Introduction :
During the analytical method transfers, from one site to other site and from one HPLC to other HPLC, some times there observes retention times change, lack of Resolution and gradient separations.
These challenges are due to the different instrument platforms which intern affected by the Dwell volumes.
Lets understand what are different Volume terms in Analytical in relation to HPLC.
Dwell volume :
Dwell volume is also known as gradient delay volume. This can be defines as below
It is the volume of liquid in the system, from the point where the gradient is formed to the inlet of the column.
It is the volume of liquid
in the system from the point where the gradient is formed to the inlet of the column
Different instrument makes have different dwell volumes
When there is a difference of Dwell volumes between the HPLC, there rises the difference in programmed gradient vs Actual gradient composition.
Now lets understand the pumps of HPLC. There are 2 types of pumps in HPLC
- Binary pumps
- Quaternary pumps
Binary Pumps ( High pressure Mixing) :
In this type of binary pump HPLC , mixing occurs after the pumps where 2 solvents can be blended.
Lets see the challenges that can be rise in this case binary pumps during method development or transfer
When the HPLC required to deliver 0.2mL flow with gradient , at some point the gradient will be 90:10. This indicates that the pump B is asked to deliver 0.02mL/min which would be very tough to deliver accurately.
Quaternary pumps (Low pressure mixing) :
Here there will be 2 - 4 solvents which are blended before they reach the pump hence this is called low pressure mixing.
How to measure the Dwell volume :
Dwell volume measurement is most important during the method transfers where the methods have critical pairs for resolution criteria.
Following the below simple steps make the measurement of dwell volume easy
- the column should be removed and placed with a capillary tubing of diameter 1m approximately of 0.125mm diameter (This dimensions will ensure the check valves work properly)
- Fix HPLC grade water as Mobile phase -A and 0.1% spiked acetone in HPLC water.
- Set the detector wavelength as 265nm.
- Use the gradient that runs for 10-30 mins at 1-2 ml/min. (2mL/min with 20 mins gradient is a good choice)
- The gradient will appear as below
6. You can print the chromatogram after that draw a best-fit line through the rising baseline. In a similar manner, extend the initial baseline until it intersects the diagonal. This is the dwell time, tD.
7. Alternatively, perform the measurement on the computer monitor. Determine the difference in signal (offset) between the initial (0% B) and final (100% B) baselines; divide this by two to find the signal corresponding to 50% B and locate this point on the curve.
8. Find the corresponding "retention" time, tR; this should occur halfway through the gradient, so subtract half this value (tG/2) and the remaining "retention" time will be equal to tD.
9.For example, if a 0–100% B gradient in run in 20 min at 2 mL/min, the midpoint should be reached at 10 min into the gradient. If the midpoint of the gradient is measured as 11.2 min, then tD = 11.2 – 20/2 = 1.2 min. Convert this to the dwell volume, VD = 1.2 min × 2 mL/min = 2.4 mL.
Dead Volume :
It is the volume of HPLC instrument between the injection point to the detection point excluding the detector
Column Volume :
Since the HPLC columns are in cylindrical shape the formula of Pi x radius (square) x length can be used.
Note : Diameter of the column/2 = radius.
Radius X radius = Radius (square)
cm cube = 1mL
Example :
a 150x4.6mm column has the column volume shown as below
pixr2xl
= 3.14 X 0.23cm X0.23cm x 15.0 cm
= 2.49 cm3
~ 2.5 mL