Saturday, April 13, 2013

Using Smaller Diameter HPLC Columns (Calculate Linear Velocity)

Lots of 2.1mm ID chromatography columns are appearing on the market right now. Since most of us are using 4.6 mm ID columns to develop HPLC and UHPLC methods, use of these smaller ID columns requires a few adjustments be made to the method. If gradient elution is used, then the gradient profile often must be changed to compensate for changes in void volume of the column and dwell volume of the system. Injection volume must also be adjusted in a linear fashion too. Additionally, to maintain the same initial mobile phase linear velocity through the column as we had before (to obtain the same approximate retention times), the flow rate must also be adjusted. *We will discuss how to calculate the change in flow rate in this installment.

In order to reproduce your original method, we must first adjust the flow rate for the new, narrower bore column. The formula to do this is very simple. We decrease the flow rate by using the square of the ratios of the column diameters times the flow rate.

( C1  / C2 )2 x original flow rate (ml/min) = new flow rate (ml/min).

Where:  C=  Diameter (mm) of new (smaller) column;
C2 =   Diameter (mm) of original column.
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Example #1: Find the new linear flow rate if we use a 2.1 mm ID column in place of a 4.6mm column with an initial flow rate of 1.000 ml/min.

( 2.1 / 4.6 ) 2 x 1.000 = 0.208 (208 ul/min)

Example #2: Find the new linear flow rate if we use a 2.1 mm ID column in place of a 4.6mm column with an initial flow rate of 2.000 ml/min.

( 2.1 / 4.6 ) 2 x 2.000 = 0.416 (416 ul/min)

Example #3: Find the new linear flow rate if we use a 1.0 mm ID column in place of a 4.6mm column with an initial flow rate of 1.500 ml/min.

( 1.0 / 4.6 ) 2 x 1.500 = 0.071  (71 ul/min)

If we assume that the original flow rate is 1.000 ml/min then we can also use this table to get an idea of how the flow rate changes with decreasing column diameter.

Column I.D. (mm)                 Flow Rate (ul/min)
4.6                                          1,000
2.1                                             208
1.0                                               47
0.3                                                 4
0.15                                               1