The four main liquid chromatography (LC) modes and normal and reversed phase operating conditions and their respective mechanisms have been presented in previous posts.
After selection of the LC mode, the selection of the proper column (stationary phase, particle size, stability of the adsorbent) and operating parameters is the next step in the development of a separation.
Based on the outcome of the first trial chromatogram(s), a number of questions have to be answered:
- Is there sufficient resolution of the desired sample components? If the components of interest are sufficiently resolved for qualitative, quantitative analysis or sample collection (in preparative LC chromatography) the chromatographer’s work is almost complete.
- Is the analysis obtained in a reasonable length of time? If there is sufficient resolution of the peaks then the flow rate of the mobile phase may be increased, or the column may be shortened so that the time required for elution will be shortened. Techniques for increasing speed, such as gradient elution can be used.
- Does the sample size permits detection of the smallest desired peak or a sufficient quantity of the desired component(s) for collection? If not, a longer column and/or higher capacity may be required.
A general approach for column selection and optimization of operating parameters for better separations will be presented in this post. The main steps are the following:
Selection of the column and of the column packing
The selection of the proper column is a crucial step in the separation process. The choice of the packing is dictated by the requirements of the separation method. For example, in analytical separations optimum resolution is required while sample capacity is of less importance. In preparative separations column packing with large sample capacity and optimum resolution is required at the expense of speed.
An ideal column would give excellent resolution for all sample components in the shortest time and have the highest sample capacity required. However, since the three attributes of chromatographic separations are resolution, speed and column capacity the analyst must compromise. Generally, resolution is optimized by sacrificing speed and sample size.
The basic column parameters are given below:
Length: The column lengthis an important design parameter. Doubling the length doubles the retention time. However, resolution is only proportional to the square root of the column length.
Diameter: Small diameter columns are used to obtain better resolution and to improve speed. Typical HPLC columns have a diameter 1-6 mm.
Solid support – Column packing: Column packing especially for HPLC falls into one of two main categories – porous and pellicular porous.
Porous packing materials are either spherical or irregularly shaped, have deep pores, and are available in a wide range of particle sizes. As the diameter of the porous particle decreases, the depth of the pore also decreases. The resulting shorter diffusion path permits an increased rate of mass transfer so that column efficiency and resolution increase.
The pellicular porous packing materials provide an alternate way of decreasing diffusion paths. They consist of a solid, non-porous core and a thin, porous outer shell (Fig. 1). Rapid solute mass transfer occurs in the thin shell.
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| Fig. 1: Pellicular porous packing material. The solid core is non-porous while the thin outer shell is porous. |
Sample capacity of the column: The sample capacity of the column is directly related to the quantity of available stationary phase. In LSC, the capacity is proportional to the surface area of the adsorbent. In LLC, to the volume of liquid phase. In ion-exchange chromatography, to the number of ion exchange sites.
Porous particles have larger surface areas than non-porous ones. Columns of porous particles have high sample capacity. A typical porous LSC adsorbent has a surface area of 200-400 m2/g. It can handle samples in the range of mg/g of adsorbent without overloading.
Pellicular porous packing has a low surface area and therefore a low sample capacity. A typical pellicular porous support has a surface area of 7 m2/g.
The choice of the column packing – porous or pellicular – is dependent on the sample capacity requirement:
- If a sample contains six or more components and/or 0.5 mg or more of total sample, a porous packing should be used
- If a sample contains less than six components and /or less than 0.5 mg of total sample, a pellicular porous packing should be used.
In general, the smaller the particle size the faster the rate of mass transfer and therefore better efficiency. The minimum particle diameter which can be used is dependent on the length and internal diameter of the column and the pressure capability of the LC/HPLC pumping system.
In liquid chromatography the particle size used is normally > 150 μm while in HPLC <40 μm ( in practice 3-8 μm).
References
L.R. Snyder. J.J. Kirkland, “Introduction to Modern Liquid Chromatography”, 2nd edition, Wiley, 1979
A. Weston and P. Brown, “HPLC and CE Principles and Practice”, Academic Press, 1997
C.F. Poole, S.K. Poole, “Chromatography Today”, Elsevier, New York, 1991
Nina Hadden et al., “Basic Liquid Chromatography”, Varian Aerograph, 1971
Nina Hadden et al., “Basic Liquid Chromatography”, Varian Aerograph, 1971
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