate through the prep column can be increased so that the retention times on the analytical and prep columns match. Flow rate(prep) =(Vprep/Vanal)*Flow rate(anal).

## Sample Volume

The volume of sample that can be injected onto the preparative or semi-preparative column can be scaled up from the analytical column. The formula below will provide a starting point for determining the sample volume that can loaded on the larger column.

Vp = Va * (Dp^{2}/Da^{2}) * (Lp/La)

Where Vp = Injection volume on the preparative column

Va = Injection volume on the analytical column

Dp = Diameter of the preparative column

Da = Diameter of the analytical column

Lp = Length of the preparative column

La = Length of the analytical column

This volume of sample solution on the preparative column should result in substantially the sample elution profile as was obtained with the analytical conditions on the analytical column. If the desired compound is well separated from impurities, the concentration of the sample solution and/or the volume of sample solution injected can be increased. As long as adequate separation is maintained the column can even be overloaded. This will allow the quantity of sample to be purified in less time and utilizing less solvent.

## Isolating the Purified Peptide

Fractions should be collected when the desired product starts to elute from the preparative column. These fractions should be tested by analytical HPLC and all fractions with sufficient purity can be combined and lyophilized. Purified fractions should be lyophilized as quickly as possible. As lyophilized powders, peptides can be stored in a freezer for lengthy periods, but peptides in solution can rapidly deteriorate.