|
Figure 1 below shows the direction of physical optimization in quantitative HPLC:
It is NOT the largest plate number or the smallest “height of a theoretical plate” (HETP) as too often stated in the literature. A small HETP value only tells about the physical quality of a column packing and - if well packed - about the quality of the packing material itself if in addition the pressure drop in the column is as small as possible. For a minimum danger for producing wrong quantitative date we need a high separation power at best expressed by the Trennzahl. MOST important is to select such a chemical selectivity, that the most critical pair of to be separated substances in the analysis have such a wide peak distance, that there is baseline separation. Only NOW we can optimize the separation speed: it should be so high, that the baseline separation around the substances in question is acceptable, the mobile phase back pressure is no problem for the life time of the HPLC pump and the PCreal number is as high as possible. This brings together data quality and economy. Figure 1 below shows, that with a column as mentioned earlier the Trennzahl and the real peak capacity (as number = PCreal) does drop only slightly with increasing phase flow speed. The analytical data productivity however rises drastically and thus isocratic HPLC should run at much higher flow speeds than proposed by classical theoretical concepts.
Figure 1 below shows how strong the fundamental ABT data change with the phase flow speed. As ALL chromatography values are based on “a, b0 and tm” - with other words all separation quality numbers can be calculated with “a, b0 and tm” - we understand, how dynamically is chromatography. It is time dependent. The residence time in the mobile and stationary phase controls everything. The mobile phase flow speed must be very constant in isocratic HPLC and must be very well reproducible in programmed HPLC. But also the programming values must be either highly constant or very well reproducible.
The reason for these mentioned conditions is easy to understand:
The peak height of a given substance depends on the mobile phase flow speed. The peak position depends on the peak residence time values tm and ts. tm changes with the flow speed, ts with temperature, flow speed, elution power of the mobile phase and retention power of the stationary phase. The peak height is a measure of the grams substance per ml mobile phase. This value is non linearly correlated with the substance concentration in the sample. In order to end up with a correct analytical result like weight-%, mole-%, gr per liter etc. we need a substance specific correction factor found by non linear calibration. If anything in HPLC changes the peak position we would need a “corrected” correction factor, that means a new calibration. But because the quantitative calibration function is non linear, we also need a very well reproducible and constant sampling process.
HPLC users should understand one critical statement given at a large international HPLC symposium and not rejected by any of the top experts: “Analytical data measured by (instrumentalized) PLC are less prone to quantitative errors than HPLC or HPLC/MS or -MS/MS data. The closest technical and methodological cooperation of HPLC with PLC (HPTLC) helps to find and reduce possible serious analytical errors qualitatively and quantitatively.”
This is the reason why we treat all chromatography modes using a uniform and as much as possible equal theoretical concept. Therefore we had to leave the classical basics.
The “error detector sf4” works also in HPLC.
With respect to the physical separation power and trace detectability HPLC is far behind GC, thus a combined use of capillary GC together with HPLC is powerful. HPLC acts in this case as sample preparation method.
With respect to the chemical separation power by the use of mobile phase programming, differing stationary phases and multi phase separation capabilities, HPLC is reasonably far behind modern PLC / HPTLC, especially in its circular mode, thus a combined use of both is advisable.
With respect to the molecular weight range of applicability HPLC is unbeatable and its further development into packed capillary versions and into the electrical driven separation as well as in micro column switching techniques has a wide field of coming developments. We remind to a forgotten mode of physical polarity switching in micro HPLC: click “here” as it is working also with packed HPLC columns and offers best possibilities in trace analysis. Reason: not the mobile phase is programmed but the stationary phase effects are electronically changed by local temperature fields. There is no need to mechanically “move an oven”. One can also change the temperature of two or more series coupled columns locally by electronics and thus have physical polarity programming controlled by software.
These are main reasons to reject method regulation. The latter means “stop development” but development is not only important. It is vital in modern bio chemistry analysis.
|