Basis knowledge about HPLC

[duynn2210]

When reducing inner diameter of LC column and reducing the velocity (linear with diameter), the retention time of the peak is remained constant, pressure is remained too. But the peak height, which will increase if the same amount of sample is injected while the band-spreading in the column will be reduced. I don't understand about this phenomenon. Can somebody help me?

[tom jupille]

First of all, if you keep the column length constant and change the mobile phase *velocity* (which has units of cm/min), the retention time *will* change.

If you want to keep the same velocity, you have to change the *flow rate* (which has units of mL/min) in proportion to the square of the diameter.

Decreasing the diameter of the column decreases its volume (in proportion to the square of the diameter). If you inject the same mass of sample into a smaller volume system, it will be more concentrated = a higher peak. This is, of course, an approximation that ignores things like extra-column volume, injection volume, etc.

[HPLCaddict]

In addition to Tom's answer, if you inject the same amount of sample on a smaller-diameter column using a scaled-down flow-rate, not only peak height will increase but also peak area (assuming you're using a concentration-dependend detecor like UV/VIS).

[lmh]

I'd guess the bit of this that's puzzling you is the question of the peak-width/band-spreading in the column being reduced.

I'm not 100% convinced about this. I'm guessing that what was meant by whoever said this, is that when the material reaches the top of the column (and when solvent reaches the top of the column) it has to be spread across the 4.6mm diameter of column (or whatever) from an inlet tube that has a diameter of only 0.2mm or so. Naturally material will arrive at the outside edges of the column later than the middle unless the inlet end of the column is very carefully designed, and this is also a piece of fluid path whose volume should be kept small. The same happens at the outlet. So if you had a very wide column, you could end up with different elution times for analyte going straight down the middle of the column, and analyte that chose the route down the outside.

On the other hand, to get the same linear flow (cm/min) down a wider column, you need a greater flow in uL/min. This means that unless you also change the HPLC tubing, you will have a greater linear flow in the post-column HPLC tubing, which will reduce band-spreading there.

So my personal feeling is that peak-widths are actually quite a complicated function of flow-rate, column-diameter, and post-column volumes in tubing and flow-cells, and I wouldn't like to make a general, global statement on how they'll behave.

[duynn2210]

Thank you for your information. I've understand about it.