Properties of HPLC Columns
Modes of Separation
Normal-phase chromatography, or NP, is the classic form of liquid chromatography using polar stationary phases and non-polar mobile phases. The analyte is retained by the interaction of its polar functional groups with the polar groups on the surface of the packing. Analytes elute from the column starting with the least polar compound followed by other compounds in order of their increasing polarity. Normal-phase chromatography is useful in the separation of analytes with low to intermediate polarity and high solubility in low-polarity solvents. Water-soluble analytes are usually not good candidates for normal-phase chromatography.
Reversed-phase chromatography, or RP, has become the most common mode of liquid chromatographic separation. In RP the stationary phase is non-polar and the mobile phase is polar. The analytes are attracted to the surface by their non-polar functional groups. The most polar analyte elutes from the RP column first followed by other analytes in order of decreasing polarity. RP chromatography is useful for the separation of compounds having high to intermediate polarity.
Ion-exchange chromatography separates analytes by their ionic functionality. Ion-exchange stationary phases are usually comprised of ionic species attached to the surface of the silica substrate or ionic functional groups evenly distributed throughout a polymeric media. Weak ion-exchange phases are usually pH dependent. Strong ion-exchange phases are always charged and therefore independent of typical pH changes. Ion-exchange columns with low capacity are used for ion chromatographic applications where low ionic strength mobile phases are required for conductivity detection.
Affinity chromatography is based on specific interactions in a lock-and-key paradigm between analytes and matrix-bound ligands. Dependent on the secondary structures of biological macromolecultes for retention of selected sample components, affinitiy chromatography is without question the most highly specific, and consequently the most powerful, mode of chromatography.
Porous silica particles are the most common substrate material used for HPLC column packings. Silica-based columns can withstand high pressures, are compatible with most organic and aqueous mobile-phase solvents, and come in a wide range of bonded phases. Silica-based columns are often used for separations of low molecular weight analytes using mobile phase solvents and samples with a pH range of 2 to 7.5. Some new-generation silica materials, such as EVEREST™, DENALI™, and GENESIS™, have extended pH ranges.
Highly cross-linked styrene-divinylbenzene based packings are compatible with most mobile phase solvents and samples with a pH of 1 to 14. Polymer-based columns tend to have lower efficiencies for small molecules compared to silica-based columns due to their smaller average surface area. In addition, polymer-based columns typically have lower mechanical strength and therefore cannot withstand the highest system backpressures. A polymer-based packing is often a good alternative if the sample requires a mobile phase pH outside the normal operating range of standard silic-based columns. Polymeric packings are often used for ion-exchange separations, and are also useful in non-aqueous GPC size-exclusing analyses and ion exclusion analyses of organic acids and carbohydrates.
The first available HPLC columns were packed using irregularly shaped silica particles. Because of this, many standard analytical methods are still based on these materials. Irregular particles are also used in large- scale preparative applications because of their high surface area, capacity and low cost.
The majority of new HPLC methods are performed on spherical shaped or spheroidal (almost spherical) particles. Spherical particles provide higher efficiency, better column stability and lower back-pressures compared to irregularly shaped particles.
Particle size for HPLC column packings refers to the average diameter of the packing particles. Most HPLC packings contain a narrow range of particle diameters. Particle size affects the back-pressure of the column and the separation efficiency. Column back-pressure and column efficiency are inversely proportional to the square of the particle diameter. This means that as the particle size decreases, the column back-pressure and efficiency increase. A well packed column with 3 μm packings produces almost twice the separation efficiency of a comparable 5 μm column. However, the 3 μm column will have about a three-fold higher back-pressure compared to the 5 μm column when operated with the same mobile phase and at the same flow rate. Highly efficient, small-particle (3 μm and 4 μm) columns are ideal for complex mixtures with similar components. Fast, high-resolution separations can be achieved with small particles packed in short (10-50 mm length) columns. Grace Vydac offers SHORTFAST™ and LIGHTNING™ HPLC columns specifically for these fast-HPLC applications.
Larger particle (5 μm and 7 μm) columns are typically used for routine analyses where analytes have greater structural differences. Large 10 μm packings have only moderate column efficiencies. Columns packed with 10 μm packings are generally used as scout columns for future preparative separations, semi- preparative applications, or routine QA/QC methods where high chromatographic efficiencies are not required. Large particles (15- 20 μm) are used for preparative-scale separations.
The pore size of a packing material represents the average size of the pores within each particle. The size of the analyte should be considered when choosing the appropriate pore size for the packing material. The molecular weight of an analyte can be used to estimate the size of the molecule. As a general rule, a pore size of 100 Å or less should be used for analytes below 3,000 MW. A pore size of 100 Å -130 Å is recommended for samples in the range of 3,000 MW - 10,000 MW. For samples above10,000MW, including peptides and proteins, a 300 Å material provides the best efficiency and peak shape.
Pore volume is a measurement of the empty space within a particle. Pore volume is a good indicator of the mechanical strength of a packing. Particles with large pore volumes are typically weaker than particles with small pore volumes. Pore volumes of 1.0 mL/g or less are recommended for most HPLC separations. Pore volumes of greater than 1.0mL/g are preferred for size-exclusion chromatography and useful for low-pressure methods.
The physical structure of the particle substrate determines the surface area of the packing material. Surface area is determined by pore size. Pore size and surface area are inversely related. A packing material with a small pore size will have a large surface area, and vice versa. High surface area materials offer greater capacity and longer analyte retention times. Low surface area packings offer faster equilibration time and are often used for large molecular weight molecules.
The carbon load is a measure of the amount of bonded phase bound to the surface of the packing. High carbon loads provide greater column capacities and resolution. Low carbon loads produce less retentive packings and faster analysis times.
Surface coverage is calculated from the carbon load and surface area of a packing material. Surface coverage affects the retention, selectivity and stability of bonded phases.
A reversed-phase HPLC column that is end-capped has gone through a secondary bonding step to cover unreacted silanols on the silica surface. End-capped packing materials eliminate unpredictable secondary interactions. Basic analytes tend to produce asymmetric tailed peaks on non end-capped columns, requiring the addition of modifiers to the mobile phase. Non end-capped materials exhibit different selectivity than end-capped columns. This selectivity difference can enhance separations of polar analytes by controlling the secondary silanol interactions.
Theoretical Maximum Column Volumes, mL
||Column Internal Diameter, mm
Typical Flow Rates
|Column Dimensions ID,mm x Length, mm
||Flow Rate, mL/min
|22.5 x 250
|10.0 x 250
|4.6 x 250
|3.0 x 250
|2.1 x 100
|1.0 x 100
Column Length, Diameter and Volume
Once a packing material has been selected, the physical dimensions of the HPLC column hardware should also be optimized for the desired separation. It is important to understand the relationship between column length, diameter and volume. The column length (L) and internal diameter (d) determine the bed volume (V) of an HPLC column by the following equation: V = L x πd2/4 This equation does not account for the reduction in liquid volume due to the packing material. It is an upper limit on the column volume – the minimum volume of mobile phase required to elute an unretained analyte from the column. Small column diameters provide higher sensitivity than larger column diameters for the same injected mass because the concentration of the analyte in the mobile phase is greater. Smaller diameter columns also use less mobile phase per analysis because a slower flow rate is required to achieve the same linear velocity through the column. HPLC instrumentation may need to be modified for columns with very small internal diameters to eliminate band broadening due to extra-column effects, i.e., mixing volumes outside the column. Longer columns often provide increased resolution. Larger-diameter columns provide greater sample loading and lower back-pressure. Column back-pressure for a given flow rate increases as the column length increases and as internal diameter decreases.
Column Flow Rate
When the column dimensions are changed to optimize a separation, or to scale a separation to a preparative or narrow-bore application, the mobile phase flow rate should be adjusted proportionally to cross-sectional area of the column to maintain consistent linear velocity and retention times. The following equation will assist in adjusting the flow rate (F) for columns of different inside diameters (d). F2 = F1 x (d2/d1)2