HPLC analysis commonly uses Isocratic or Gradient elution. In Isocratic mode the mobile phase composition remains unchanged during the analysis run whereas in gradient elution the mobile phase composition changes as per defined program during the run.
In the previous article on off-line mixing of solvents was discussed. Further mixing to attain required mobile phase composition takes place in the HPLC system depending on choice of operation.
Isocratic methods commonly use mixtures of organic solvents with water. Either pure solvents are placed in mobile phase reservoirs or pre-mixed mixtures are delivered to the system using the required pumping arrangement.
Low pressure mixing
Mixing of mobile phase components takes place on the low-pressure side before entering the pump. Proportionating solenoid valves deliver the individual solvent to the mixing chamber and the mixture is led to the chromatographic column at a constant flow rate by the pump downstream.
Low-pressure systems are capable of delivering a combination of up to 4 different solvents.
High pressure mixing
In high-pressure mode individual pumps drive each solvent to the mixing chamber where mixing takes place under higher pressures and the mobile phase blend is led thereafter to the column.
Significance of Dwell Volume
Dwell volume is the physical volume from the point of mixing of solvents to the start of column. High-pressure systems have dwell volumes ranging from 1.5 – 3.0 ml as mixing takes place after the pumps whereas in low-pressure mixing systems mixing occurs before the pump so the dwell volume can be upto 4.0 ml as it includes the volume in mixing chamber and the tubing upto the pump.
During programming the final composition reaches the column after mixing as it has to cross that dwell volume. The delay is more in low-pressure mixing systems so retention times of early eluting peaks are longer than late eluting peaks. Such differences are insignificant in isocratic separations.
High-pressure versus low-pressure mixing
Both systems have their own advantages and it cannot be claimed that one is superior to the other. However, it is appropriate to list out a brief comparison of both the systems:
- Due to heat of mixing composition can vary on mixing but present day software provides correction features.
- Use of more than one pump in high-pressure pump means increased cost and greater maintenance.
- High-pressure systems are usually limited to 2 solvents
- High-pressure mixing provides precise control and reproducibility of mobile phase composition up to 0.1%
- Low-pressure mixing can be more useful in development work as operation up to quaternary systems is possible
- Outgassing of dissolved gases can cause more problems in low-pressure mixing so degassing of mobile phase is essential
- Outgassing of dissolved gases can cause problems in HPLC analysis and subsequent article on degassing of mobile phase will briefly discuss the different degassing techniques.
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