Scale of Separations

The sample amount needed to be purified or separated for analysis will dictate which scale of chromatography is needed. In general, the scales of chromatography are microbore, analytical, semi-preparative, and preparative. Nanoscale and microscale are smaller scale ranges but are not covered in Hamilton’s product offerings. Columns with inside diameters of 1.0 mm to 2.1 mm can be classified as microbore, 4.0 mm to 4.6 mm as analytical, 7.0 mm to 10 mm as semi-preparative, and 21.2 mm and higher as preparative. Hamilton lists both microbore and analytical scale products as “analytical” products. Required sample loading needs to be considered for determining which scale to use. A good starting point is that a 4.1 x 150 mm HPLC column packed with 5 μm PS-DVB particles will typically be able to bind up to 10 mg of sample on a Hamilton column. With this in mind, a linear scale up or down can be calculated to determine which column size is needed for more or less sample. The scale of chromatography needed for a given application will determine the appropriate column hardware size and particle size.

It may be necessary to increase the size of a chromatography column in order to increase the production capacity of a process. When scaling a separation from microbore to analytical, or analytical to semipreparative or preparative scale, it is important to maintain linear flow rates. This ensures that gradients are accurately replicated with increased scale, thus maintaining the desired elution time of analytes. As the sample amount and flow rate are increased with increasing scale, the particle size of the stationary phase will also need to be increased to prevent over-pressuring the system.

Scale-Up Calculations

Column hardware scale-up based on sample loading:

(load₂)/(load₁) = [(radius₂)/(radius₁)]²

Linear scale-up flow rate calculation:
[volume flow₂/volume flow₁]= [load₂/load₁]

Why Choose PEEK or Stainless Steel (SS) Hardware

Hamilton offers both stainless steel and PEEK column hardware options. PEEK is typically used in cases where biocompatibility is a serious concern. Stainless steel hardware is unlikely to leach into samples, but PEEK can be used to further reduce the likelihood of leaching.

Column Length

Column length is one parameter that can be used to optimize a separation. Longer columns give better separation efficiency at the cost of time. Shorter columns can be used for faster separations with lower resolution. Depending on the complexity or number of analytes in the sample, a shorter column may be sufficient for a good separation.

Column Diameter

Column diameter can also be used to optimize a separation. Columns with a smaller diameter (inner diameter, or ID) should be chosen when sample amounts are limited. Since detection methods are sensitive to sample concentration, the amount of mobile phase needs to be reduced if a smaller amount of sample will be analyzed. Employing a smaller diameter column will give a stronger detector signal with limited sample amounts. Columns with a larger diameter are used to increase sample capacity or when more product yield is needed.

Particle Size

Smaller particles provide more efficient separations with sharper peaks but result in higher backpressure. Complex mixtures with many similar components should be separated with a high efficiency column with 5 μm particles or smaller. Simpler mixtures of structurally different sample components can be separated on columns with 7 μm or 10 μm or larger particles.