Heating or Cooling
Ignoring temperature in 374 A
A N A LY T I C A L C H E M I S T R Y / J U LY 1 , 2 0 0 2
LC Columns
Tyge Greibrokk University of Oslo (Norway)
eparation scientists say that temperature plays a significant role in all chromatographic techniques. Although it is recognized as the most important variable in GC, many chromatographers have traditionally regarded temperature, or variations in temperature, as a mere nuisance in LC. This attitude may have been a shortsighted mistake. Because of the miniaturization of column dimensions, from capillary GC to capillary LC to electrochromatography on microchips, controlling temperature has recently become easier and more profitable. In this article, the importance of temperature in separations, particularly in LC and applications ranging from chiral drugs to polymers, will be discussed.
S
Too hot to handle? Whereas elevated temperatures increase solubility and diffusivity, they decrease viscosity. Retention, peak shape, column efficiency, and total analysis time are affected by temperature because both the thermodynamics and kinetics of adsorption processes are functions of temperature. Solvent strength, a major variable for controlling retention in LC, can be affected by temperature. Pressure (to a minor degree) and pH are other important variables. If controlling pH has become common practice in LC for separating basic and acidic analytes, why have so many chromatographers neglected to optimize the effects of temperature?
LC might be a mistake. J U LY 1 , 2 0 0 2 / A N A LY T I C A L C H E M I S T R Y
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Effects
Amitriptyline 10 8 6 4 2 0 –2 –4 –6 –8 pH
Temp
Temp*pH
Effects
Propranolol
Retention, selectivity, and peak shape Changes in the column temperature, often combined with changes in the solvent strength, may improve both peak shape and resolution. The solute retention is related to temperature through Equations 1 and 2:
6 4 2 0 –2 –4 –6 pH
Temp
Temp*pH
Effects
Amphetamine 6 4 2 0 –2 –4 –6 pH
Temp
Temp*pH
FIGURE 1. Retention effect plots of the temperature (10–90 °C), pH (2–7), and interaction between temperature and pH on an octadecylsilane column with 30–70% acetonitrile in water mobile phase. (Adapted with permission from Ref. 6.)
The answer is partly that LC is also a preparative technique, and radial temperature gradients make temperature control slower and more difficult to implement on large-size columns. Therefore, many chromatographers have been satisfied with room-temperature operations, even though ambient room temperature varies. With narrow-bore columns, radial temperature effects will be much smaller, even negligible (1, 2). However, with good temperature control of the incoming solvent, conventional, large-bore columns can be maintained at reasonably stable conditions. Column ovens equipped with Peltier elements, which allow cooling as well as heating, have become available, but only in the more expensive instruments. Chromatographers may also shy away from using elevated temperatures in LC for fear of reducing the long-term stability of stationary phases or creating a safety hazard if leaking organic solvents are heated past the flash point. Although some stationary phases are stable in aqueous solvents and won’t degrade with increasing temperature, overcoming traditional skepticism toward elevated temperatures will require further improvements in column stability. The safety aspect of using flammable solvents at elevated temperatures must be considered. A leak within the narrow dimen376 A
sions of a packed capillary column contained in a vented column oven is not expected to be a problem. As long as there are no leaks from connections inside the column oven, there is no danger of autoigniting the solvent. Although the risks are higher if a leak occurs in a standard-size column, the polymer industry has used high-temperature size-exclusion chromatography for decades without any accidents, to my knowledge. Furthermore, an oven equipped with a device that replaces air with carbon dioxide for rapid cool-down will limit risks.
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G° = –RT ln (k/) ln k = –H °/RT + S °/R + ln
(1) (2)
where G° is the Gibbs free energy, k is the retention factor, is the phase ratio, H ° is the enthalpy, and S ° is the entropy for phase transfer from the mobile to the stationary phase. Selectivity is not affected by temperature changes when a linear relationship exists between H and ln k; however with nonlinear relationships and in entropy-dominated situations, changes in temperature will affect selectivity (3). Several studies have directly compared the effects of changing the solvent composition and column temperature. After examining a series of homologous n-alkylbenzenes, Bowermaster and McNair found that a 1% increase in methanol concentration had approximately the same effect as a temperature increase of 4 °C (4). Chen and Horváth found a similar relationship (1%:5 °C) with alkylbenzenes in acetonitrile/water (5). Acenaphthene and diethylphthalate showed similar effects in our studies in reversed-phase systems. A 1% increase of organic solvent was comparable with a 4–5 °C increase in acetonitrile and a 3 °C increase in methanol. However, basic compounds, such as the secondary amine propranolol, demonstrate that a 2 °C increase in the column temperature had the same effect as a 1% increase in the acetonitrile concentration in an acetonitrile/ water solvent composition. Thus, some test substances, particularly the basic compounds, give different results (6). Figure 1 shows results from a multivariate experiment designed for the reversed-phase study of three basic compounds (amitriptyline, amphetamine, and propranolol), which demonstrate that temperature variations within the 10–90 °C range had approximately the same effect as pH variations within the pH 2–7 range. This means that the resolution of neutral and basic compounds can be changed by manipulating solvent strength and temperature, even though McCalley demonstrates that temperature effects with basic compounds are a function of pH and difficult to predict (7 ). Because solvent strength affects solute retention more than temperature, direct comparisons between solvent gradients and temperature programs are of little interest. The effects of solvent strength and temperature are often complementary (8). Partic-
ularly for larger molecules, such as peptides and proteins, simultaneous optimization of temperature and gradient steepness is a powerful and convenient means of controlling band spacing and separation (8). In general, entropy-dominated separations are expected to benefit more from variations in temperature than solvent strength in relation to changes in selectivity and resolution. However, asymmetric peak shapes of compounds with basic groups are occasionally obtained with silica-based columns, usually through secondary interactions with residual silanol groups and sometimes by slow kinetics. Figure 2 demonstrates how increased temperature can efficiently improve asymmetric peak shape.
Column efficiency Although open tubular columns work well with GC and supercritical fluid chromatography, they are not generally advantageous for LC. The higher compressibility of a gas compared with a liquid and differences in diffusivity and viscosity account for column performance (9). The viscosity of a liquid is decreased and the diffusivity is increased by elevated temperatures, resulting in improved mass transfer and increased column efficiency (10). At very high temperatures (~200 °C), the greatly reduced viscosity allows long 50-µm i.d. open tubular columns to have up to 1 million theoretical plates (11). At lower temperatures, however, the reduced diffusivity requires smaller internal diameters to achieve the same plate height, resulting in increased back pressures from increased viscosity. This is a practical hindrance to open tubular LC unless 5–10-µm i.d. columns are used at extremely high pressures. Consequently, elevated temperatures increase efficiency by allowing the use of longer columns and improving the shape of asymmetric peaks caused by slow kinetics. In a recent study, a peak capacity of 200 was obtained by a gradient run at room temperature. By increasing the temperature to 100 °C, the run time was reduced from 60 to 15 min, but the same peak capacity was maintained (12). These advantages were evident for both standard-size and narrow-bore packed columns; however, controlling the temperature in a narrow-bore column is easier because of its smaller thermal mass.
lectivity with overall intermolecular interactions (16). Some enantiomers that interconvert at room temperature require low temperatures to be separated. For example, chiral 1-naphthamides require temperatures as low as –60 °C for separation (17 ).
Instrumentation Instrumentation for narrow-bore columns is available from several major instrument manufacturers but needs to be improved. Ovens with temperature programming, from below room temperature to ~100 °C, are currently available from only one manufacturer. Chromatographers need ovens that can rapidly return to the start of a temperature program, and the Peltier elements currently used are too slow for adequate temperature reduction. Detectors must be compatible with narrow-bore columns. All the standard LC detectors (except the refractive index detector) are compatible with temperature programming (2). Occasionally, at high sensitivity, a baseline drift correction may be required, depending on the detector, flow cell, mobile phase,
Effects on shape recognition Temperatures below ambient can improve the selectivity of separations on the basis of molecular shape. For entropydominated retention processes, isomers with rigid, well-defined structures can be separated on the basis of differences in molecular shape, rather than physical or chemical properties. Shape recognition is usually increased at reduced temperature (13). The most significant temperature effects are observed for long alkyl chain and liquid crystalline stationary phases (14). Chiral separations are based on different interactions of enantiomers with a chiral selector. Chromatographic selectivity is usually increased by a decrease in temperature; however, notable exceptions, such as the drug thalidomide, show increased selectivity with increasing temperature (15). According to a recent thermodynamic study of chiral separations, it is still extremely difficult to predict or even correlate chiral se-
FIGURE 2. Peak shape and retention time improve when temperature increases from (a) 10 °C to (b) 90 °C at pH 2 with 70/30 acetonitrile/ water on an octadecylsilane column. Compounds 3 and 4 also reversed their order of elution. Peaks: 1, uracil; 2, butylparaben; 3, ibuprofen; 4, DL-leucine-DL-phenylalanine; 5, acenaphthene; 6, propranolol. (Adapted with permission from Ref. 6.) J U LY 1 , 2 0 0 2 / A N A LY T I C A L C H E M I S T R Y
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FIGURE 3. Separation of individual oligomers of a polymeric amine on a 0.32 mm 40 cm, 3-µm octadecylsilane packed column with 95/5 acetonitrile/triethylamine. Temperature program: 28–120 °C at 0.7 °C/min and evaporative light-scattering detection. (Adapted with permission from Ref. 20.)
and application. If temperatures exceed the boiling point of the mobile-phase components, the transfer line from the column or from the detector should be made of narrow-bore fused-silica tubing. The narrow tubing applies a small back pressure on the column outlet and keeps the solvents from vaporizing.
Applications Temperature-assisted LC or temperature-programmed LC is a supplement to isocratic and solvent gradient elution at room temperature. As narrow-bore columns become more and more common, this supplement is expected to become an integral part of development. Today, lower temperatures are used mainly for chiral separations, which are hampered by the lack of commercially available instrumentation. Reduced temperatures can also be used for large-volume sample introduction, with or without solvent focusing (18). Because elevated temperatures can potentially reduce analysis time and improve efficiency and detector compatibility, they are commonly used for many applications on reversed-phase and normal-phase systems. Temperature programming is a limited alternative to gradient elution and is particularly valuable when LC is used with MS and NMR. Separations of individual polymer oligomers with a MW of 10,000–20,000 (Figure 3) can be obtained using temperature programs and UV and lightscattering detection (19, 20). Larger polymers can be separated according to functional groups (21). In comprehensive LC with two coupled columns, temperature focusing can aid in transferring and reconcentrating each fraction from one column to another. This is a new and exciting option for proteomics research.
Limitations Columns used at higher temperatures should be selected with care, especially if they are going to be used in aqueous systems. Older, loosely packed, narrow-bore columns are not compatible with temperature variations and will collapse almost immediately. Today’s commercially available narrow-bore columns are more 378 A
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robust and can withstand temperature programming. Aqueous mobile phases present compatibility problems with some columns because either the packing structure is not stable or the stationary phase decomposes over time by hydrolytic cleavage. Cleavage has been much less of a problem in the past few years. Most manufacturers can supply the customer with columns that can withstand aqueous mobile phases at temperatures up to 70 °C. Above these temperatures, separations in aqueous solutions should be performed only on columns that the manufacturer recommends for such use. Future columns may be based on zirconia or other materials that are resistant to more aggressive conditions, such as high pH, but some silica-based materials are reported to be stable to ≥90 °C (22, 23). Applications in nonaqueous solutions present no problems as long as the packing structure and the analyte are stable. Tyge Greibrokk is a professor at the University of Oslo. His research interests include development of chromatographic techniques for miniaturized separation and detection systems. Address correspondence to Greibrokk at the University of Oslo, Department of Chemistry, P.O. Box 1033, 0315 Oslo, Norway or
[email protected].
References (1) (2) (3) (4) (5) (6) (7) (8) (9) (10) (11) (12) (13) (14) (15) (16) (17) (18) (19) (20) (21) (22) (23)
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