Figure 3.4 illustrates the most important measurements. Measure the variables in seconds or in millimeters. Many of our optimization parameters are ratios, so the units cancel out.
You can make simple measurements from a chromatogram to discover how well the columns are performing. The data most often collected are shown in this illustrative chromatogram above, and are further discussed in the text.
Figure 3.4 Typical Chromatogram Measurements.
It's not possible to measure the actual peak width at the baseline because the peak gradually fades away. Instead, draw tangent lines along the flat sides of the peak and extend the baseline across the width of the peak. Chromatographers call this procedure triangulating the peak.
Some chromatographers prefer to measure the width of the peak at half its height. This is often easier to do than triangulating the base width, and perhaps more accurate, since no triangulation errors occur. The two width measurements are related, and either of them can be used to evaluate column performance.
Make the following measurements:
Measure the holdup time (tM) from the injection time mark to the apex of the air peak (you can triangulate the air peak if you wish).
Measure the retention time (tR) of each component peak from the injection time mark to the intersection of its tangent lines.
Measure the base width (wb) of each component peak between the intersections of the tangent lines with the extended baseline.
Alternatively, measure the width at half height (w0.5) of each component peak.
For clarity of display, Figure 3.4 shows a single wide peak. You may have many peaks, and most likely they will be narrower than that. It's difficult to measure the width of a narrow peak. To obtain a good measurement, you may have to expand the time base on a computer display, or increase the chart speed on a recorder.
Typical calculations
These few measurements are enough to evaluate the performance of a single column. By way of example, let's calculate the plate number (N) for the peak in Figure 3.4. The math is not difficult:
(3.3)
Inserting the data from Figure 3.4 gives:
A plate number of 576 is very low and would indicate a very inefficient column, since most columns generate about 2000 plates per meter. Of course, this peak is intentionally drawn wide so the measurements are clearly seen. A real peak would be much narrower than this: a more typical peak might have one third of the width and nine times the plate number.
The alternative equation using the peak width at half height (w0.5) is:
(3.4)
Chromatographers calculate plate number as a way to evaluate the performance of a column. Any change of operating parameters that increases plate number automatically improves the separating power of a column.
Another performance indicator is the plate height (H), which is the length of a column required to generate one plate. It's usually reported in mm. Knowing the column length (L), you can easily calculate the plate height:
(3.5)
Plate height gives a measure of column performance that is independent of the column length and is the primary variable used for optimizing performance.
Knowledge Gained
It's obviously true to say that peaks come out of the column at different times.
It's not true to say that molecules move at different speeds inside the column.
For all injected molecules, there are only two speeds possible inside the column; stop or go.
When molecules are in the liquid phase, they cannot move along the column.
When molecules are in the gas phase, they move at full carrier gas speed.
The “air peak” doesn't dissolve in the liquid phase so it travels at the same speed as the carrier gas.
It's impossible for any peak to travel faster than the carrier gas.
No peak from the same injection can appear on the chromatogram earlier than the air peak.
To get through the column, all injected molecules must travel for the same time as the air peak does.
If the column had no liquid in it, all the peaks would elute together with the air peak.
Any additional retention time beyond the air peak time is the time a peak stopped in the liquid phase.
Separation is not caused by motion, it's due to peaks stopping for different times in the liquid.
The time that a peak stops in the liquid phase is directly proportional to its solubility in that liquid.
The spacing of peaks on the chromatogram is different than their spacing inside the column.
Peaks that migrate slowly along the column come out very much later on the chromatogram.
Retention time is the sum of time traveling in the gas phase and time stopped in the liquid phase.
Holdup time is the time in the gas phase; adjusted retention time is the time in the liquid phase.
Column operating performance can be evaluated from chromatogram measurements.
Chromatogram measurements may be made in seconds or millimeters.
You can measure the holdup time, peak retention time, and peak width on the chromatogram.
When measuring base width, triangulate the peak and extend the baseline under the peak.
Alternatively, measure the peak width at half the peak height.
Plate number is calculated from measurements of retention time and peak width.
Any change in operating conditions that increases plate number increases column separating power.
Did you get it?
Self‐assessment quiz: SAQ 03
These questions relate to gas‐liquid chromatography:
1 Q1. At what speed do component molecules move along the column when they are in the gas phase?
2 Q2. At what speed do component molecules move along the column when they are in the liquid phase?
3 Q3. What causes the separation between components?
4 Q4.