BIOMARK002.49 - How important is your flash column’s plate count, aka efficiency, to your purification

Bob Bickler

How important is your flash column’s plate count, aka efficiency, to your purification?

October 29, 2019 at 5:00 PM / by Bob Bickler

Plate count is a theoretical number describing the separation efficiency of a chromatography column. In short, it is a measure an eluting compound's bandwidth at the time it elutes from a column, Equation 1.

                                              N = 16 (tr/Wb)2                                         Equation 1

Where: N is the column plate count

             tr is the compound’s peak apex time

             Wb is the peak width at baseline

This value, in tandem with the column’s selectivity (a measure of separation) for a pair of compounds, provides chemists with a resolution value. So, in theory, as resolution increases, so does loading capacity and/or fraction purity.

For analytical chemists, plate count, and therefore resolution, is king. The better the resolution, the better the detection sensitivity and quantitation.

In preparative chromatography, plate count’s role in a separation of compounds is diminished. The reason for this is that higher efficiency columns, typically those packed with smaller diameter media, overload more quickly than columns packed with larger particles.

Equation 2 shows that loading capacity (Mi) is inversely proportional to the column’s plate count (n) so that as column efficiency increases, loading capacity decreases. I know, this seems to counter what was stated earlier, and it is.

                                       Mi = Am*(1.9*r2*L)*(1+k)/n1/2[1]                  Equation 2

Where: Mi is the mass injected

             Am is a constant

             r is the column radius

             L is the column bed length

             k is the capacity factor (compound retention value)

             n is the column plate count

Figure 1 graphically shows this relationship with a plot of predicted loading capacity vs. particle size. As mentioned earlier, smaller particles generate higher plate counts.

 

Load vs plates

Figure 1. Impact of particle size on loading capacity shows that smaller particles (higher efficiency) columns have reduced benefit in a preparative mode.

This phenomenon is one of the reasons preparative chromatography has historically been performed using larger particles, the other reason being lower pressure.

That being said, there is a role for plate count in flash chromatography. Since most of us use flash chromatography for intermediate purification, yield is the priority, with 80+% product purity as a secondary goal. By using slightly smaller particles, say 20 µm vs. 50 µm, the separation performance remains high, even with slightly higher loads and does not decline at the same rate as smaller particles, e.g. <20 µm, Figure 2.

 

Load vs particle size-1

Figure 2. Sample load impact on separation efficiency (plate count) shows marginal performance loss with 20-40 µm media compared to typical 32-63 µm (nominal 50 µm) media.

So what should you use to purify your crude mixture? That depends on your purification goals and the separation difficulty but certainly, a 20-40 µm media will generate a good separation at high loads.

[1] www.Chromatography-online.com, Preparative Chromatography - The Loading Capacity of a Column

Interested in learning more about flash chromatography? If so, please download our white paper, Successful Flash Chromatography.

Download the White Paper Here

Topics: Optimization, Media

Bob Bickler

Written by Bob Bickler