System suitability

Let us discuss first column compatibility with available HPLC system.

The key limitations are the detector flow cell volume and injection volume. Pump parameters are usually bring no limitations unless you are planning to do either micro-column separations or ultra-fast analyses.

What kind of limitation injection volume could bring?

Let us discuss what is going to happen with the mixture of two analytes (A, and B) injected on the 150x4.6 mm 5 m particle column (further I will call this type column "conventional"). Injection was 200 ml and let say that component A is almost nonretained, but component B is highly retained. Flow rate is 1 ml/min.

This type of the column usually shows Vo (void volume) around 1.7 ml

Component A will "fly" trough the column and will come out of the column into the detector with the same or higher peak with as it was injected. Let calculate an apparent efficiency for that peak.

where w is the peak width. Let assume nonrealistic ideal case when column did not add any additional broadening to the injected volume, so w=0.2 ml, VR=Vo=1.7 ml, and N will be equal 1156 theoretical plates. As you can see that despite our assumption of no column broadening affect (which means infinite column efficiency) we got extremely poor system efficiency. There is a graph on the left where we had shown how the apparent efficiency changes with the volume injected.

What will than happened to the component B, which is highly retained? When you inject your mixture at the flow rate of 1 ml/min it will take 12 seconds to load your sample (200 ml) on the column. Component B tend to stick on the adsorbent surface since it is strongly retained, and it will be accumulated on the very top of the column. That component will actually experience, so called, adsorption compression, and will go through the column as relatively narrow zone. Its peak width will correspond to the actual efficiency of the column.

This means, that the injection of the large volumes will significantly decrease the efficiency for the early eluting components, and if you want to develop a fast separation method, you have to consider small injection volumes.

If you compare three chromatograms of the same mixture made on the columns of 16,000 theoretical plates (TP), 10,000 TP, and 4,000 TP, you will not notice any significant difference for the first two columns, but the chromatogram on the third column will irritate you eyes. Let now look at the graph above, the efficiency exceeds 10,000 at the injection volume of 30 ml or less. It is better to inject less than 25 ml to avoid any noticeable contribution of the injection volume in the apparent efficiency.

Detector flow cell volume plays the important role also. If the geometry of the flow cell is not optimized to diminish chromatographic band dilution you may get significant additional band broadening. But even if it has ideal shape and did not show any band broadening you may experience a significant quantification error if it is too big.

Schematically it could be explained by the following. Quantification in HPLC usually has done by integrating the peak area, since this measure less dependant on the chromatographic condition than peak height. Peak area is the sum of the areas of peak slices. It is obvious that the more slices we have on the peak the more accurate our peak area will be calculated. Ideal chromatographic peak has Gaussian peak shape, and for accurate area measurements we have to have more than 15 "independent" slices ("area counts"). Each cell volume represents the single independent measurement on the peak. If, for example, we have 10,000 TP column with Vo=1.7 ml, and our flow cell has 20 ml volume we can easy calculate the number of our independent measurements on the less retained peak, VR=2 ml (eluted close to void volume).

   where w = 80 ml for given column. So we will have only 4 independent measurements for quantification. This won't be seen on the plotted chromatogram the same way as it is shown in green area since peak passing through the cell gradually. But the recorded and integrated peak shape will be different from what it should be (red line).

Flow-cell volume should be at least 100 times less than the column dead volume. In general it is better to work with the flow-cell of 10 ml or less.

Cell volume and shape have some additional requirements they are briefly discussed in the Instrumentation chapter.

Slide 3