# Selection of Purifiation Equipment for Hydrophobic Interaction and Reversed Phase Chromatography

Appendix 1, extracted from Hydrophobic Interaction and Reversed Phase Chromatography Principles and Methods (PDF), GE Healthcare, 2014

A chromatography system is required in order to achieve a high-resolution separation using accurately controlled linear gradient elution, to take advantage of the high flow rates of modern media, or when the same column is to be used for many runs. The simplest HIC separations, such as elution by a step-gradient, can be performed using a syringe or peristaltic pump with prepacked HiTrap™ columns. ÄKTAdesign™ systems can be chosen to suit separation needs ranging from a simple step-gradient elution in the laboratory through to separations that must meet cGMP requirements. Ettan™ MDLC is a multidimensional liquid chromatography system for protein identification and characterization in proteomic research, ideally used in combination with an electrospray tandem mass spectrometer (ESI-MS/MS).

### Converting from linear flow (cm/hour) to volumetric flow rates (ml/min) and vice versa

It is convenient when comparing results for columns of different sizes to express flow as linear flow (cm/hour). However, flow is usually measured in volumetric flow rate (ml/min). To convert between linear flow and volumetric flow rate use one of the formulae below.

 Volumetric flow rate (ml/min)  = Linear flow (cm/h) 60 x column cross sectional area (cm2) = Y 60 x π x d2 4

where

Y = linear flow in cm/h

d = column inner diameter in cm

Example:

What is the volumetric flow rate in an XK 16/70 column (i.d. 1.6 cm) when the linear flow is 150 cm/hour?

Y = linear flow = 150 cm/h

d = inner diameter of the column = 1.6 cm

 Volumetric flow rate (ml/min)  = Linear flow (cm/h) 60 x column cross sectional area (cm2) = 150 x π x 1.6 x 1.6 60 x 4 x ml  min = 5.03 ml/min

From volumetric flow rate (ml/min) to linear flow (cm/hour)

 Linear flow (cm/h) = Volumetric flow rate (ml/min) x 60 column cross sectional area (cm2) = Z x 60 x 4  π x d2

Z = volumetric flow rate in ml/min

d = column inner diameter in cm

Example:

What is the linear flow in a Tricorn 5/50 column (i.d. 0.5 cm) when the volumetric flow rate is 1 ml/min?

Z = Volumetric flow rate = 1 ml/min

d = column inner diameter = 0.5 cm

 Linear flow = 1 x 60 x 4  π x 0.5 x 0.5 cm/h = 305.6 cm/h

1 ml/min = approximately 30 drops/min on a HiTrap™ 1 ml column

5 ml/min = approximately 120 drops/min on a HiTrap™ 5 ml column