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Chapter 16 Supercritical Fluid Chromatography
Chapter 16
Supercritical fluid chromatography Mary Ellen P. McNally
16.1
INTRODUCTION
What is a supercritical fluid? The discovery of supercritical fluids occurred in 1879, when Thomas Andrews actually described the supercritical state and used the term critical point. A supercritical fluid is a material above its critical point. It is not a gas, or a liquid, although it is sometimes referred to as a dense gas. It is a separate state of matter defined as all matter by both its temperature and pressure. Designation of common states in liquids, solids and gases, assume standard pressure and temperature conditions, or STP, which is atmospheric pressure and 01C. Supercritical fluids generally exist at conditions above atmospheric pressure and at an elevated temperature. Figure 16.1 shows the typical phase diagram for carbon dioxide, the most commonly used supercritical fluid [1].
Critical point p Liquid Solid Gas 1 atm
T
Fig. 16.1. Phase diagram for carbon dioxide critical temperature 31.31C critical pressure 72.9 atm. Comprehensive Analytical Chemistry 47 S. Ahuja and N. Jespersen (Eds) Volume 47 ISSN: 0166-526X DOI: 10.1016/S0166-526X(06)47016-1 r 2006 Elsevier B.V. All rights reserved.
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M.E.P. McNally TABLE 16.1 Comparison of physical properties of liquids, gases and supercritical fluids Phase type
Density (g/cm3)
Diffusion (cm2/s)
Viscosity (g/cm s)
Gas at 1 atm, 211C Supercritical fluid Liquid
10–3 0.3–0.8 1
101 103–104 o 105
104 104–103 102
Source: Van Wassen et al. [2]
The critical point of a material is the temperature and pressure conditions at which the liqiud state ceases to exist. As a liquid is heated, it becomes less dense and starts to form a vapor phase. The vapors being formed becomes more dense, with continued heating the liquid and vapor densities become closer to each other until the critical temperature point is reached. At this same point, the liquid-line or phase boundary disappears. This critical point was first discovered and reported in 1822 by Baron Charles Cagniard de la Tour. As a fluid, the supercritical state generally exhibits properities that are intermediate to the properties of either a gas or a liqiud. In Table 16.1, the physical properties of liquids, gases and supercritical fluids are compared [2]. Examination of the values in Table 16.1 makes the intermediate nature of a supercritical fluid more obvious. The density of a supercritical fluid approaches the levels of a liquid as does its diffusivity, while its viscosity is similar to a typical gas. These properties offer rapid movement (equilibration) as in a gas, but solvation or solubilization as found in a liquid. The best of both worlds from a chromatographic viewpoint. This is because as a solute travels through a chromatographic column the number of equilibration points it reaches, as defined by the Van Dempter equation, defines how effective the separation will be, either through the number of theoretical plates, N, the resolution, Rs, or the alpha value, a, also known as a separation factor. For a detailed description of the theory of chromatography, the reader is refered to Snyder and Kirkland’s text Modern Practice of Liquid Chromatography [3] or Chapters 12, 14, 15 of this text. The ease of solubilizing the analyte is a key factor also. This is because the easier it is to get the solute to these potential equilibration points inside the chromatographic column when the fluid is moving with high diffusivity, the faster the equilibrations or separation can take place. Compared to a gas where the solubilities of the analytes of 562
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interest is almost non-existent, the liquid-like density of a supercritical fluid offers enhanced solubilization. In supercritical fluid chromatography, fluids above their critical point are used as mobile phases. This chapter discusses the principles of operation, mobile phase considerations, parameters that can be adjusted in method development as well as an overview of instrumentation required and a few pertinent examples from current literature. Not everything can be illustrated, but the advantages of this diverse technology will be highlighted.
16.2
HISTORY OF SFC
In terms of chromatography, the first individual credited with the use of supercritical fluids as the mobile phase is Ernst Klesper when in 1962 he reported on the separation of metal porphyrins using dense-gas chromatography (GC) or SFC [3]. But it was not until the 1980s that the analytical community took hold of the abilities and advantages of the technique with the advent of several commercial instrumentation ventures. Two approaches were taken during this time, one by gas chromatographers and the other by liquid chromatographers. Practicing gas chromatographers who experimented with supercritical fluid chromatography for its enhanced solvating powers pursued the first approach. They coupled supercritical fluids, in general pure carbon dioxide; with small narrow bore capillary columns, similar to leading GC columns of the time. With that coupling they were able to get enhanced resolution of compounds that are too difficult to analyze by GC, because they were not soluble in the nitrogen and helium mobile phases commonly used in GC. The second approach was taken by practicing liquid chromatographers. They routinely dealt with thermally labile, highly polar molecules and frequently sacrificed resolution, and speed in their separations because of the aqueous mobile phases that were required. With the enhanced diffusion and decreased viscosity of supercritical fluids over liquids, chromatographic run-time and resolution could be improved when supercritical fluids were used. But solubility in pure carbon dioxide mobile phases, which has the solvating powers from hexane to methylene chloride under normal density ranges, was a problem for these polar molecules. To compensate for this, experimentalists started working with mixed mobile phases. These mixed phases were based on 563
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the addition of polar modifiers or additives to the carbon dioxide mobile phase. Similar to the first approach, this second group of scientists continued to use the columns with similar dimensions to liquid chromatography (LC) columns prevalent in the 1980s that they were already using, i.e., 25 cm by 4.6 mm; the columns were also functionalized with the common C-18, C-8 and phenyl phases as found in LC. With both these approaches rapidly being pursued by a wide variety of academic, industrial and instrument company laboratories, the growth and publication rate in supercritical fluid chromatography during the mid-late 1980s and the early 1990s was exponential. A wide variety of applications are available from that time period as well as several references that explain the advantages of optimizing a supercritical fluid separation using all the parameters available in SFC compared to GC or LC [4–8].
16.3
BASIC PRINCIPLES IN SFC
The most common and widely used supercritical fluid in SFC is carbon dioxide. It is inert, in that it is non-toxic and non-flammable, it also has mild critical parameters, a low critical temperature of 31.31C and a critical pressure of 72.8 atm [1]. Using pure, supercritical carbon dioxide eliminates organic solvent waste and with it waste disposal costs and concerns. This is extremely practical advantage in the industrial environment where the generation of waste requires special handling and significant cost. Beyond this practical consideration, the advantages of SFC from a technical perspective are in the wider range of parameters that can be optimized to achieve the best separation [4]. Taken from the same two approaches described above, the parameter that is most commonly adjusted to change resolution and retention time in GC is the temperature, after that the only choice an experimentalist might have is to change the column used for separation. However, from the liquid chromatographers perspective, the mobile phase composition is the principal component that can be changed to effect a better separation, the mobile phase components are generally the second choice and then finally the choice of chromatographic column. Using supercritical fluid chromatography gives all the parameters optimized in LC as well as the ability to see drastic retention changes due to a temperature gradient, as in GC. In addition, SFC has the added parameters of pressure and/or density that can be selected to achieve the best separation conditions. 564
Supercritical fluid chromatography
From an overall perspective, both the temperature and pressure of the mobile phase control the density or elution power. A change in either temperature or pressure changes the mobile phase density and will alter the chromatographic elution. It should be noted that increasing the temperature in SFC increases the retention time, generally an undesirable effect and the opposite effect that is exhibited in GC. Because of this, reverse temperature gradients, from a high temperature to a low temperature, are utilized in SFC to decrease retention. With decreasing temperature, density of the fluid increases; these higher density mobile phases solubilize the analytes of interest and results in earlier elution of the compounds of interest. The solvating ability of carbon dioxide in SFC is significant when compared to gases used in GC. This is because as a pure component, a change in density of carbon dioxide causes a change in the material’s Hildebrand solubility parameter. In carbon dioxide, this range of solubilities can be considered equivalent to the solubilities seen from hexane to methylene chloride. However, in terms of solvating moderately polar or highly polar molecules, this solubility parameter range is not sufficient and modifiers or additives to carbon dioxide must be included. Once a modifier has been added to the mobile phase, the critical parameters of the original solvent are no longer valid and a two-phase system can exist. As an example, a 10% methanol in carbon dioxide solution has a critical temperature of 51.51C with a critical pressure of 74.2 atm. The critical parameters of pure methanol are 240.51C, temperature, and 78.9 atm, pressure. From these values it can easily be determined that there is no linear relationship for critical point change when the components of a two-phase system are mixed together. What should be noted though is that above the critical point of a mixture, the mobile phase is one phase. This is extremely important in chromatography, an equilibrium-based process, where by definition the movement of the analyte is between two phases. If one of the phases is a not a pure component, equilibrium is difficult to reproduce precisely, the analyte is not transferring back and forth between the stationary phase and the mobile phase, but instead between three phases, the stationary phase and a two-component mobile phase. This discussion emphasizes that, from a practical standpoint, one of the key considerations, often neglected by initial practitioners, is the need to maintain the mobile phase above its critical point at all times during the chromatographic separation. This may be difficult with the wide range of parameters that are adjusted to optimize a separation. 565
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Parameter optimization
Being able to change the density, via either changes in pressure or temperature, is the key difference in SFC over GC and LC separations. Typical density ranges are from 0.3 to 0.8 g/ml for pure carbon dioxide. Table 16.2 shows data obtained from ISCO’s SF-Solver Program for the calculation of density (g/ml), Hildebrand Solubility Parameter and a relative equivalent solvent for pure carbon dioxide at a constant pressure of 6000 psi, approximately 408 atm. But the real advantage in SFC is not just the ability to adjust the density of the mobile phase but the ability to adjust it from two different directions. Density changes achieved by a change in pressure can yield different separation factors then density changes achieved by adjusting the temperature. This offers an advantage in method development by SFC not available in GC or LC. Beyond the density changes that can be used to control method modifications in SFC, the mobile phase composition can also be adjusted. Typical LC solvents are the first choice, most likely because of their availability, but also because of their compatibility with analytical detectors. The most common mobile phase modifiers, which have been used, are methanol, acetonitrile and tetrahydrofuran (THF). Additives, defined as solutes added to the mobile phase in addition to the modifier to counteract any specific analyte–column interactions, are frequently included also to overcome the low polarity of the carbon dioxide mobile phase. Amines are among the most common additives. TABLE 16.2 Calculation of density, Hildebrand solubility parameter using ISCO’s SF solver program at constant pressure of 6000 psi (408 atm) Temperature (1C)
Density (g/mL)
Hildebrand solubility
Equivalent solvent
40 49 55 64 70 73 79 85 94
0.967 0.937 0.917 0.886 0.865 0.855 0.834 0.815 0.785
8.248 7.902 7.814 7.554 7.375 7.287 7.113 6.944 6.694
C6H12 C2H4 CF4 C8H16 C6H16 O2 C4H10 C8H18 F2
566
Cyclohexane Ethylene Carbon tetrafluoride n-Octane n-Hexane Oxygen n-Butane 2-Trimethyl pentane Fluorine
Supercritical fluid chromatography
Modifiers and gradient compositions in LC are used broadly. A gradient that runs with 30–80% methanol or acetonitrile is not uncommon. This amount of modifier is generally not needed in supercritical fluid chromatography to affect the same separation. Typical modifier composition in SFC is 1.0–10% and would achieve higher Hildebrand Solubility Parameter adjustment overall than the broader gradients found in LC. 16.3.2
Instrument requirements
The major difference in supercritical fluid chromatography and conventional LC equipment is the pumping systems as well as the safety features installed to maintain higher pressure. Unique SFC equipment differences are: 1. 2. 3. 4. 5. 6.
Carbon dioxide tank for mobile phase supply a. Equipped with a pressure relief value and rupture disk High-pressure pump a. Chiller to maintain mobile phase in liquid state High-speed injector Pressure restrictor a. High-pressure tubing High-pressure flow cell for UV detection Solvent collection device with ability to vent to a laboratory hood or elephant trunk.
Carbon dioxide is usually purchased in a tank, inside the tank the mobile phase exists as a liquid. Typically, the tank does not come with a pressure gauge but is hooked up to a pressure relief valve and rupture disk, which are set above the tank pressure should a tank leak occur. High-pressure pumps used in SFC can come in a variety of types; most of them are modifications of pumps used in LC. Piston, diaphragm and syringe pumps are used. The pumps deliver the most accurate flow of the carbon dioxide if it is pumped in the liquid state. Since that is the case, if there is a great distance between the tank and the pump then a chiller is usually placed so that the tubing containing the carbon dioxide from the tank can be cooled maintaining the carbon dioxide in the liquid state. Most commercially available pumps operate at high enough pressures that the pump heads or pump bodies do not need ancillary cooling, but older models frequently required a chiller that provided a circulation of cold 567
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fluids to the parts of the pump where the carbon dioxide has a potential to vaporize out of the liquid state. A high-speed injector is required in supercritical fluid chromatography. This is to prevent loss of pressure during the injection process. There are a variety of restrictor types that control the pressure of the fluid during the chromatographic process. Each manufacturer has patented their individual restriction devices. It is beyond the scope of this text to go into the detail about all of these restrictors. However, by way of explanation, most of the mechanical restrictors that are used in commercial equipments operate under the principle of decreasing a volume or a space that the mobile phase must pass through by some mechanical means. This decrease in volume, if metered accurately, increases the pressure in the system in a controlled manner. As a safety precaution, for any equipment where elevated pressures are used, tubing should be rated with a safety factor of at least 1.5 times the maximum pressure the SFC system can achieve. Because the density is a controlling factor in achieving the separation, maintaining the elevated pressure used in the separation is necessary up to the point of detection. For UV measurements, this requires a high-pressure cell also rated to the maximum pressure the SFC system can achieve with a desired safety factor of 1.5 times. For other detection methods, i.e., flame ionization detection and mass spectrometry, where the sample is nebulized into the detector, the output of the restrictor is generally right at the nebulization point. This positioning of the column outlet eliminates any peak merging that could occur under low- or no- pressure movement. 16.4
CURRENT EXAMPLES
The examples illustrated herein are not all-inclusive but should give a representation of the advantages of the technique and its most common uses. 16.4.1
Chiral separations
The use of supercritical fluids to separate enantiomers is one of the most important tasks in several areas of research, especially pharmaceuticals and agrochemicals. This is because it is well known that the two enantiomeric forms of a molecule can display dramatically different biological activity. The use of supercritical fluids to separate, with higher efficiencies and shorter retention times, enantiomers is 568
Supercritical fluid chromatography
extremely advantageous in this difficult work. In their article entitled ‘‘Chiral separation of some triazole pesticides by supercritical fluid chromatography, Toribio and coworkers, separated six triazole pesticides and showed the effects of different organic modifiers on the resolution and retention via k0 value [9]. The modifiers they examined were methanol, ethanol and 2-propanol. The addition of additives was also examined. Table 16.3 shows some of their modifier results. In general, separations could be conducted in less than 10 min and the best modifier for the individual separations was compound dependent. This TABLE 16.3 Values of capacity factors and resolutions obtained for diniconazole, tetraconazole, hexaconazole and tebuconazole using different modifiers [9] Compound Hexaconazole Methanol (%, v/v) 5 10 15 Ethanol (%, v/v) 5 10 15 2-Propanol (%, v/v) 5 10 15 Tetraconazole Methanol (%, v/v) 5 10 15 Ethanol (%, v/v) 5 10 15 2-Propanol (%, v/v) 5 10 15
k10
k20
Rs
5.35 5.68 0.67 2.33 2.59 1.02 1.43 1.61 0.87 5.14 6.23 2.41 2.01 2.28 1.14 1.19 1.32 0.65 4.38 5.01 0.54 2.71 2.90 0.48 1.45 1.45 0
4.30 5.23 1.55 2.05 2.41 1.27 1.06 1.83 1.02 4.26 5.29 3.05 2.24 2.84 2.49 1.68 1.98 2.06 5.86 8.75 4.98 3.28 5.46 4.01 2.40 3.69 3.43
Compound
k10
k20
Rs
Tebuconazole Methanol (%, v/v) 5 13.23 14.74 1.28 10 5.26 5.82 1.17 15 3.11 3.42 1.03 Ethanol (%, v/v) 5 7.16 7.16 0 10 5.04 5.04 0 15 2.69 2.69 0 2-Propanol (%, v/v) 5 20.36 22.53 0.98 10 7.61 8.35 1.2 15 3.54 3.91 1.15 Diniconazole Methanol (%, v/v) 5 7.29 12.44 6.08 10 2.81 5.69 6.90 15 1.63 3.43 5.87 Ethanol (%, v/v) 5 6.97 10.63 4.55 10 2.53 3.87 3.77 15 1.37 2.08 3.11 2-Propanol (%, v/v) 5 12.46 21.06 1.23 10 4.09 4.09 0 15 2.04 2.04 0
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is consistent with the understanding of modifier interactions with the solutes and their effect on the overall equilibrium between the stationary and mobile phases in SFC as previously described. Recalling, acceptable resolution, Rs, values for quantitation purposes are greater than 1.5, values that are greater than 1.5 for resolution are highlighted in Table 16.3. Figure 16.2 shows an example chromatogram of a typical separation. These separations were conducted on a Hewlett-Packard 1205A supercritical fluid chromatograph equipped with a photodiode array detector; detection was at 220 nm. A Chiralpak AD 25 cm 4.6 mm column packed with the 3, 5-dimethylphenylcarbamate derivative of amylose, coated on a 10 mm silica support was used. The mobile phase was carbon dioxide modified as outlined in Table 16.3 for each of the individual separations. Other chromatographic conditions were 351C, 2 ml/min flow rate and a pressure of 200 bar (2960 atm) (Fig. 16.3). (Note: Because of the low temperature of the separations, the mobile phase was not likely in the supercritical state for all of the modifier concentrations examined in this report.) 16.4.2
Polymer separations
mAU
The advantages of supercritical fluid chromatography for polymer separations have been illustrated in the literature for many years. A recent example is the separation of long-chain polyprenols using SFC with matrix-assisted laser-desorption ionization TOF mass spectrometry [10]. The generic name for 1,4-polyprenyl alcohols is polyprenol; these compounds generally have smaller polymerization chains of less (A)
200 150 100 50 0
mAU
0
2
4
6 Min.
8
10
2
4
6 Min.
8
10
(B)
200 150 100 50 0 0
Fig. 16.2. Enatiomeric separation of hexaconazole at 200 bar, 351C, 2 ml/min and 10 v/v 2-propanol. (A) Without using additives; (B) using 0.1% (v/v) triethylamine and 0.1% (v/v) trifluoroacetic acid [9].
570
Supercritical fluid chromatography
Fig. 16.3. Enatiomeric separation at 200 bar, 351C, 2 ml/min. (A) Tetraconazole with 4%(v/v) ethanol; (B) diniconazole 15% (v/v) ethanol [9].
than 30 monomers. Chromatographic separation of these chains was conducted using a Jasco Super-201 chromatograph. The system has two separate pumps, one delivers the carbon dioxide, and the other delivered the THF modifier. The column temperature was 801C, the pressure, controlled by a backpressure regulator was controlled at 19.6 MPa. An Inertsil Ph-3 (25 cm 4.6 mm), 5 mm column was used. A flow gradient was used to introduce the THF modifier, this is not typical, but it is possible with the two pumps on this instrument. The consistent flow rate of the carbon dioxide is 3.0 ml/min; the THF flow rate started at 0.8 ml/min and was adjusted over 30 min- to 2.0 mL/min and then held constant. Figure 16.4 illustrates the resultant chromatogram obtained for Eucimmia ulmoides leaves, a plant producing fibrous rubber; over 100-mer (MW: 6818) components were completely separated using the conditions described above.
16.4.3
High throughput screening of pharmaceuticals
At Abbott Laboratories, Hochlowski and coworkers used preparatory scale SFC to screen the output of high throughput organic synthesis (HTOS) for purity levels and classify chemical structures into libraries of similar analogs [11]. These scientists used a Berger Instruments SFC self-modified for this particular use. Figure 16.5 shows the schematic of this modified system. In this application, the authors reported adding SFC capability to their repertoire of instrumentation to solve their analytical challenges. Figure. 16.6 shows the comparison of a typical reaction mixture analyzed by both high performance liquid chromatography (HPLC) and SFC. 571
M.E.P. McNally 38
100 20
0
10
20
30
40
50
Min
Fig. 16.4. SFC chromatogram of long-chain polyprenols in E. ulmoides leaves. The sample had Mn 3.99 103 (calibrated against cis-1,4-polyisoprene standards) and Mw/Mn 1.41. The numbers in the chromatogram represent degrees of polymerization for polyprenol homologues [10].
Autosampler
CO2 delivery system
Custom software
Methanol wash system
Dual arm fraction collector
Fig. 16.5. Preparative supercritical fluid chromatographic system, customized for high throughput organic synthesis (HTOS) screening [11].
Actual operating conditions can be found in the figure caption. A given limitation of SFC, relative to HPLC, as described is the ability to dissolve samples in a solvent system compatible with the methanol/ carbon dioxide mobile phase. For this particular mobile phase, other compatible sample diluents that worked effectively are pure methanol, 572
Supercritical fluid chromatography 1000
1
mAU
4
2
750
3
500 250 0 0
3
2
1
(a)
5 4 Minutes
6
7
7
8
8
9
10
2
1100 mAU
8250 6500
4
1
3
2750 0 0
1
(b)
2
3
4 5 Minutes
6
Fig. 16.6. (a) HPLC chromatograms for reaction mixture A. HPLC conditions: C18 (20 100 mm Nova–Pak TM), gradient 5–95% acetonitrile/water, with 0.1% TFA; (b) SFC chromatogram for reaction mixture A. SFC conditions: Diol column (21.2 150 mm, Berger Instruments), gradient 5–60% Methanol with 0.5% dimethylethylamine in carbon dioxide [11].
or mixtures of either methanol and acetonitrile or methanol and dichloromethane. Dimethyl sulfoxide, DMSO, on the other hand was not compatible with the carbon dioxide mobile phase used.
16.5
CONCLUSIONS
Supercritical fluids offer properties intermediate to gas and liquids. As such, supercritical fluid chromatography is an alternative technique to both LC not liquid and or GC. The distinct advantages of supercritical fluids, because of gas like densities yields faster chromatographic elution and therefore shorter overall run times than in LC. Greater solvating power than gases, makes it more applicable to a wider variety of analytes that can typically be analyzed by GC. Ability to optimize a wider variety of parameters than GC or LC is also advantageous; density, temperature and pressure, mobile phase composition, gradient elution and the addition of additives to the mobile phase are all available. But, the challenge to the analytical chemist or technical operator is that there is more to understand to utilize the technique correctly, work in 573
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the optimized range of a phase diagram and deal with the higherpressure equipment and safety precautions that are necessary. Because of this complication, SFC has not replaced LC or GC in the mainstream analytical laboratory although it has the capability to do so.
REFERENCES Supercritical Fluid Extraction: Principles and Practice, M. A. McHugh and V. Krukonis, 2nd Edition, Butterworths-Heinemann, 1994, 608. ISBN: 0750692448 2 U. Van Wassen, I. Swaid and G.M. Schneider, Angew. Chemie Int. Ed. Engl., 19 (1980) 575. 3 Introduction to Modern Liquid Chromatography, L.R. Snyder and J.J. Kirkland, 2nd Edition, Wiley Interscience, 1979 4 Supercritical Fluids in Chromatography and Extraction, R. M. Smith and S.B. Hawthorne, Elsevier, 1997, 414, ISBN: 0-444-82869-9 5 J.A. Cros and J.P. Foley, Anal. Chem., 62 (1990) 378–386. 6 M.E. McNally and J.R. Wheeler, J. of Chromatogr, 477 (1988) 53–63. 7 M.E. McNally and J.R. Wheeler, LC/GC Magazine, 6(9) (1988). 8 M.E. McNally, Anal. Chem., 67(9) (1995) 308A–314A. 9 L. Toribio, M.J. del Nozal, J.L. Bernal, J.J. Jimenez and C. Alonso, J. Chromatogr A., 1046 (2004) 249–253. 10 T. Bamba, E. Fukusaki, Y. Nakazawa, H. Sato, K. Ute T. Kitayama and A. Kobayashi, J. Chromatogr. A., 995 (2003) 203–207. 11 J. Hoxhlowski, J. Olson, J. Pan, D. Sauer, P. Searle and T. Sowin, J. Liq. Chromatgr. & Rel. Tech., 26(3) (2003) 3333–3354. 1
REVIEW QUESTIONS 1.
2. 3. 4. 5.
574
Describe the parameters that are available to the practicing chromatographer in LC, GC and SFC to conduct method development and obtain the best resolution of the components of a mixture. What is the principal requirement in SFC instrumentation to control the pressure? Define the critical point of a substance. Describe the change that occurs in the critical point of a substance if a second component is added. What is the main advantage of SFC over LC and GC? How is this obtained?
Supercritical fluid chromatography Mary Ellen P. McNally
16.1
INTRODUCTION
What is a supercritical fluid? The discovery of supercritical fluids occurred in 1879, when Thomas Andrews actually described the supercritical state and used the term critical point. A supercritical fluid is a material above its critical point. It is not a gas, or a liquid, although it is sometimes referred to as a dense gas. It is a separate state of matter defined as all matter by both its temperature and pressure. Designation of common states in liquids, solids and gases, assume standard pressure and temperature conditions, or STP, which is atmospheric pressure and 01C. Supercritical fluids generally exist at conditions above atmospheric pressure and at an elevated temperature. Figure 16.1 shows the typical phase diagram for carbon dioxide, the most commonly used supercritical fluid [1].
Critical point p Liquid Solid Gas 1 atm
T
Fig. 16.1. Phase diagram for carbon dioxide critical temperature 31.31C critical pressure 72.9 atm. Comprehensive Analytical Chemistry 47 S. Ahuja and N. Jespersen (Eds) Volume 47 ISSN: 0166-526X DOI: 10.1016/S0166-526X(06)47016-1 r 2006 Elsevier B.V. All rights reserved.
561
M.E.P. McNally TABLE 16.1 Comparison of physical properties of liquids, gases and supercritical fluids Phase type
Density (g/cm3)
Diffusion (cm2/s)
Viscosity (g/cm s)
Gas at 1 atm, 211C Supercritical fluid Liquid
10–3 0.3–0.8 1
101 103–104 o 105
104 104–103 102
Source: Van Wassen et al. [2]
The critical point of a material is the temperature and pressure conditions at which the liqiud state ceases to exist. As a liquid is heated, it becomes less dense and starts to form a vapor phase. The vapors being formed becomes more dense, with continued heating the liquid and vapor densities become closer to each other until the critical temperature point is reached. At this same point, the liquid-line or phase boundary disappears. This critical point was first discovered and reported in 1822 by Baron Charles Cagniard de la Tour. As a fluid, the supercritical state generally exhibits properities that are intermediate to the properties of either a gas or a liqiud. In Table 16.1, the physical properties of liquids, gases and supercritical fluids are compared [2]. Examination of the values in Table 16.1 makes the intermediate nature of a supercritical fluid more obvious. The density of a supercritical fluid approaches the levels of a liquid as does its diffusivity, while its viscosity is similar to a typical gas. These properties offer rapid movement (equilibration) as in a gas, but solvation or solubilization as found in a liquid. The best of both worlds from a chromatographic viewpoint. This is because as a solute travels through a chromatographic column the number of equilibration points it reaches, as defined by the Van Dempter equation, defines how effective the separation will be, either through the number of theoretical plates, N, the resolution, Rs, or the alpha value, a, also known as a separation factor. For a detailed description of the theory of chromatography, the reader is refered to Snyder and Kirkland’s text Modern Practice of Liquid Chromatography [3] or Chapters 12, 14, 15 of this text. The ease of solubilizing the analyte is a key factor also. This is because the easier it is to get the solute to these potential equilibration points inside the chromatographic column when the fluid is moving with high diffusivity, the faster the equilibrations or separation can take place. Compared to a gas where the solubilities of the analytes of 562
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interest is almost non-existent, the liquid-like density of a supercritical fluid offers enhanced solubilization. In supercritical fluid chromatography, fluids above their critical point are used as mobile phases. This chapter discusses the principles of operation, mobile phase considerations, parameters that can be adjusted in method development as well as an overview of instrumentation required and a few pertinent examples from current literature. Not everything can be illustrated, but the advantages of this diverse technology will be highlighted.
16.2
HISTORY OF SFC
In terms of chromatography, the first individual credited with the use of supercritical fluids as the mobile phase is Ernst Klesper when in 1962 he reported on the separation of metal porphyrins using dense-gas chromatography (GC) or SFC [3]. But it was not until the 1980s that the analytical community took hold of the abilities and advantages of the technique with the advent of several commercial instrumentation ventures. Two approaches were taken during this time, one by gas chromatographers and the other by liquid chromatographers. Practicing gas chromatographers who experimented with supercritical fluid chromatography for its enhanced solvating powers pursued the first approach. They coupled supercritical fluids, in general pure carbon dioxide; with small narrow bore capillary columns, similar to leading GC columns of the time. With that coupling they were able to get enhanced resolution of compounds that are too difficult to analyze by GC, because they were not soluble in the nitrogen and helium mobile phases commonly used in GC. The second approach was taken by practicing liquid chromatographers. They routinely dealt with thermally labile, highly polar molecules and frequently sacrificed resolution, and speed in their separations because of the aqueous mobile phases that were required. With the enhanced diffusion and decreased viscosity of supercritical fluids over liquids, chromatographic run-time and resolution could be improved when supercritical fluids were used. But solubility in pure carbon dioxide mobile phases, which has the solvating powers from hexane to methylene chloride under normal density ranges, was a problem for these polar molecules. To compensate for this, experimentalists started working with mixed mobile phases. These mixed phases were based on 563
M.E.P. McNally
the addition of polar modifiers or additives to the carbon dioxide mobile phase. Similar to the first approach, this second group of scientists continued to use the columns with similar dimensions to liquid chromatography (LC) columns prevalent in the 1980s that they were already using, i.e., 25 cm by 4.6 mm; the columns were also functionalized with the common C-18, C-8 and phenyl phases as found in LC. With both these approaches rapidly being pursued by a wide variety of academic, industrial and instrument company laboratories, the growth and publication rate in supercritical fluid chromatography during the mid-late 1980s and the early 1990s was exponential. A wide variety of applications are available from that time period as well as several references that explain the advantages of optimizing a supercritical fluid separation using all the parameters available in SFC compared to GC or LC [4–8].
16.3
BASIC PRINCIPLES IN SFC
The most common and widely used supercritical fluid in SFC is carbon dioxide. It is inert, in that it is non-toxic and non-flammable, it also has mild critical parameters, a low critical temperature of 31.31C and a critical pressure of 72.8 atm [1]. Using pure, supercritical carbon dioxide eliminates organic solvent waste and with it waste disposal costs and concerns. This is extremely practical advantage in the industrial environment where the generation of waste requires special handling and significant cost. Beyond this practical consideration, the advantages of SFC from a technical perspective are in the wider range of parameters that can be optimized to achieve the best separation [4]. Taken from the same two approaches described above, the parameter that is most commonly adjusted to change resolution and retention time in GC is the temperature, after that the only choice an experimentalist might have is to change the column used for separation. However, from the liquid chromatographers perspective, the mobile phase composition is the principal component that can be changed to effect a better separation, the mobile phase components are generally the second choice and then finally the choice of chromatographic column. Using supercritical fluid chromatography gives all the parameters optimized in LC as well as the ability to see drastic retention changes due to a temperature gradient, as in GC. In addition, SFC has the added parameters of pressure and/or density that can be selected to achieve the best separation conditions. 564
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From an overall perspective, both the temperature and pressure of the mobile phase control the density or elution power. A change in either temperature or pressure changes the mobile phase density and will alter the chromatographic elution. It should be noted that increasing the temperature in SFC increases the retention time, generally an undesirable effect and the opposite effect that is exhibited in GC. Because of this, reverse temperature gradients, from a high temperature to a low temperature, are utilized in SFC to decrease retention. With decreasing temperature, density of the fluid increases; these higher density mobile phases solubilize the analytes of interest and results in earlier elution of the compounds of interest. The solvating ability of carbon dioxide in SFC is significant when compared to gases used in GC. This is because as a pure component, a change in density of carbon dioxide causes a change in the material’s Hildebrand solubility parameter. In carbon dioxide, this range of solubilities can be considered equivalent to the solubilities seen from hexane to methylene chloride. However, in terms of solvating moderately polar or highly polar molecules, this solubility parameter range is not sufficient and modifiers or additives to carbon dioxide must be included. Once a modifier has been added to the mobile phase, the critical parameters of the original solvent are no longer valid and a two-phase system can exist. As an example, a 10% methanol in carbon dioxide solution has a critical temperature of 51.51C with a critical pressure of 74.2 atm. The critical parameters of pure methanol are 240.51C, temperature, and 78.9 atm, pressure. From these values it can easily be determined that there is no linear relationship for critical point change when the components of a two-phase system are mixed together. What should be noted though is that above the critical point of a mixture, the mobile phase is one phase. This is extremely important in chromatography, an equilibrium-based process, where by definition the movement of the analyte is between two phases. If one of the phases is a not a pure component, equilibrium is difficult to reproduce precisely, the analyte is not transferring back and forth between the stationary phase and the mobile phase, but instead between three phases, the stationary phase and a two-component mobile phase. This discussion emphasizes that, from a practical standpoint, one of the key considerations, often neglected by initial practitioners, is the need to maintain the mobile phase above its critical point at all times during the chromatographic separation. This may be difficult with the wide range of parameters that are adjusted to optimize a separation. 565
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Parameter optimization
Being able to change the density, via either changes in pressure or temperature, is the key difference in SFC over GC and LC separations. Typical density ranges are from 0.3 to 0.8 g/ml for pure carbon dioxide. Table 16.2 shows data obtained from ISCO’s SF-Solver Program for the calculation of density (g/ml), Hildebrand Solubility Parameter and a relative equivalent solvent for pure carbon dioxide at a constant pressure of 6000 psi, approximately 408 atm. But the real advantage in SFC is not just the ability to adjust the density of the mobile phase but the ability to adjust it from two different directions. Density changes achieved by a change in pressure can yield different separation factors then density changes achieved by adjusting the temperature. This offers an advantage in method development by SFC not available in GC or LC. Beyond the density changes that can be used to control method modifications in SFC, the mobile phase composition can also be adjusted. Typical LC solvents are the first choice, most likely because of their availability, but also because of their compatibility with analytical detectors. The most common mobile phase modifiers, which have been used, are methanol, acetonitrile and tetrahydrofuran (THF). Additives, defined as solutes added to the mobile phase in addition to the modifier to counteract any specific analyte–column interactions, are frequently included also to overcome the low polarity of the carbon dioxide mobile phase. Amines are among the most common additives. TABLE 16.2 Calculation of density, Hildebrand solubility parameter using ISCO’s SF solver program at constant pressure of 6000 psi (408 atm) Temperature (1C)
Density (g/mL)
Hildebrand solubility
Equivalent solvent
40 49 55 64 70 73 79 85 94
0.967 0.937 0.917 0.886 0.865 0.855 0.834 0.815 0.785
8.248 7.902 7.814 7.554 7.375 7.287 7.113 6.944 6.694
C6H12 C2H4 CF4 C8H16 C6H16 O2 C4H10 C8H18 F2
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Cyclohexane Ethylene Carbon tetrafluoride n-Octane n-Hexane Oxygen n-Butane 2-Trimethyl pentane Fluorine
Supercritical fluid chromatography
Modifiers and gradient compositions in LC are used broadly. A gradient that runs with 30–80% methanol or acetonitrile is not uncommon. This amount of modifier is generally not needed in supercritical fluid chromatography to affect the same separation. Typical modifier composition in SFC is 1.0–10% and would achieve higher Hildebrand Solubility Parameter adjustment overall than the broader gradients found in LC. 16.3.2
Instrument requirements
The major difference in supercritical fluid chromatography and conventional LC equipment is the pumping systems as well as the safety features installed to maintain higher pressure. Unique SFC equipment differences are: 1. 2. 3. 4. 5. 6.
Carbon dioxide tank for mobile phase supply a. Equipped with a pressure relief value and rupture disk High-pressure pump a. Chiller to maintain mobile phase in liquid state High-speed injector Pressure restrictor a. High-pressure tubing High-pressure flow cell for UV detection Solvent collection device with ability to vent to a laboratory hood or elephant trunk.
Carbon dioxide is usually purchased in a tank, inside the tank the mobile phase exists as a liquid. Typically, the tank does not come with a pressure gauge but is hooked up to a pressure relief valve and rupture disk, which are set above the tank pressure should a tank leak occur. High-pressure pumps used in SFC can come in a variety of types; most of them are modifications of pumps used in LC. Piston, diaphragm and syringe pumps are used. The pumps deliver the most accurate flow of the carbon dioxide if it is pumped in the liquid state. Since that is the case, if there is a great distance between the tank and the pump then a chiller is usually placed so that the tubing containing the carbon dioxide from the tank can be cooled maintaining the carbon dioxide in the liquid state. Most commercially available pumps operate at high enough pressures that the pump heads or pump bodies do not need ancillary cooling, but older models frequently required a chiller that provided a circulation of cold 567
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fluids to the parts of the pump where the carbon dioxide has a potential to vaporize out of the liquid state. A high-speed injector is required in supercritical fluid chromatography. This is to prevent loss of pressure during the injection process. There are a variety of restrictor types that control the pressure of the fluid during the chromatographic process. Each manufacturer has patented their individual restriction devices. It is beyond the scope of this text to go into the detail about all of these restrictors. However, by way of explanation, most of the mechanical restrictors that are used in commercial equipments operate under the principle of decreasing a volume or a space that the mobile phase must pass through by some mechanical means. This decrease in volume, if metered accurately, increases the pressure in the system in a controlled manner. As a safety precaution, for any equipment where elevated pressures are used, tubing should be rated with a safety factor of at least 1.5 times the maximum pressure the SFC system can achieve. Because the density is a controlling factor in achieving the separation, maintaining the elevated pressure used in the separation is necessary up to the point of detection. For UV measurements, this requires a high-pressure cell also rated to the maximum pressure the SFC system can achieve with a desired safety factor of 1.5 times. For other detection methods, i.e., flame ionization detection and mass spectrometry, where the sample is nebulized into the detector, the output of the restrictor is generally right at the nebulization point. This positioning of the column outlet eliminates any peak merging that could occur under low- or no- pressure movement. 16.4
CURRENT EXAMPLES
The examples illustrated herein are not all-inclusive but should give a representation of the advantages of the technique and its most common uses. 16.4.1
Chiral separations
The use of supercritical fluids to separate enantiomers is one of the most important tasks in several areas of research, especially pharmaceuticals and agrochemicals. This is because it is well known that the two enantiomeric forms of a molecule can display dramatically different biological activity. The use of supercritical fluids to separate, with higher efficiencies and shorter retention times, enantiomers is 568
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extremely advantageous in this difficult work. In their article entitled ‘‘Chiral separation of some triazole pesticides by supercritical fluid chromatography, Toribio and coworkers, separated six triazole pesticides and showed the effects of different organic modifiers on the resolution and retention via k0 value [9]. The modifiers they examined were methanol, ethanol and 2-propanol. The addition of additives was also examined. Table 16.3 shows some of their modifier results. In general, separations could be conducted in less than 10 min and the best modifier for the individual separations was compound dependent. This TABLE 16.3 Values of capacity factors and resolutions obtained for diniconazole, tetraconazole, hexaconazole and tebuconazole using different modifiers [9] Compound Hexaconazole Methanol (%, v/v) 5 10 15 Ethanol (%, v/v) 5 10 15 2-Propanol (%, v/v) 5 10 15 Tetraconazole Methanol (%, v/v) 5 10 15 Ethanol (%, v/v) 5 10 15 2-Propanol (%, v/v) 5 10 15
k10
k20
Rs
5.35 5.68 0.67 2.33 2.59 1.02 1.43 1.61 0.87 5.14 6.23 2.41 2.01 2.28 1.14 1.19 1.32 0.65 4.38 5.01 0.54 2.71 2.90 0.48 1.45 1.45 0
4.30 5.23 1.55 2.05 2.41 1.27 1.06 1.83 1.02 4.26 5.29 3.05 2.24 2.84 2.49 1.68 1.98 2.06 5.86 8.75 4.98 3.28 5.46 4.01 2.40 3.69 3.43
Compound
k10
k20
Rs
Tebuconazole Methanol (%, v/v) 5 13.23 14.74 1.28 10 5.26 5.82 1.17 15 3.11 3.42 1.03 Ethanol (%, v/v) 5 7.16 7.16 0 10 5.04 5.04 0 15 2.69 2.69 0 2-Propanol (%, v/v) 5 20.36 22.53 0.98 10 7.61 8.35 1.2 15 3.54 3.91 1.15 Diniconazole Methanol (%, v/v) 5 7.29 12.44 6.08 10 2.81 5.69 6.90 15 1.63 3.43 5.87 Ethanol (%, v/v) 5 6.97 10.63 4.55 10 2.53 3.87 3.77 15 1.37 2.08 3.11 2-Propanol (%, v/v) 5 12.46 21.06 1.23 10 4.09 4.09 0 15 2.04 2.04 0
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is consistent with the understanding of modifier interactions with the solutes and their effect on the overall equilibrium between the stationary and mobile phases in SFC as previously described. Recalling, acceptable resolution, Rs, values for quantitation purposes are greater than 1.5, values that are greater than 1.5 for resolution are highlighted in Table 16.3. Figure 16.2 shows an example chromatogram of a typical separation. These separations were conducted on a Hewlett-Packard 1205A supercritical fluid chromatograph equipped with a photodiode array detector; detection was at 220 nm. A Chiralpak AD 25 cm 4.6 mm column packed with the 3, 5-dimethylphenylcarbamate derivative of amylose, coated on a 10 mm silica support was used. The mobile phase was carbon dioxide modified as outlined in Table 16.3 for each of the individual separations. Other chromatographic conditions were 351C, 2 ml/min flow rate and a pressure of 200 bar (2960 atm) (Fig. 16.3). (Note: Because of the low temperature of the separations, the mobile phase was not likely in the supercritical state for all of the modifier concentrations examined in this report.) 16.4.2
Polymer separations
mAU
The advantages of supercritical fluid chromatography for polymer separations have been illustrated in the literature for many years. A recent example is the separation of long-chain polyprenols using SFC with matrix-assisted laser-desorption ionization TOF mass spectrometry [10]. The generic name for 1,4-polyprenyl alcohols is polyprenol; these compounds generally have smaller polymerization chains of less (A)
200 150 100 50 0
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200 150 100 50 0 0
Fig. 16.2. Enatiomeric separation of hexaconazole at 200 bar, 351C, 2 ml/min and 10 v/v 2-propanol. (A) Without using additives; (B) using 0.1% (v/v) triethylamine and 0.1% (v/v) trifluoroacetic acid [9].
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Fig. 16.3. Enatiomeric separation at 200 bar, 351C, 2 ml/min. (A) Tetraconazole with 4%(v/v) ethanol; (B) diniconazole 15% (v/v) ethanol [9].
than 30 monomers. Chromatographic separation of these chains was conducted using a Jasco Super-201 chromatograph. The system has two separate pumps, one delivers the carbon dioxide, and the other delivered the THF modifier. The column temperature was 801C, the pressure, controlled by a backpressure regulator was controlled at 19.6 MPa. An Inertsil Ph-3 (25 cm 4.6 mm), 5 mm column was used. A flow gradient was used to introduce the THF modifier, this is not typical, but it is possible with the two pumps on this instrument. The consistent flow rate of the carbon dioxide is 3.0 ml/min; the THF flow rate started at 0.8 ml/min and was adjusted over 30 min- to 2.0 mL/min and then held constant. Figure 16.4 illustrates the resultant chromatogram obtained for Eucimmia ulmoides leaves, a plant producing fibrous rubber; over 100-mer (MW: 6818) components were completely separated using the conditions described above.
16.4.3
High throughput screening of pharmaceuticals
At Abbott Laboratories, Hochlowski and coworkers used preparatory scale SFC to screen the output of high throughput organic synthesis (HTOS) for purity levels and classify chemical structures into libraries of similar analogs [11]. These scientists used a Berger Instruments SFC self-modified for this particular use. Figure 16.5 shows the schematic of this modified system. In this application, the authors reported adding SFC capability to their repertoire of instrumentation to solve their analytical challenges. Figure. 16.6 shows the comparison of a typical reaction mixture analyzed by both high performance liquid chromatography (HPLC) and SFC. 571
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100 20
0
10
20
30
40
50
Min
Fig. 16.4. SFC chromatogram of long-chain polyprenols in E. ulmoides leaves. The sample had Mn 3.99 103 (calibrated against cis-1,4-polyisoprene standards) and Mw/Mn 1.41. The numbers in the chromatogram represent degrees of polymerization for polyprenol homologues [10].
Autosampler
CO2 delivery system
Custom software
Methanol wash system
Dual arm fraction collector
Fig. 16.5. Preparative supercritical fluid chromatographic system, customized for high throughput organic synthesis (HTOS) screening [11].
Actual operating conditions can be found in the figure caption. A given limitation of SFC, relative to HPLC, as described is the ability to dissolve samples in a solvent system compatible with the methanol/ carbon dioxide mobile phase. For this particular mobile phase, other compatible sample diluents that worked effectively are pure methanol, 572
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500 250 0 0
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5 4 Minutes
6
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1100 mAU
8250 6500
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Fig. 16.6. (a) HPLC chromatograms for reaction mixture A. HPLC conditions: C18 (20 100 mm Nova–Pak TM), gradient 5–95% acetonitrile/water, with 0.1% TFA; (b) SFC chromatogram for reaction mixture A. SFC conditions: Diol column (21.2 150 mm, Berger Instruments), gradient 5–60% Methanol with 0.5% dimethylethylamine in carbon dioxide [11].
or mixtures of either methanol and acetonitrile or methanol and dichloromethane. Dimethyl sulfoxide, DMSO, on the other hand was not compatible with the carbon dioxide mobile phase used.
16.5
CONCLUSIONS
Supercritical fluids offer properties intermediate to gas and liquids. As such, supercritical fluid chromatography is an alternative technique to both LC not liquid and or GC. The distinct advantages of supercritical fluids, because of gas like densities yields faster chromatographic elution and therefore shorter overall run times than in LC. Greater solvating power than gases, makes it more applicable to a wider variety of analytes that can typically be analyzed by GC. Ability to optimize a wider variety of parameters than GC or LC is also advantageous; density, temperature and pressure, mobile phase composition, gradient elution and the addition of additives to the mobile phase are all available. But, the challenge to the analytical chemist or technical operator is that there is more to understand to utilize the technique correctly, work in 573
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the optimized range of a phase diagram and deal with the higherpressure equipment and safety precautions that are necessary. Because of this complication, SFC has not replaced LC or GC in the mainstream analytical laboratory although it has the capability to do so.
REFERENCES Supercritical Fluid Extraction: Principles and Practice, M. A. McHugh and V. Krukonis, 2nd Edition, Butterworths-Heinemann, 1994, 608. ISBN: 0750692448 2 U. Van Wassen, I. Swaid and G.M. Schneider, Angew. Chemie Int. Ed. Engl., 19 (1980) 575. 3 Introduction to Modern Liquid Chromatography, L.R. Snyder and J.J. Kirkland, 2nd Edition, Wiley Interscience, 1979 4 Supercritical Fluids in Chromatography and Extraction, R. M. Smith and S.B. Hawthorne, Elsevier, 1997, 414, ISBN: 0-444-82869-9 5 J.A. Cros and J.P. Foley, Anal. Chem., 62 (1990) 378–386. 6 M.E. McNally and J.R. Wheeler, J. of Chromatogr, 477 (1988) 53–63. 7 M.E. McNally and J.R. Wheeler, LC/GC Magazine, 6(9) (1988). 8 M.E. McNally, Anal. Chem., 67(9) (1995) 308A–314A. 9 L. Toribio, M.J. del Nozal, J.L. Bernal, J.J. Jimenez and C. Alonso, J. Chromatogr A., 1046 (2004) 249–253. 10 T. Bamba, E. Fukusaki, Y. Nakazawa, H. Sato, K. Ute T. Kitayama and A. Kobayashi, J. Chromatogr. A., 995 (2003) 203–207. 11 J. Hoxhlowski, J. Olson, J. Pan, D. Sauer, P. Searle and T. Sowin, J. Liq. Chromatgr. & Rel. Tech., 26(3) (2003) 3333–3354. 1
REVIEW QUESTIONS 1.
2. 3. 4. 5.
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Describe the parameters that are available to the practicing chromatographer in LC, GC and SFC to conduct method development and obtain the best resolution of the components of a mixture. What is the principal requirement in SFC instrumentation to control the pressure? Define the critical point of a substance. Describe the change that occurs in the critical point of a substance if a second component is added. What is the main advantage of SFC over LC and GC? How is this obtained?