|Advantages of using microcolumns in HPLC|
Packed capillary columns have a smaller inner diameter (ex: 0.32 mm) than conventional HPLC columns (ex: 4.6 mm).
Hence, the volumes of the columns are different.
Volume of an "empty" column: V = pi × r2 × L
By comparing a packed capillary column and a conventional column, and assuming equal lenght (L) and stationary phase, the volumetric relationship between the two columns can be expressed:
Vconv / Vcap = (pi × rconv2 × L) / (p × rcap2 × L) = rconv2 / rcap2 = dconv2 / dcap2 = (dconv / dcap)2
where dconv = 4.6 mm og dcap = 0.32 mm
-> dconv2 / dcap2 = (4,6 mm / 0,32 mm)2 = 207
Hence, the conventional column has a volume that is 207 times larger than the packed capillary column.
As a consequense, the mobile phase flow in the packed capillery LC system must be 207 times lower than in the conventional LC system, in order to obtain the same retention time and efficiency. Hence, the mobile phase consumption is drastically reduced from typically 1 ml/min in the conventional system to typically 5 µL/min in the miniaturized system.
With regard to coupling of LC to detectors based on mobile phase nebulization/elimination, the low flow of miniaturized LC systems is benefical. A typical example is the coupling of conventional LC to the mass spectrometer requires flow splitting that gives sample loss and reduced sensitivity.
When injecting an identic absolute amount of sample (ex: 10 ng) in both the capillary and the conventional system, the peak is expected to be 207 times larger in the miniaturized system. This is due to the smaller volume of the packed capillary column, consequently giving less dilution of the chromatographic band and a more concentrated peak volume. Hence, increased mass sensitivity is obtained.
However, the injection volume of the miniaturized system (typically 0.05 µL) should be 207 times lower than in the conventional system (typically 10 µL) to obtain identical chromatography. In practice, this means that the peak height and peak area will be equal when injecting the same sample solution on both systems.
But, it is important to notice the fact that large sample volumes can be injected on packed capillary columns, too. The analyte can be enriched at the column inlet by dissolving the analyte in a solvent or a solvent mixture with non-eluting properties, allowing large volume injection without efficiency loss. It is of course possible to do this on the conventional column too, but then the injection volume must be 207 times larger to obtain the same peak height.
Hence, using miniaturized LC is very advantageous when separating compounds of low concentrations in limited sample volumes such as many biological samples.