Column efficiency:

DISTRIBUTION

As mentioned previously, compounds will spend some time in the stationary phase, and some time in the mobile phase. The time spent by an individual molecule in each of the two phases is called the capacity factor or retention factor k. The ratio of time spent in the two phases is equal to the ratio of the mass of the compound in the two phases.

k=

time spent in the stationary phase time spent in the mobile phase

= mass in the stationary phase mass in the mobile phase

The amount of time a molecule spends in one of the phases is directly related to the concentration of the compound in that phase. The ratio of the concentration of a compound in the two phases is commonly called the partition coefficient (K) which is a measure of a compounds affinity for the stationary phase. This value is sometimes called the partition ratio, distribution constant, or equilibrum constant.

K=

molar concentration in stationary phase molar concentration in mobile phase

The factors k and K are related to each other based on another parameter called the phase ratio.

volume of mobile phase Volume of stationary phase

and the relationship k and K is given as:

k = K/

This last equation simply shows that the mass ratio is a function of the concentration in the two phases and the relative volume of the two phases. The value of k which is based on mass is a better factor than K(which is based on concentration), because it is the mass of the compound that is moving through the column.

Movement through the Column The retention factor (k) is the ratio of the mass of a compound in the stationary and mobile phases. This is really the same as the ratio of the time the compound spends in the two phases. Put another way, the more mass of a compound in the mobile phase, the faster it will move through the column. The time it takes for a compound to travel through the column (from when an analyte is injected to when it reaches the detector) is known as the retention time(tr). If a compound is not retained at all, it will still take time to travel through the column. Therefore, in order to make a relationship between k and retention time, an adjustment must be made for this travel time. The time it takes for an unretained compound to travel through the column is often known as the dead time(to). Using the dead time, the retention factor for a compound can be related to the retention time.

k = (tr to)/ to
Where tr = the retention time of the compound, and to = the dead time

Selectivity The retention time of a compound is based on its k value. When more than one compound is injected into the column, they will travel through the column at different rates based on their k values. The ratio between the k values of two compounds is known as the selectivity factor and is an indication of how well the compounds will separate. Selectivity Factor :

= kB/kA
Where kB is the retention factor for the more strongly retained compound and kA is the retention factor for the less strongly held compound. (in other words, is always greater than or equal to 1).

Efficiency The selectivity factor is important to determining how compounds separate, but it is not enough by itself. Figure 2 shows portions of two different chromatograms where the selectivity value () is the same (the

a)

b)

peaks are the same distance apart), but in Fig 2a, the peaks are separated and in Fig. 2b the peaks are not separated.

Figure 2. Illustration of the separation of 2 pairs of peaks with the same value. a) peaks are fully separated because they are narrow. b) peaks are not separated because they are wide. The important difference between Fig. 2a and Fig. 2b is the width of the peaks. If peaks are too wide, they wont separate well. Therefore, we need a measure of peak width also known as efficiency. The term that is generally used to describe column efficiency is number of theoretical plates or N. The value of (N) is related to the column length (L) and the size or height (H) of an individual plate by the following equation:

N = L/H
Where L =column length, H = plate height (both in the same units)

Chromatography columns with high numbers of theoretical plates produce very narrow peaks resulting in better separation. The above equation shows that efficiency (N) can be increased by using longer columns, or using columns with small plate height The value for N is related to the width of a peak by the following formula:

N = 5.54 (tr/w1/2)2
Where tr = the retention time of the compound, And w1/2 is the peak width at half height

The values of tr and w1/2 are often both measured on the x axis of a chromatogram. They could both typically have units measured in minutes or actual distance measured with a ruler, in which case the units would be in cm or a similar unit. It is important that both are measured in the same units! The width of a chromatographic peak is typically measured at a point half way between the baseline and the top of the peak. This is because the peak broadens rapidly near the baseline and it is difficult to measure accurately (The fact that the peak width is measured at height instead of at the baseline, is accounted for by the constant 5.54 in the equation ). If the width at the baseline is measured the equation becomes:

N = 16(tr/w)2
Where tr = the retention time of the compound, And w is the peak width at the baseline.

Resolution The purpose of chromatography is to separate or resolve compounds. The separation or distance between two peaks is known as their resolution and is a function of the 3 factors discussed previously: retention (the time it takes for the analytes to elute, related to k), selectivity (how different the analytes are from each other and related to ), and efficiency (how good the column is, related to N). Therefore, these three factors can be brought together to describe resolution as follows:

Rs = ((-1)/) (k/k+1) N
There are a number of way to express the value of Rs. Another common equation is sometimes used to calculate Rs from actual measurements of peak retention times and measured peak widths. This equation is:

Rs = 2 (tR-B tR-A)/ wb-A + wb-B

Where: A and B are the two peaks , tR = retention time and wb = the peak width at the base of each peak

With a resolution value of 1.0, two peaks overlap by about 4%. Values less than 1.0 indicate peaks that overlap, while at a resolution of 1.5, the peaks are considered fully separated.