Basic Concepts of High-performance Liquid Chromatography

The two basic questions in high-performance liquid chromatography focus on (a) how particular compounds can be separated, and (b) why particular compounds were separated by the liquid chromatographic method used. The answers can be obtained by the consideration of some simple representative chromatograms of the separation of well-known compounds. Such separations can be easily understood according to common principles of physics and chemistry.

A separation is described by the following equation, which indicates the degree of resolution between two peaks in a chromatogram, Rs. A complete separation requires Rs > 1.2 units.

Rs = 1/4(α - 1/α)[square root of N]

The resolution can be improved by increasing the column plate number, N, and/ or the separation factor, α(α = the ratio of the retention factors of the two compounds). N is the physical parameter and α is the chemical parameter for the separation. Higher N and α values give a better separation.

The physical and chemical aspects of liquid chromatography, in addition to mechanical aspects, are briefly described in this chapter. Theoretical approaches are explained in detail in later chapters. The effect of stationary phase materials on the chemical selectivity is described in Chapter 3, and the influence of the eluent components is covered in Chapter 4. The plate number theory is discussed in Chapter 5. Quantitative optimization is explained in Chapter 6.

1 Physical Parameters for High-speed Separations

It was thought that high-speed separations would be achieved by the development of a physically stable pumping system and highly sensitive detectors; however, the main contribution to high-speed separation is made by small-size stationary phase materials. A shorter separation time with complete resolution cannot be achieved simply by increasing the flow rate or by using a small column. The theoretical plate number of a small column must be the same as that of a larger column to obtain the same separation.

For example, the separation of a mixture of benzene, acetophenone, toluene, and naphthalene has been completed within 5.5 min using a 15 cm long, 4.6 mm i.d. column, packed with 10 µm porous octadecyl-bonded silica gel, whose theoretical plate number was 38 000 m-1, as shown in Figure 1.1 A. Increasing the flow rate 4-fold reduced the separation time to 1.5 min, because this mixture was well separated (Figure 1.1B). The same mixture was separated within 4.5 min using a 10 cm long, 4.6 mm i.d. column packed with 3 µm octadecyl-bonded porous silica gel with a theoretical plate number of 117 000 m-1 (Figure 1.1C). Doubling the flow rate resulted in completion of the separation within 2 min, as shown (Figure 1.1D).

Comparison of these four chromatograms suggests that a fast separation can be performed either using a longer column with 10 µm stationary phase material with a high flow rate of the eluent, to give high resolution, or by a smaller column packed with 3 µm stationary phase material. However, a high flow rate through the 3 µm stationary phase material is limited by a high column back pressure. The separation could also be completed within 1.2 min on the short column packed with 3 µm stationary phase material by using a stronger eluent, as shown in Figure 1.2. Furthermore, the sensitivity was also improved by using the smaller-size stationary phase material because the sample is less spread out in the eluent and is more concentrated when it reaches the detector. The actual peak height in Figure 1.1C is 1.6 times that in Figure 1.1 A. A small column packed with small particle-size stationary phase materials promises high performance and a high-speed separation both in theory and in practice. The following equation describes the relationship of the column length (L) to the column efficiency: N = L/H. The high plate number N required for good separation is proportional to the longer column length L and small H value. The term H is the height equivalent to a theoretical plate (HETP), which is the length of column needed to generate one theoretical plate. A good column has a high plate number for its length, and, thus, a good column has a low H value. The value of H can also be described by the following equation (which is described in detail in Chapter 5):

[MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII]

This equation indicates that the particle size, dp, is the main contributor to the H value. The smaller the particles, the higher the theoretical plate number. The optimum condition is obtained by the relationship between the theoretical plate height and the flow velocity.

2 Physical Considerations

High-speed separations can be achieved with a short column packed with 3 µm stationary phase material, as shown in Figure 1.2. The sensitivity was also improved by the use of smaller-size stationary phase materials, due to less sample diffusion inside the column. The following conditions are required to obtain such a separation.

a. small-diameter, spherical stationary phase materials that have high physical strength;

b. a high pressure pump with controlled flow rate;

c. a system that limits sample diffusion, by considering the column design, using small inner diameter connecting tubing, and a small volume detector flow cell; and

d. a detector and recorder capable of a high-speed response.

The theoretical plate number N of peak B can be calculated from the chromatogram given in Figure 1.3 by the following equation:

N = 16(VR/w)2

where VR is the retention volume and w is the peak width at the base (measured in volume units). However, the retention volume includes the hold-up volume VM (also called dead volume). The hold-up volume is the sum of the void volume of the column (V0 = YA), the volume of the injector (OX) and the volume of the detector and connecting tubing (XY) as shown in Figure 1.3. The actual separation efficiency is defined as the effective theoretical plate number Neff, which excludes the hold-up volume:

Neff = 16(VR - VM/w)2

Commercial instruments have a reasonable balance between the recommended column size and the volume of the column and connecting tubing (XY). However, the theoretical plate number of a single column may give different values on different instruments, and even on replacement of the components and parts of a single instrument. Such discrepancies can be understood in terms of differences in the mechanics of the instruments and the design of their parts.

The normally acceptable extra-column dead volume (OY in Figure 1.3) before there is a significant effect on the...