We’ve been looking for the last few weeks at some of the
calculations that are at the foundation of measuring the performance of
an HPLC run. This week the topic is chromatographic selectivity.
Selectivity is the ability of an HPLC method to separate two analytes
from each other.Selectivity usually is abbreviated with the Greek letter
α, and is calculated as: α = k2 / k1
where k1 and k2 are the retention factors, k, of the first and second peaks of a peak pair. The calculation of α is shown in Figure 1. (Refer to HPLC Solutions, Issue #28 for measurement of k.) For the two peaks with k-values of 1.95 and 2.15, α = 1.10. Although this chromatogram looks like a good separation, with a little baseline space between the two peaks, α is a poor way to determine the quality of the chromatogram. The reason for this is that it does not take the peak width into account. It is easy to imagine that if the two peaks of Figure 1 were twice as wide, the valley between the two peaks would not reach baseline. This would be a much poorer separation, yet the α-value would be unchanged. This means that we need a way to measure the width of the peaks, as will be covered in next week’s discussion.
What Influences Selectivity?
We might wonder of what use α is, if it doesn’t give us a measure of chromatographic quality. In general, if α ≥ 1.1, we should be able to get baseline separation for a good quality column, but there are better ways to measure the separation, as we’ll see in later discussions. For the moment, let’s look at some of the things that can be used to change α – that is, how can we move peaks around relative to each other?
Selectivity is changed when we change the chemistry of the chromatographic system. Changes in the chemistry of the system influence how a sample solute interacts with the stationary phase and mobile phase. If two compounds interact with the column or mobile phase in a sufficiently different manner, we can separate them – if they interact in the same manner, a single peak results. Some of the more important variables that affect selectivity are the solvent strength and type, the temperature of the column, the buffer and other additives, and the type of column packing. Let’s look briefly at the way each of these variables influences reversed-phase separation under isocratic (constant solvent-strength) conditions.
By solvent strength, usually we are referring to the ratio of the aqueous and organic components of the mobile phase. Stronger solvents are those that elute compounds more quickly so that retention is smaller. Thus, the less acetonitrile (ACN) or methanol (MeOH) in the mobile phase, the larger will be the retention times and longer the run. This will also cause peaks to broaden, and because area is constant, to be shorter. Weaker mobile phases also tend to improve the separation, but this is not true in every case.
The solvent type also affects the peak spacing. For mobile phases of equal strength, that is ones that give the same average retention times, peak spacing usually will differ with different type solvents. The three most common organic solvents used for reversed-phase HPLC are acetonitrile, methanol, and less commonly tetrahydrofuran (THF). Although changing from one of these solvents to another is almost guaranteed to change the α-value, at least for some of the peaks in a chromatogram, there is no way of knowing in advance if a specific change will improve or worsen a separation.
Column temperature works in a similar manner to mobile-phase strength in that higher column temperatures reduce retention times and lower temperatures decrease them. Selectivity often changes with a change in column temperature, but it is not possible in advance to predict whether the separation of one pair of peaks will improve or get worse. The changes in separation with a change in temperature often are different than those when solvent type or solvent strength is changed.
The mobile-phase pH is an important variable when ionic or ionizable compounds are present. Ionized analytes tend to have smaller retention times than non-ionized ones or ones for which ionization is suppressed. Buffer strength, or molarity, does not have a major effect in most reversed-phase separations, but if too little buffer is present, peak tailing can be worse. Other additives, such as ion-pairing reagents, also can affect the separation of two peaks under the proper circumstances.
Finally, the column packing type can influence the peak spacing in a chromatogram. There are numerous stationary phases available, including C18, C8, C4, cyano, phenyl, amino, embedded polar phases, and fluoro phases. Each of these has different chemical characteristics, so a change in column type is likely to change peak spacing for at least some peaks in a chromatogram. Even a c aahange from one brand to another within a type of packing can change chromatographic selectivity, as we saw for C18 columns in the very first installment of HPLC Solutions (HPLC Solutions, Issue #1).
So What?
We have seen above that there are many different ways to change the relative peak spacing in a chromatogram. Often the challenge is how to pick which variable to choose when you desire to make such a change. In contrast, with so many ways to change selectivity, we should also be aware that this means that there are many ways to ruin a satisfactory separation by unintentionally changing a variable if we aren’t careful to control the chromatographic conditions. The susceptibility of a chromatogram to changes in conditions gives us myriad topics for future discussions.
Read more at:
where k1 and k2 are the retention factors, k, of the first and second peaks of a peak pair. The calculation of α is shown in Figure 1. (Refer to HPLC Solutions, Issue #28 for measurement of k.) For the two peaks with k-values of 1.95 and 2.15, α = 1.10. Although this chromatogram looks like a good separation, with a little baseline space between the two peaks, α is a poor way to determine the quality of the chromatogram. The reason for this is that it does not take the peak width into account. It is easy to imagine that if the two peaks of Figure 1 were twice as wide, the valley between the two peaks would not reach baseline. This would be a much poorer separation, yet the α-value would be unchanged. This means that we need a way to measure the width of the peaks, as will be covered in next week’s discussion.
What Influences Selectivity?
We might wonder of what use α is, if it doesn’t give us a measure of chromatographic quality. In general, if α ≥ 1.1, we should be able to get baseline separation for a good quality column, but there are better ways to measure the separation, as we’ll see in later discussions. For the moment, let’s look at some of the things that can be used to change α – that is, how can we move peaks around relative to each other?
Selectivity is changed when we change the chemistry of the chromatographic system. Changes in the chemistry of the system influence how a sample solute interacts with the stationary phase and mobile phase. If two compounds interact with the column or mobile phase in a sufficiently different manner, we can separate them – if they interact in the same manner, a single peak results. Some of the more important variables that affect selectivity are the solvent strength and type, the temperature of the column, the buffer and other additives, and the type of column packing. Let’s look briefly at the way each of these variables influences reversed-phase separation under isocratic (constant solvent-strength) conditions.
By solvent strength, usually we are referring to the ratio of the aqueous and organic components of the mobile phase. Stronger solvents are those that elute compounds more quickly so that retention is smaller. Thus, the less acetonitrile (ACN) or methanol (MeOH) in the mobile phase, the larger will be the retention times and longer the run. This will also cause peaks to broaden, and because area is constant, to be shorter. Weaker mobile phases also tend to improve the separation, but this is not true in every case.
The solvent type also affects the peak spacing. For mobile phases of equal strength, that is ones that give the same average retention times, peak spacing usually will differ with different type solvents. The three most common organic solvents used for reversed-phase HPLC are acetonitrile, methanol, and less commonly tetrahydrofuran (THF). Although changing from one of these solvents to another is almost guaranteed to change the α-value, at least for some of the peaks in a chromatogram, there is no way of knowing in advance if a specific change will improve or worsen a separation.
Column temperature works in a similar manner to mobile-phase strength in that higher column temperatures reduce retention times and lower temperatures decrease them. Selectivity often changes with a change in column temperature, but it is not possible in advance to predict whether the separation of one pair of peaks will improve or get worse. The changes in separation with a change in temperature often are different than those when solvent type or solvent strength is changed.
The mobile-phase pH is an important variable when ionic or ionizable compounds are present. Ionized analytes tend to have smaller retention times than non-ionized ones or ones for which ionization is suppressed. Buffer strength, or molarity, does not have a major effect in most reversed-phase separations, but if too little buffer is present, peak tailing can be worse. Other additives, such as ion-pairing reagents, also can affect the separation of two peaks under the proper circumstances.
Finally, the column packing type can influence the peak spacing in a chromatogram. There are numerous stationary phases available, including C18, C8, C4, cyano, phenyl, amino, embedded polar phases, and fluoro phases. Each of these has different chemical characteristics, so a change in column type is likely to change peak spacing for at least some peaks in a chromatogram. Even a c aahange from one brand to another within a type of packing can change chromatographic selectivity, as we saw for C18 columns in the very first installment of HPLC Solutions (HPLC Solutions, Issue #1).
So What?
We have seen above that there are many different ways to change the relative peak spacing in a chromatogram. Often the challenge is how to pick which variable to choose when you desire to make such a change. In contrast, with so many ways to change selectivity, we should also be aware that this means that there are many ways to ruin a satisfactory separation by unintentionally changing a variable if we aren’t careful to control the chromatographic conditions. The susceptibility of a chromatogram to changes in conditions gives us myriad topics for future discussions.
Read more at:
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