Saturday, July 12, 2014

USP Guideline Note: HPLC Column Diameter Changes to Maintain Linear Velocity

USP Allowed Variations in HPLC Column Diameter (*USP 32, Second Supplement, Dec 1, 2009). In the previous USP version, a change of up to 50% of the flow rate was allowed. This has been changed in the more recent version. A wide range of column diameter changes are now allowed, provided that the linear velocity is kept constant. *We addressed the effect of changing column diameter on flow rate in a previous blog post, but this time I have also expanded on the calculation by including the extra variable for column length (L1 and L2) as well.

Linear Velocity Formula:

New Linear Flow Rate2 = Flow Rate1 x (L2 x D22) / (L1 x D12)

Flow Rates are in ml/min.
L1 = Column Length (original) in mm.
L2 = Column Length (proposed) in mm.
D1 = Column Diameter (original) in mm.
D2 = Column Diameter (proposed) in mm.

Example #1:
Original column is a 150mm x 4.6mm (L x ID) used at 1.000 ml min. We would like to determine what the equivalent flow rate (F2) would be for a column which is 150mm x 2.1mm (L x ID) to maintain the same linear velocity. This is a proposed change in column diameter of > 50% so it would not have been allowed under the old guidelines. The newer guidelines take into account that with the same particle size, changing the column diameter will not change the chromatography if the linear velocity is maintained as before. Let’s calculate the new flow rate using the formula above.
1.000 x (150 x 2.12) / (150 x 4.62) = F2
1 x (661.50 / 3,174) = F2
0.208 ml/min = F2