Superheated water chromatography – A green technology for the future

Available online at www.sciencedirect.com

Journal of Chromatography A, 1184 (2008) 441–455

Review

Superheated water chromatography – A green technology for the future Roger M. Smith Department of Chemistry, Loughborough University, Loughborough, Leics LE11 3TU, UK Available online 5 July 2007

Abstract Reversed phase liquid chromatography using superheated water as the mobile phase, at temperatures between 100 and 250 ◦ C, offers a number of advantages for the analyst. It is an environmentally clean solvent, reducing solvent usage and disposal costs. It has advantages in detection, allowing UV spectra to be monitored down to short wavelengths, as well as a compatibility with universal flame ionisation detection and mass spectroscopy. By employing deuterium oxide as the eluent, solvent free NMR spectra can be measured. The development of newer more thermally stable stationary phases, including hybrid phases, have expanded the analytes that can be examined and these now range from alkylbenzenes, phenols, alkyl aryl ketones and a number of pharmaceuticals to carboxylic acids, amino acids, and carbohydrates. Very few compounds have been found to be unstable during the analysis. The separation methods can be directly coupled to superheated water extraction providing a totally solvent free system for sample extraction and analysis. © 2007 Elsevier B.V. All rights reserved. Keywords: Superheated water chromatograph; High temperature liquid chromatography; NMR spectroscopy; Flame ionisation detection; Thermally stable stationary phases

Contents 1. 2. 3. 4.

5.

6.

7. 8.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Early work on SHWC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Water as a mobile phase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. Water as a chromatographic mobile phase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Instrumentation for superheated water chromatography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1. Pumps and ovens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2. Sample Injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.3. Eluent preheating . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Stationary phases for superheated water chromatography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.1. Column materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.2. Column dewetting with high proportions of water in the eluent . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.3. PS-DVB column materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.4. Silica-based column materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.5. Hybrid silica phases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.6. Zirconia-based columns . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.7. Carbon-based columns . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Detection in superheated water chromatography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.1. Spectroscopic detectors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.2. GC-based detectors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.3. NMR and MS spectrometric detection. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Applications of superheated water chromatography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.1. Hyphenated systems with SHWE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

E-mail address: [email protected]. 0021-9673/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.chroma.2007.07.002

442 442 443 443 443 444 444 445 445 446 447 447 447 448 448 448 449 449 449 450 451 453 453 454

442

R.M. Smith / J. Chromatogr. A 1184 (2008) 441–455

1. Introduction In reversed phase liquid chromatography, water is usually regarded as a weak eluent, primarily employed to reduce the elution strength of the stronger non-polar organic modifiers, such as methanol, acetonitrile and tetrahydrofuran. However, on heating under pressure to temperatures between 80 and 250 ◦ C, the polarity of liquid water decreases markedly to the extent that it can replace the organic modifier and act as the sole eluent. Potentially, this can provide an environmentally friendly green method of analysis [1,2], with savings both in the use and disposal of organic-based eluents. This mode of separation has been termed superheated water chromatography (SHWC), pressurised water chromatography, or subcritical water chromatography (as the temperatures used are lower than the critical temperature for water of 374 ◦ C). Earlier reviews by Smith et al. [3] and by Coym and Dorsey [4] have provided an introduction to the technique as it emerged and method development has been described by Su et al. [5]. The same enhanced solvation strength also enables superheated water to be used as a clean extraction solvent [6]. A number of studies have also examined the use of steam as an eluent for gas chromatography [7] and this ability to use a single eluent for different modes of chromatography has been discussed in the concept of unified chromatography [8]. The current state of SHWC, the equipment required and applications of this approach will be considered in this review. Although it has a number of attractive features the adoption of superheated water chromatography has been slow over the last 10 years. This can be attributed to a hesitation on the part of users concerned about analyte and/or stationary phase stability at elevated temperatures, the limited range of thermally stable stationary phases and a lack of specialised instrumentation and possibly a lack of gas chromatographic experience in many current HPLC users, which could have demonstrated that many even complex analytes can be thermally stable. Many studies and reviews [9–11] have examined the effects of temperature on conventional normal phase and on reversed phase separations with aqueous–organic eluents and by 2002, Dolan [12] was able to observe that temperature was now a recognised variable to change selectivity in HPLC. The use of elevated temperatures and temperature programming in conventional liquid chromatography has been recently reviewed by Vanhoenacker and Sandra [13].

Fig. 1. Separation of benzene, toluene and styrene using water at 165 ◦ C on a Spherosil XOA 600-D/C18 column with refractive index and UV spectroscopic detection [15]. Reproduced with permission of the publisher.

and styrene (Fig. 1). They also linked the separations to a flame ionisation detector (FID); however, the paper unfortunately lacked an experimental description of the equipment used and appears to have not been followed up. The more recent interest in HPLC with superheated water started with the work of Hawthorne et al. [16] in 1994, who were interested in water as a clean solvent for the extraction of non-polar analytes from environmental samples. They noted the marked changes in the relative permittivity of water with temperature from nearly ε = 80 at room temperature to ε = 20 at 300 ◦ C. In a subsequent study, they reported that the solubility of polynuclear aromatic hydrocarbons increased by five orders of magnitudes on raising the temperature by 200 ◦ C [17]. Because the relative permittivity of water at about 200 ◦ C was similar to that of methanol (ε = 33) and acetonitrile (ε = 37) (Fig. 2) [5,17], it appeared that by controlling the temperature

2. Early work on SHWC The potential use of water as a supercritical fluid for separations was initially noted by Lovelock in 1958 [14]. The first description of separations using only hot water as the mobile phase, with the title thermal aqueous liquid chromatography (TALC), was reported by Guillemin et al. in 1981 [15]. They described the separation of lower alkanols at ambient temperature and then showed that by heating the column to 150 ◦ C they could separate higher alcohols up to decanol using a Spherosil XOA 600 D/C18 column, with both UV and RI detection. These high temperature conditions would also resolve benzene, toluene

Fig. 2. Effect of temperature and pressure on the permittivity of water at different pressures compared to relative permittivity of representative organic solvents: (), 33 bar; (), 129 bar; and (♦), 322 bar. The drop to low values corresponds to the formation of superheated steam. Based on data from [18].

R.M. Smith / J. Chromatogr. A 1184 (2008) 441–455

of water over that range it should be able to mimic the elution strengths of the organic–water mixtures used for HPLC at room temperature. This lead to work by Smith and Burgess [19,20], who demonstrated the SHWC of a series of phenols on a polystyrene divinylbenzene (PS-DVB) column. They showed that the retention times were reduced by raising the temperature and that programming the temperature could achieve an effective gradient elution. Shortly afterwards Miller and Hawthorne [21] reported the separation of a series of homologous alkanols on a PS-DVB column. In both cases, the chromatography behaved as a typical reversed phase hydrophobic separation and thus offered a method that should be widely applicable for moderately polar analytes in fields, such as pharmaceutical and food analysis. 3. Water as a mobile phase The physical and chemical properties of high temperature water and steam have been extensively studied for many years. As well as its use in separation techniques, superheated water is also of interest in many other areas of chemistry, including waste remediation and as a green solvent for organic synthesis [22,23] and the properties of supercritical water as a solvent have been reviewed by Weing¨artner and Franck [24]. Three factors change markedly as the temperature of water is increased. As already noted (Fig. 2) the relative permittivity decreases. The viscosity at 25 MPa also decreases from 888 ␮Pa s at 25 ◦ C to 140 ␮Pa s at 200 ◦ C [18] and hence the back-pressure in the column decreases. The vapour pressure increases, although the changes are modest (Fig. 3) and at 250◦ only reaches 40 bar [18]. This is well within the operating condition of most HPLC systems and is considerably lower than the pressures employed for supercritical fluid chromatography with carbon dioxide. Over this range the density of water is effectively constant and only changes from 0.9989 cm3 /g at 0 ◦ C under a pressure of 25 bar to 1.1347 cm3 /g at 200 ◦ C under a pressure of 250 bar [18]. This low compressibility has the advantage that the modest pressures used in SHWC do not need to be controlled precisely and have been shown to have a negligible effect on retention. However, at higher pressures up to 30,000 psi (2000 bar), Kephart and Dasgupta [25] noted that there are changes in the relative permittivity and suggested that

443

the properties of the water would vary along the length of a column. 3.1. Water as a chromatographic mobile phase The solvation parameters of superheated water as a solvent for chromatography from 75 to 180 ◦ C have been determined by Pawlowski and Poole [26]. They concluded that the feature of room-temperature water was its high cohesive energy and hydrogen bonding capacity. It retained many of these characteristics even at 180 ◦ C and was thus still a weaker eluent than organic solvents. They suggested that the selectivity changes on heating water were not equivalent to the effect of adding an organic modifier, such as methanol, acetonitrile or propan-2-ol. However, high temperature water could provide a complementary selectivity and was most suited for polar analytes. A comparison of the retention properties of superheated water and organic–water eluents was carried out by Kondo and Yang [27] using a series of aromatic analytes. They concluded that a 3.5 ◦ C rise in water temperature corresponded to a 1% increase in methanol and a 5–8 ◦ C rise corresponded to a 1% increase in acetonitrile. Coym and Dorsey [28] also examined the relationship between temperature and retention to determine if the retentions in water at elevated temperatures could be used to estimate the retention at 35 ◦ C. They concluded that this was not a suitable method because the entropy of transfer between the mobile and stationary phases appeared to differ between ambient and temperatures greater than 100 ◦ C. There is a change in the pKa of water with temperature but in practice this has a limited effect on retention. However, buffers can be used and despite initial concerns that inorganic salts might precipitate as the polarity decreased no problems have been reported. For example, Chienthavorn and Smith [29] demonstrated that a wide range of buffers from pH 3 to 13 could be used on a PLRP-S column up to 190 ◦ C. If the changes in retention with ionisation were used to calculate the pKa values of a series of sulfonamides, up to 100 ◦ C the values were comparable to those reported for room temperature. At higher elution temperatures the values differed by no more than one pKa unit. Teutenberg et al. [30] also used pH at 3.5 or 11.5 to control the separation of a series of anticancer drugs. The relative retentions of analytes with different substituents can change as the temperature is altered. A recent study by Edge et al. [31] used three coupled 10 cm 1.7 ␮m Acquity columns from 40 to 180 ◦ C at an initial pressure of 15,000 psi and suggested that temperature and pressure could be used as a method for optimising the separation of drug compounds. 4. Instrumentation for superheated water chromatography

Fig. 3. Vapour pressure of water at different temperatures [3] based on data from [18]. Reproduced with permission.

The instrumentation required to carry out SHWC (Fig. 4) is essentially the same as that for conventional HPLC, except for the addition of a high temperature oven and a method of controlling the column back-pressure. However, the limited availability of commercial systems is probably one the reasons that has restricted the widespread adoption of the method.

444

R.M. Smith / J. Chromatogr. A 1184 (2008) 441–455

Fig. 4. Example of equipment used for superheated water chromatography. Components: 1, HPLC pump; 2, injection valve; 3, pre-heating coil; 4, column; 5, gas chromatographic oven; 6, cooling coil; 7, UV detector; 8, back-pressure regulator; 9, computerised data acquisition system.

4.1. Pumps and ovens One advantage of SHWC is that as water is used as the sole mobile phase component only a single pump is required, which is operated in an isocratic mode so no mixer or gradient control is required. The deionised water or distilled water being used as a mobile phase is usually degassed or sparged with nitrogen or helium. The aim is to reduce the oxygen level and hence minimise the potential for rusting of the instrument or oxidation of the analyte. The main change compared to conventional HPLC instrumentation is in the oven as normally liquid chromatography column ovens have a range only up to 80 or 100 ◦ C. They are primarily designed to maintain the column isothermally at a constant temperature, although the effective column temperature can differ in different designs of oven [32]. Most SHWC systems have been built around GLC ovens [19], which can usually go up to 350 ◦ C and have a built-in capability for temperature programming. Few special precautions are needed with water as the mobile phase as there is no fire hazard from mobile phase leaks. Currently, two specialised high temperature ovens capable of use up to 200 ◦ C are marketed, one isothermal and one with temperature gradient capability. Because the thermal mass of most LC columns is significant, for both standard 4.6 mm I.D. and narrower bore columns, a frequent concern is the ability of the oven to heat the column during temperature programming. The temperature lag has been monitored in a number of studies [33] and one guide to the thermal equilibration is to monitor the column back-pressure as the viscosity of water is highly temperature sensitive. A review of temperature programming in HPLC by Jones [34] examined the ability of different types of ovens to achieve the desired setting within the column and the importance of also controlling the inlet temperature. To achieve a rapid temperature programme, a close heater to column contact is necessary. A specially designed instrument for superheated water chromatography has been reported by

Teutenberg et al. [35], which included facilities for rapid column heating up to 225 ◦ C and circulating oil cooling to enable fast cycle times. An alternative with a closely fitted resistively heated jacket has been used by Harvey-Doyle et al. [36] and columns directly wrapped with a resistive heater coil were used by Fogwill and Thurbide [37] to achieve heating rates of up to 57 ◦ C/min from 70 to 200 ◦ C. Compared to HPLC, the other change which is often needed in SHWC is to generate a back-pressure at the end of the column to prevent the water from boiling, however, the pressures needed are low (30–40 bar) and often can be accommodated by standard detector flow cells. Some groups have used specialised restrictors, made of capillary tubing, or employed active or static back-pressure regulators taken from SFC systems. Alternatively a simple length of narrow bore PEEK tubing (typically 3 m × 0.13 mm I.D.) can be sufficient at a flow rate of 1 ml/min. 4.2. Sample Injection Except for narrow bore columns, conventional HPLC rotary injection valves are usually employed with 10–20 ␮l injections. Normally these are mounted externally to the oven so the samples can be loaded under ambient condition to prevent boiling or evaporation. Ideally the sample solvent should be water, because it can be assumed that stronger solvent would cause band broadening. However, there can be problems if the analyte has a low solubility in cold water and on occasions some organic solvent may be required. In contrast, a recent paper [38] has suggested that for water mobile phases at room temperature, there may be an advantage in using a stronger eluent as the injection solvent as long as it is more retained on the column than the analytes. It has also proved possible to directly link superheated water extractions to SHWC by using a cold sorbent trap to focus the sample, that no solvent is used in the extraction or separation stage. The trap is then thermally desorbed either on-line [39,40] (Fig. 5) or off- line [41] to inject the sample.

R.M. Smith / J. Chromatogr. A 1184 (2008) 441–455

445

Fig. 5. On-line superheated water extraction trapping and chromatographic system used to extract, trap, clean-up and assay triazine pesticides from compost. SWE–SWC system. 1, Water pump; 2, injector; 3, UV detector; 4, back-pressure regulator; EC, extraction cell; TC, Xterra trap column; AC, PGC analytical column; V1, V2, V3, V4, switching valves-1, 2, 3 & 4; P1, P2, P3, pre-heating coils; C1, C2, C3, cooling coils [107]. Reproduced with permission.

4.3. Eluent preheating A large difference between the mobile phase and the column temperatures can cause problems because the thermal mass of the cold water entering the column can result in a considerable cooling effect. In most SHWC either an extended pre-heater coil within the oven (as in Fig. 4) or a heater on the inlet line have been used to raise the mobile phase temperature. Thompson et al. [42] demonstrated that problems with a thermal mismatch were more severe at high eluent flow rates and suggested that the temperature difference between the incoming eluent and the column should not exceed 5 ◦ C. They also suggested that the length of any preheating coil was important and should be matched to the flow rates and column I.D. being used. The length of the preheating coil was also studied by Fields et al. [43] who found that short coil of only 15 cm (with a volume of 3.4 ␮l) gave distorted peaks at more than 0.7 ml/min whereas consistent good shape were obtained with a 140 cm long preheating coil up to 1.5 ml/min. Guillarme et al. [44] illustrated an improvement in peak shape on increasing the length of the preheating coil (Fig. 6). However, if a heated metal block in close contact with the inlet tubing is employed then the heat transfer is enhanced and only a short length of 15 cm is sufficient even up to 190 ◦ C [35]. However, work on conventional HPLC [45,46] has reported that a difference of 10–20 ◦ C between the eluent and column oven, can result in an improvement in the column efficiency, by apparently inverting the temperature profile across the column. This counteracts the frictional heating caused by the eluent flowing through the packed bed. A detailed study [47] has found that the optimum difference varied with the stationary phase and in an extreme example reached 40 ◦ C [32]. After the separation many SHWC systems cool the eluent before the detector with either Peltier coolers or heat exchanger coils. This is primarily to achieve temperature stability in the detector flow cell. Failure to control the temperature can lead to an unstable baseline and increased noise.

5. Stationary phases for superheated water chromatography The general effect of temperature on column efficiency has been comprehensively studied in recent years and will not be

Fig. 6. Comparison of different lengths of preheating tubing on efficiency of benzene, toluene, ethylbenzene, propylbenzene, butylbenzene, pentylbenzene on a ZirChrom-DB-C18 column. Flow rate 4 ml/min; temperature 150 ◦ C; I.D. preheating tube 0.127 mm. Length: a, 2 m efficient; b, 1 m inefficient [44]. Reproduced with permission.

446

R.M. Smith / J. Chromatogr. A 1184 (2008) 441–455

covered here. Usually there is a linear van’t Hoff relationship between the retention factor and the inverse of the absolute temperature in both conventional and in SHWC, for example, in a study by Sanagi and See [48] of the retentions of aromatic test compounds and barbiturates on a PLRP-S column from 100 to 200 ◦ C. In contrast, Guillarme et al. [44] found that although the classic linear model was valid over limited temperature ranges (typically up to 80 ◦ C) in SHWC, over wider ranges a quadratic fit was usually needed. However, a recent paper by Gritti and Guichon [49] suggests that there are too many parameters involved in separations on a C18 bonded silica for a linear relationship to be anything other than accidental. 5.1. Column materials Probably the major constraint on the use of high temperatures has been the poor stability of many stationary phases. Although ODS-bonded silica phases are widely accepted in conventional ambient HPLC, these columns are usually degraded when the temperatures rise above about 80 ◦ C. As a result most of the early work with superheated water employed thermally stable PS-DVB materials. However, these columns have a high retention capacity and it can be difficult to elute even moderately non-polar analytes below 220 ◦ C, their effective upper limit. Alternative column materials used for SHWC have included porous graphitised carbon- and zirconia-based phases and in recent years a number of hybrid silica type materials have been studied. A number of these materials can be used readily for separations up to 200 ◦ C (Table 1). The interactions between potential stationary phase materials and analytes in superheated water were investigated by Yang et al. [50] over the temperature range 50–250 ◦ C. They concluded that supercritical water could elute both polar and non-polar analytes from normal and reversed phase packings, because of the lower polarity and surface tension as the temperature was increased. The energy and hence the temperature required for elution from a reversed phase material was higher than for a normal phase material and was even higher when aromatic interactions were present. Many of the studies have used conventional HPLC column sizes (100–250 mm × 4–4.6 mm I.D with 3–5 ␮m particles), although the low viscosity of water at elevated temperature should enable the use of small particle sizes or multiple linked

columns [31]. Some studies have used narrower bore (2–3 mm) columns, because their lower mass should enable them to respond more rapidly to temperature programming. Some of the practical problems with SHWC are caused by the tendency of column manufacturers to employ PEEK mounted frits or internal PEEK ferrules, which are hidden from the user, as part of specially designed column fittings. However, under the temperatures and pressures used in SHWC, the PEEK creeps and after a single run can start to leak or in the worst instances can coat a frit blocking the eluent flow. Normally metal column fittings are used on connecting tubing in the oven but recently a high temperature PEEK coupling has been marketed. Although the eluents are usually degassed (see earlier) a few authors have reported pre-saturating the mobile phase with a silica guard column to reduce attacks on the stationary phase. However, Kephart and Dasgupta [25] working with silica capillary columns, found a guard column essential to prevent the column walls from being attacked, otherwise the columns burst within 3 days at 250 ◦ C. There have been a number of studies of the stability and life time of columns of different types, including work by Claessens and van Straten [51] who found a short life time for ODS silica materials even under modest conditions in methanol–water eluents but higher stability for hybrid and polydentate bonded silicas, polymer and zirconia-based materials. Wilson [52] compared PGC, PS-DVB, ZirChrom PBD, Zirchrom CARB, and silica bonded materials using superheated water conditions for a range of pharmaceuticals. However, although the conventional ODS bonded silicas gave good results they were not robust and degraded after 2 days use, whereas the hybrid Xterra material was stable. Marin et al. [53] reported that PRP-1, and Hypercarb columns were stable up to 200 ◦ C without evidence of column degradation with conventional eluents. However, ZirChrom PBD, Zirchrom CARB and Diamondbond columns were less satisfactory as there was an increase in the background signal at 254 nm when the temperature was programming from 50 to 200 ◦ C. He and Yang [54] looked at the stable lifetimes of silica, polymer, and zirconia-based columns. They found that all five columns studied, ZirChrom-PS, PRP-1, Zorbax C8, Nucleosil C18AB and Hypersil BDS C18, were unaffected after 6000 column volumes at 100 ◦ C and after extended studies concluded that PRP-1 columns appeared to be especially suitable for SHWC. In another study, Yang et al. [55] exam-

Table 1 Stationary phases employed for high temperature superheated water chromatography Column materials

Typical Phase

Temperature limit (◦ C)

Reference

PS-DVB Porous graphitised carbon Zirconia supported columns Zirconia supported columns Alkyl bonded silica Polydentate bonded silica Encapsulated silica Methyl-bridged hybrid Ethylene-bridged hybrid

PRP-1, PLRP-S Hypercarb ZirChrom PBD ZirChrom CARB ZirChrom-PS Diamondbond C18 Numerous Blaze Pathfinder Xterra C8, C18 Phenyl Xbridge C18 Phenyl

220 >200 140 180 80 100 200 140 200

[3] [81] [76] [53] [51,52] [65] [64] [68] [70]

The temperatures limits are indicative and suggest a value at which results have been reported although the column may have limited stability.

R.M. Smith / J. Chromatogr. A 1184 (2008) 441–455

447

ined the effect of temperature from 60 to 160 ◦ C on the column efficiency for four different columns. Up to 100–120 ◦ C at a constant flow rate the efficiency increased but it then decreased at higher temperatures, which they ascribed to increased longitudinal diffusion. More recently Teutenberg et al. [56] compared the stability of a number of bonded and unbonded zirconia and titania columns to 10 temperature cycles up to 185 ◦ C (and silica up to 120 ◦ C). The metal oxide columns appeared to be very rugged but the silica columns rapidly degraded even at the lower temperature. 5.2. Column dewetting with high proportions of water in the eluent A number of groups have noted that when separations are carried out with eluents containing more than 95% water, there can be sudden shortening of retention times, often without loss of efficiency, which can be reversed on returning to eluents with higher proportions of organic modifiers. The changes seemed to occur after a period when the columns were not operating and were not under pressure. They were originally attributed to “phase collapse” [57,58] in which the C18 alkyl chains flatten onto the surface, excluding the polar solvent and effectively reduced the volume of stationary phase available for retention. This has led manufacturers to develop specialised water compatible phases, termed polar embedded or aqua phases which usually contained a polar linkage between the C18 chain and the silica surface to retain the polar eluent at the base of the bonded chains [59,60]. These retention changes have also been noted in SHWC with both ODS-silica and ODS-Xbridge materials, especially when columns have been cooled and left overnight without a flow. Although the loss of retention can be reversed by elution with an organic–aqueous eluent and the column activity restored, the changes may reoccur quite suddenly, even from one run to another. Methods to overcome the problem, such as the use of polar embedded phases, were discussed by Walter et al. [61], who also tried to predict when the problem might occur. More recently it has been suggested that this effect is a dewetting process in which water is expelled from the pores of the stationary phase, thereby reducing the effective volume of the static layer of mobile phase on the surface of the stationary phase [62]. Pettersson et al. [63] proposed the use of a back-pressure of 40–60 bar to maintain wetting when injecting peptides onto room temperature columns in 100% aqueous samples. 5.3. PS-DVB column materials Most of the early studies used polystyrene divinylbenzene (either PLRP-S or PRP-1) columns (Fig. 7), which were known to be stable to at least 160 ◦ C and have a high chemical stability to pH extremes. These proved suitable for SHWC, although the column efficiency was not high, but they could be used up to 220 ◦ C. However, the main limitation was the high retention capacity of the columns, which mean that high temperatures were often needed to achieve elution of moderately polar compounds, such as alkyl aryl ketones.

Fig. 7. Water–FID chromatograms of substituted phenols on PRP-1 column. Flow rate 200 ␮l/min, temperature 175 ◦ C. Reprinted with permission from [21]. Copyright 1997 American Chemical Society.

5.4. Silica-based column materials The stability of alkyl-bonded silica phases has been studied by a number of groups and generally limits of 70–80 ◦ C have been suggested with conventional mobile phases. In a number of studies C18 phases have been used successfully for SHWC but the life times were short [52]. In unpublished observations, the changes in retention on the bonded phases columns with use were generally small, probably caused by a loss of bonded phase, but were followed by a catastrophic collapse of the stationary phase materials to leave a large cavity of 15–20% of the column volume. This suggested that the silica had been thinned until it could no longer support the back-pressure. To provide columns with additional stability, other groups examined polymer encapsulated silicas, such as Aquatherm Pathfinder C18 [64], which were reported to be stable at 200 ◦ C, but they appear to have been little used. The polydentate-bonded stationary phase Blaze C8 was tested to 100 ◦ C [52] and a more recent study suggest that although it can tolerate higher temperatures, up to 200 ◦ C, in the presence of organic modifiers, with 100% water the recommended stability limit is 100 ◦ C [65]. Young et al. [66] suggested the use of a bonded non-porous silica substrate which would have a low retention capacity. It was used to separate alkylbenzenes at room temperature with water as the eluent or could be linked to superheated water extractions.

448

R.M. Smith / J. Chromatogr. A 1184 (2008) 441–455

Fig. 9. Fast temperature gradient separation of pharmaceuticals on ZirChromPS column. Programme 40–130 ◦ C in 4 min. Eluent, water + 0.1% formic acid at 2 ml/min; detection, 254 nm. Elution order: 1, cytarabine; 2, 5-fluorouracil; 3, sulfadiazine; 4, sulfathiazole; 5, sulfamethoxypyridazine; 6, chloramphenicol; 7, etoposide [35]. Reproduced with permission.

Fig. 8. Separation of (A) alkylbenzenes at 190◦ at 4 ml/min and (B) phenylalkanols at 170◦ at 1 ml/min on C18 bonded ethylene bridged hybrid column [70]. Reproduced with permission.

5.5. Hybrid silica phases An alternative method to improve both chemical and thermal stability have been the merging of polymer and silica technologies to prepare organic–inorganic hybrid phases with enhanced thermal and chemical stability [67] and these phases look particularly attractive as future phases for SHWC. These supports, which can carry C18 , C8 or phenyl bonded phases, include methylsiloxane-based Xterra columns [68], which have been successfully used in SHWC up to about 140 ◦ C, and the ethylenebridged hybrid phase (Xbridge) [69,70] (Fig. 8) columns. The ethylene-bridged material has been reported [69] to be useable at up to 200 ◦ C for the separation of anilines, alkylbenzenes and phenylalkanols and to show a linear van’t Hoff relationship. Other studies by Al-Khateeb and Smith [71] on the separation of phenols on a phenyl-bonded Xbridge column over a wide temperature range have suggested a possible change in interaction around 100 ◦ C. 5.6. Zirconia-based columns To overcome the thermal instability of silica, other metal oxides have been studied as potential reversed-phase substrates,

including alumina, zirconia and titania and these have been reviewed by Nawrocki et al. [72–74]. However, making alkylbonded reversed-phase materials has proved to be more difficult than with silica and the amphoteric properties of the metal oxide has required the use of mobile phase additives, such as fluoride ions. Only zirconia-based columns are widely available commercially, mainly as polybutadiene (PBD) or polystyrene (PS) encapsulated zirconia (Fig. 9), carbon coated (CARB) zirconia, or as secondary bonded C18 zirconia columns (Diamondbond). The columns have been extensively studied with a range of mobile phases. The initial studies on zirconia PBD columns suggested a high temperature stability up to 200 ◦ C [75], however, the commercially marketed columns have a recommended limit of 150 ◦ C. The stability of these phases was confirmed by Wu et al. [76], who found a PBD zirconia monolithic capillary column to be stable at 150 ◦ C for over 200 h but could be used up to 260 ◦ C. Because of the low viscosity of water at 120 ◦ C, Yan et al. [77] were able to use high flow rates up to 12 ml/min to achieve the separation of five phenols in less than 30 s on a polystyrene coated zirconia column. Fields et al. [43] found that the order of elution of a number of steroids on a zirconia PBD column at 160 ◦ C was similar to that using acetonitrile–water 35:65 at room temperature on an ODS silica column. ZirChrom PBD and CARB packed capillary columns at up to 370 ◦ C and 300 ◦ C, respectively, and pressures up to 11000 psi were used by Kephart and Dasgupta [25] for the separation of phenols and alkylbenzenes. The relative elution order of nitrobenzene and toluene changed with temperature on the CARB column but not on the PBD material. 5.7. Carbon-based columns Porous graphitised carbon (PGC) stationary phases [78–80] are produced at high temperatures and should have high thermal stability and they have been successfully used for SHWC (Fig. 10) [81]. However, PGC columns have a highly active surface and are subject to contamination problems and peak shapes are often asymmetric [3]. Despite their high temperature stability towards degradation, the column performance does deteriorate

R.M. Smith / J. Chromatogr. A 1184 (2008) 441–455

449

Fig. 11. Separation of oligomeric ethylene glycols at 125 ◦ C on PRP-1 column with UV detection at 195 nm [84]. Reproduced with permission.

6.1. Spectroscopic detectors

Fig. 10. Chromatograms showing the separation of the triazines released from X-Terra trap in dynamic mode at (A) 180 ◦ C and (B) 200 ◦ C, separated on PGC (100 mm × 2.1 mm i.d) analytical column with gradient temperature from 160 to 260 ◦ C, 15 ◦ C/min. Experimental conditions: mobile phase, 100% water; flow rate, 1.0 mL/min: detection, 222 nm. Peaks: 1, propazine; 2, atrazine; 3, simazine; 4, ametryn; 5, terbutryn [111]. Reproduced with permission.

with time, apparently due to mechanical stress because of the differences in the thermal expansion of the PGC and the stainless steel column material [81]. 6. Detection in superheated water chromatography Detection methods in conventional liquid chromatographic methods are often constrained by the presence of the organic solvent in the mobile phase. This has lead to the dominance of UV-visible and fluorescence spectroscopic methods of detection, with the alternatives of refractive index and electrochemical method playing a minor role. More universal methods, such as evaporative light scattering, mass spectrometry, and charged aerosol detection, are attractive but have limitations often determined by the volatility of the analytes. One of the interests in using an organic solvent-free eluent is that it makes alternative detection techniques feasible, in particular flame ionisation detection, and simplifies others, such as mass spectrometry by maintaining a constant eluent composition. Because thermal programming can be used in SHWC, compositional gradients are not required to elute analytes with a wide range of polarities. The separation can therefore be carried out with a constant eluent composition, which is an advantage in MS, where solvent changes can alter the ionisation process, and for the nebuliser systems, such as the ELSD where the spray properties remain constant. As well as the detectors described below for SHWC, refractive index detection has been used for carbohydrates and aliphatic acids [82] and electrochemical detection for phenols (A. Orta, R.M. Smith unpublished work). Guillarme and Heinisch [83] have recently reviewed detection methods for high temperature liquid chromatography, including SHWC.

Spectroscopic, especially UV/visible absorbance, detection has been widely used in SHWC. The use of 100% water as the eluent has the advantage that there is no background eluent absorbance. This was exploited by Yarita et al. [84] to detect polyethylene glycols at 190 nm (Fig. 11). The only limitation in SHWC is the need to ensure that the detector flow cell can withstand any back-pressure that has been applied to the column to prevent the water from boiling. If the pressure control has to be placed before the detector flow cell it can contribute to band broadening. It is also desirable to control the eluent temperature after the column before it reaches the flow cell, because temperature changes can cause baseline noise. Fluorescence detection has been used for vitamins [85] and salicylamide [86]. 6.2. GC-based detectors The absence of an organic solvent from the eluent means that flame-based gas chromatographic detectors can be used and these have been reported by a number of research groups. Earlier attempts to couple LC and the flame ionisation detector required a method to eliminate the organic component of the eluent, usually with some form of transport system, and had limited success. As with linking HPLC to MS, the main developments have been interfacing the superheated water separation and detection system to handle involatile analytes and controlling the quantity of water vapour that can be passed to the FID flame without overloading the combustion. In the earliest SHWC study, Guillemin et al. [15] linked an ambient temperature water separation of aromatic compounds, carbohydrates and iprodione, to an FID with good results, however, no experimental details were provided. An early example of the linkage of the FID for volatile analytes from an ambient aqueous eluent was reported by Bruckner et al. [87], who used a drop-headspace interface in which the analytes were blown from droplets at the end of the column into the flame with a steam of helium. When SHWC was examined in more detail the potential of the linkage to the FID was rapidly recognised. The first studies usually employed a heated capillary tube (at 300–400 ◦ C) to provide both the back-pressure for the column and the interface

450

R.M. Smith / J. Chromatogr. A 1184 (2008) 441–455

to the flame ionisation detector, in a similar way to the use of the thermospray interface for conventional LC–MS. To maintain the FID flame, it was necessary to markedly increase the hydrogen flow, typically to 300 ml/min, and to optimise the air supply to compensate for the high volume of water vapour passing through the flame. In addition it was often necessary to restrict the mobile phase flow. For example, Miller and Hawthorne [21] reported flow rates were limited to 50–200 ␮l/min. They found detection limits of 1 ng for the volatile alkanols but also reported the determination of involatile compounds, including caffeine, guiacol, hydroxyphenols and a number of amino acids. Smith et al. [3] reported the detection of parabens in both isothermal and thermal gradient elution, with a similar interface but in later work found problems with blocking of the heated capillary, especially when analysing involatile analytes, such as carbohydrates. By separately thermostatting a 50 ␮m capillary restrictor at 75 ◦ C, Ingelse et al. [88] prevented the superheated water from sputtering in the restrictor which gave improved signal stability during the detection of alcohols and aldehydes separated 175 ◦ C. An alternative approach was taken by Yang et al. who used a conventional column and then split the effluent flow so that only 40 ␮l/min passed into the flame. They reported the determination of carbohydrates, carboxylic acids and amino acids with a linear response and detection limits of 200–600 ng/␮l of which about around 50–60 ng reached the detector [89]. In a later study [87], they employed a microbore column and were able to pass the complete flow of 20 ␮l/min to the detector, which was held at 400 ◦ C. They obtained detection limits of 0.3–3 ng for the amino acids and were also able to separate and detect carbohydrates. Finell et al. [91] found that in a split flow system only 0.12 ml/min could be directed to the FID, which had sensitivity for aromatic esters approximately 100-fold less than a DAD at 192 nm. They also had problems with blocking and/or corrosion of both silica and metal restrictors. A number of other groups also examined volatile analytes with a split flow, including Nakajima et al. [92], who determined the lower alkanols, and in a study of alcoholic beverages [93] found equivalent values to GC–FID measurements. In a similar study Guillarme et al. [94] separated the C1 –C6 alcohols on a narrow bore column with flow rate from 20 to 100 ␮l/min and found a FID sensitivity comparable to GC–FID detection. They carried out a detailed optimisation of the gas and liquid flow rates and the detector temperature. Direct vaporisation through a heated metal capillary at greater than 390 ◦ C was used by Wu et al. [76] to detect alkanols and phenols. Fogwill and Thurbide [37] used a post-column splitter consisting of two fused silica capillaries to reduce the flow to an FID. In all these studies, the temperature of the detector was important and position of the tip of the restrictor relative to the flame had to be optimised. Rather than an open capillary column jet, Shen et al. [95] used a restrictor consisting of metal beads sintered inside a capillary tube to control the split flow into a FID held at 400 ◦ C for the detection of a series of anilines. As part of a series of studies of the potential applications of GC detectors to LC Hooijschuur et al. [96] interfaced an eluent-jet with an inverted FID flame for the FIA–FID

Fig. 12. Separation of a mixture of aliphatic and aromatic alcohols on PS-DVB column at 140–180 ◦ C at 7◦ C/min with UV and FID detection [1]. Reproduced with kind permission of Springer Science and Business Media.

detection of phenols, carboxylic acids and amino acids in water and for the micro aqueous LC of alkanols and bis(2hydroxyethylthio)alkanes at around 10 ␮l/min. An alternative approach was proposed by Smith and coworkers [97,98] in which an ambient temperature nebuliser and spray chamber were used to generate a mist, which carried the analytes into the flame. This method was compatible with column flow rates of up to 1 ml/min and could be used to examine wide range of aliphatic and aromatic volatile and involatile analytes (Fig. 12), including carbohydrates, amino and carboxylic acids. Linear responses over a wide range were obtained in each case. A different approach was taken by Kephart and Dasgupta [25], who used a capillary column linked to an FID with no final restrictor for the separation of alkylbenzenes. This configuration allowed the water to vaporise at some point along the column during a temperature ramp from 50 to 250◦ at 50 ◦ C/min. 6.3. NMR and MS spectrometric detection The information available from modern NMR spectrometry makes the combination with HPLC [99] a very powerful method for structural identification, especially in areas such as natural product chemistry [100] and even more so when linked to mass spectrometry. Initially, NMR spectrometers were relatively insensitive, requiring typically 100–40 ␮g on-column for flow studies but subsequent improvements in cell design and operation have reduced this limit. In addition, stop–flow techniques are available that enable the spectra to be measured over a longer time scale. However, the presence of organic solvents and water in the eluent can cause interfering peaks even though some can be suppressed by using pulse techniques. The use of SHWC firstly removes the need for expensive deuterated organic solvents and also offers the possibility of

R.M. Smith / J. Chromatogr. A 1184 (2008) 441–455

451

Fig. 13. IR, UV, NMR and MS spectra obtained from 113 ␮g antipyrine following chromatography on Oasis HLB column at 185 ◦ C [105]. Reproduced by permission of the Royal Society of Chemistry.

using only moderately expensive deuterium oxide as the eluent, which has a negligible background signal. Because the SHWC system has to be outside the magnetic field, the connecting tubing provides sufficient back-pressure and cooling so that as the eluent reaches the spectrometer flow cell, it is at ambient pressure and temperature. This means that if the flow is stopped the sample remains in the flow cell and can be subject to prolonged examination. This coupling was first demonstrated for a series of barbiturates by Smith et al. [101] and was used to examine a number of polar vitamins [85]. Possible applications to more complex mixtures were demonstrated for a mixture of kava lactones [102] and an extract of ginger [103]. By splitting the eluent flow, it was possible to link both NMR spectrometry and MS to SHWC. This method was used for a number of model drug compounds [86] and also demonstrated that the COSY spectra of salicylamide could be readily obtained in a stop–flow mode. During the separation, the weakly acidic protons of the methyl groups in sulfamerazine and sulfamethazine underwent an unexpected deuterium exchange, which could be easily identified from the results from the two detectors [104]. In a number of studies, Wilson and co-workers [105] showed that the use of multiple detectors could be extended to SHWC with LC–NMR–MS–IR–UV for the examination of pharmaceuticals (Fig. 13) and for the identification of ecdysteroids [106]. A recent study [107] has examined high temperature separations on porous graphitic carbon with mass spectrometry and reported the separation of purines, pyrimidines and nucleic acids with 100% water using isothermal and temperature gradient conditions. They reported the effect of the eluent temperature on the sensitivity and analysis times.

7. Applications of superheated water chromatography One of the initial concerns with working at the high temperatures employed in SHWC was that compounds would be labile and would degrade or rearrange. With a few exceptions these worries have proved ungrounded and confirmed the expectations from GLC. Firstly, the sample exposure time is typically short, from 5 to 30 min and typical thermal reaction involves dehydration, which is unlikely to occur in an aqueous environment. Oxidation reactions will usually be avoided because the eluent has been degassed or flushed with nitrogen to avoid rusting. For example, although it might be expected that the alkyl p-hydroxybenzoates (parabens) could suffer either oxidation or hydrolysis when they were examined at up to 200 ◦ C, no degradation was observed [20]. Only a few cases of degradation have been noted in our laboratory. Thiamine could be chromatographed at 50 ◦ C but an initial study at 160 ◦ C yielded a number of breakdown products, including 4-methyl-5-thiazole-ethanol, which was identified by on-line MS and NMR spectroscopy [85]. Nitrobenzene was degraded when it was examined at >220 ◦ C on a PS-DVB column but could be readily assayed at a lower temperature on a less retentive column. Andersson et al. [108] studied the degradation of polycyclic aromatic hydrocarbons in pressurised hot water for up to 4 h to determine possible losses on superheated water extraction. At 300 ◦ C, most of the analytes degraded in 10 min and there were some losses even at 100 ◦ C. Thompson and Carr [109] examined the temperature stability of a number of basic drugs and alkaloids in a reversed phase eluent and found that the stability reflected the degradation rate at ambient conditions and the residence time on the column as expressed

452

R.M. Smith / J. Chromatogr. A 1184 (2008) 441–455

Table 2 Examples of superheated water separations of aliphatic, aromatic and polymeric compounds Compound Alkanol, alkylbenzenes Alkanols, phenols, amino acids Alkanols Alcohols, aldehydes Aliphatic acids and alkylphosphoric acids Alkanols, phenols Alkanols Alkanols (C1 –C6 ) Aliphatic and aromatic test compounds Alkylbenzenes, phenylalkanols Alkylbenzenes Alkylbenzenes Amino acids, aminophenols Aromatic standards and alkyl aryl ketones Carbohydrates, amino acids and carboxylic acids Diethyl phthalate and halogenated alkylbenzenes Nitro, halo, and alkyl substituted anilines Phenols, anilines, parabens Phenols, aryl amides, aryl aldehydes, alkyl aryl ketones, parabens Phenols Phenols and chlorophenols Phenols, polyhydroxybenzenes, pyridine, aniline Phenols, aniline, alkylbenzenes C18 columns Phenols Polyethylene glycols

Column

Temperature

Detector

Reference

Spherosil XOA 600 PRP-1 PRP-1 column and C30 Silica gel ODS silica, Hypercarb and PS-DVB Kovasil MS-H

150 ◦ C

RI FID FID FID FID

[15] [21] [92,93] [88] [96]

FID

[76]

HyperCarb or Zirchrom-PDB PLRP-S 100 C18 bonded ethylene bridged-hybrid phase ZirChrom PBD ZirChrom Carb capillary columns Zirchrom-DB-18 PRP-1 PLRP-S

Up to 260 ◦ C Up to 200 ◦ C At 57◦ C/min 120 ◦ C Up to 180◦ Up to 190 ◦ C Up to 370 ◦ C 150 ◦ C Up to 100 ◦ C 100–200 ◦ C

FID FID UV UV UV or FID UV 254 nm FID UV 254

[37] [94] [26] [69] [25] [44] [90] [48]

Hypercarb and PRP-1

80–160 ◦ C

FID

[89]

ZirChrom-PDB

175 ◦ C

UV

[28]

Xbridge C18 Various PLRP-S

Up to 200 ◦ C Up to 200 ◦ C Up to 210 ◦ C

FID and UV UV/FID/NMR UV

[95] [3] [19,20]

Zorbax RXC8 PRP-1 Hypersil ODS Zirchrom PBD PRP-1 PRP-1 Zorbax RX C18 Chromatorex C-18

Up to 140◦ 100–150 ◦ C Up to 200 ◦ C

UV UV UV

[55] [111] [27]

PRP-1 and Nucleosil C18 AB

100–200 ◦ C

UV

[112]

PS-Zirchrom PRP-1

120 ◦ C 125 ◦ C

UV 195 nm

[77] [84]

100–175 ◦ C 125 ◦ C Various

PBD encapsulated zirconia PBD coated zirconia monolith PRP-1

by the Damk¨ohler number. Only norpseudoephedrine showed any significant degradation. They concluded that many complex compounds should be sufficiently stable to be analysed at high temperature.

The SHWC of a wide range of analytes have been reported using different columns and conditions and with different methods of detection. The initial studies tended to concentrate on polar analytes, such as alkanols and phenols, as these were rela-

Table 3 Superheated water separations of typical pharmaceuticals Compound

Column

Temperature

Detector

Reference

Analgesics, salicylamide, caffeine, paracetamol, and phenacetin Anti-cancer drugs Barbiturates Barbiturates Caffeine, phenacetin Paracetamol, caffeine, antipyrine, dimethylantipyrine, phenacetin

PLRP-S or Novapak C18

80–130 ◦ C at 8◦ C/min

UV–F–NMR

[86]

Nucleogel RP (PS-DVB) PSRP-S PLRP-S XTerra C8 Oasis HLB PLRP-S Oasis 40 ZirChrom PDB ZirChrom CARB Hypercarb BDS Hypersil Hypercarb PLRP-S PLRP-S ZirChrom-PBD ZirChrom PBD PLRP-S Acquity C18

160 ◦ C 200 ◦ C 100–200 ◦ C Up to 185 ◦ C Various up to 225 ◦ C

UV 254 nm UV–NMR UV 254 nm NMR–IR–UV–MS UV

[30] [101] [48] [105] [52]

100–200 ◦ C 70–190 ◦ C at 2 ◦ C/min 160–200 ◦ C at 2◦ C/min 160 ◦ C 100–150 ◦ C Various 50–200 ◦ C 40–180 ◦ C

UV 254 UV 254 UV, NMR, MS UV UV 195 nm UV–F–MS–NMR UV

[107] [29] [104] [43] [113] [85] [31]

Purines, pyrimidines Sulfonamides Sulfonamides Steroids, including testosterone Triazole fungicides Vitamins, pyridoxine, riboflavin, thiamine Range of pharmaceuticals

R.M. Smith / J. Chromatogr. A 1184 (2008) 441–455

453

Table 4 Separation of natural product extracts with superheated water chromatography Samples Kava lactones Ginger extracts Ecdysteroids

Column Zirconia PBD Xterra RP18 Xterra C8

Temperature 2 ◦ C/min

80–100–160 at 50–130 ◦ C at 4◦ C/min 160 ◦ C

Detector

Reference

UV–NMR UV and LC–NMR (D2 O) UV–IR–NMR–MS

[102] [103] [106]

Table 5 Examples of hyphenated on-line and off-line superheated water extraction and superheated water chromatographic system Sample

Trap (normally on-line)

Column

Column temperature

Detection

Reference

Isoflavanoids Pharmaceuticals Anilines, phenols Flavones Triazine herbicides on compost Alkylbenzenes

MCI Gel PLRP-S ZirChrom-PS (Off-line) Xterra (unbonded) Direct connection

PLRP-S PLRP-S ZirChrom-PS Discovery HS PEG Hypercarb Bonded non-porous silica

170 ◦ C 75–195 at 15 ◦ C/min 80 ◦ C 100 ◦ C 130–160 ◦ C Ambient

UV UV UV 254 nm UV 254 nm UV 222 nm UV 200 nm

[40] [39] [41] [41] [111] [66]

tively easy to elute from the PS-DVB columns. Later studies expanded the range to include esters, such as the parabens, amides, esters and even the non-polar alkane and alkylbenzenes as less retention columns became available (Table 2). As well as examining the scope of SWHC, these methods were also used to test the compatibility of different detectors. Many studies have also examined potential application areas in particular pharmaceuticals (Table 3) where the polarity of analytes is often compatible with an aqueous eluent. One possible advantage here is that the presence of water in the sample has no effect on the chromatography and pH control of the eluent can also be used as well as highly sensitive MS detection. The other area, where SHWC is envisaged to offer an advantage, is in the environmental and food industry and this has been the subject of a number of studies (Table 4). In both cases, analyte stability is important and before any quantitative work is carried out it is necessary to check recoveries and ensure that the high temperatures don’t result in artefact peak formation. 7.1. Hyphenated systems with SHWE Analytes from aqueous solutions can be trapped on a cartridge column and then released in a more concentrated solution by thermal desorption for subsequent LC or SHWC analysis [110] and Tajuddin and Smith [39] showed that it was possible to achieve a sequential series of releases from the trap by increasing the temperature in steps (Fig. 14). The initial extraction can be carried out with superheated water and if SHWC is then used for the analysis an integrated solvent free assay can be developed [41] (Table 5). An on-line system (Fig. 4) was reported by Tajuddin and Smith [81] for the determination of triazine herbicides from compost, in which extraction, concentration, fractionation, and chromatography were carried out sequentially in a series of linked traps and columns with a single water source, in which the extraction, trapping, release, and chromatography steps are all temperature controlled.

Fig. 14. Separation of test mixture and fractions after extraction and trapping and sequential elution at temperatures. Separations on PS-DVB column at 75–185 ◦ C at 15 ◦ C/min. Analytes: 1, paracetamol; 2, salicylamide; 3, caffeine; 4, methyl paraben; 5, phenacetin; 6, ethyl paraben. Separations: a, direct injection of original mixture of 1–6 without trapping; b, fraction untrapped at ambient temperature; c, fraction released from trap at 70 ◦ C; d, released at 90 ◦ C; e, released at 110 ◦ C [39]. Reproduced by permission of the Royal Society of Chemistry.

8. Conclusions Advances in thermally stable stationary phases are making superheated water chromatography a more readily accessible

454

R.M. Smith / J. Chromatogr. A 1184 (2008) 441–455

method of liquid chromatography, with the potential to provide an efficient green and environmentally friendly separation method. It offers a number of unique possibilities for detection, in particular with coupled detectors and GC detectors, such as the flame ionisation detector. More assays are continually being demonstrated, and together with linked extraction methods, are expanding the applications area. References [1] R.M. Smith, Anal. Bioanal. Chem. 385 (2006) 419. [2] Y. Yang, J. Sep. Sci. 30 (2007) 1131. [3] R.M. Smith, R.J. Burgess, O. Chienthavorn, J.R. Stuttard, LC–GC Internat. 12 (1999) 30; LC–GC N. Am. 17 (1999) 938. [4] J.W. Coym, J.G. Dorsey, Anal. Lett. 37 (2004) 1013. [5] Y. Su, J. Jen, F.-W. Zhang, Chin. J. Chromatogr. 23 (2005) 238. [6] R.M. Smith, J. Chromatogr. A 975 (2002) 31. [7] V.G. Berezkin, Adv. Chromatogr. 41 (2001) 337. [8] T.L. Chester, J.D. Pinkston, Anal. Chem. 76 (2004) 4606. [9] T. Greibrokk, T. Andersen, J. Chromatogr. A 1000 (2003) 743. [10] E. Lundanes, T. Greibrokk, Adv. Chromatogr. 44 (2006) 45. [11] R.M. Smith, in: Y. Kazakevich, R. Lobrutto (Eds.), HPLC for Pharmaceutical Scientists, Wiley, Hoboken N.J, 2007, p. 811. [12] J.W. Dolan, J. Chromatogr. A 965 (2002) 195. [13] G. Vanhoenacker, P. Sandra, J. Sep. Sci. 29 (2006) 1822. [14] C.M. White (Ed.), Modern Supercritical Fluid Chromatography, H¨uthig, Heidelberg, 1988. [15] C.L. Guillemin, J.L. Millet, J. Dubois, J. High Resolut. Chromatogr. 4 (1981) 280. [16] S.B. Hawthorne, Y. Yang, D.J. Miller, Anal. Chem. 66 (1994) 2912. [17] D.J. Miller, S.B. Hawthorne, A.M. Gizir, A.A. Clifford, J. Chem. Eng. Data 43 (1998) 1043. [18] C.A. Meyer, Steam Tables: Thermodynamic and Transport Properties of Steam, American Society of Mechanical Engineers, New York, 1993. [19] R.M. Smith, R.J. Burgess, Anal. Commun. 33 (1996) 327. [20] R.M. Smith, R.J. Burgess, J. Chromatogr. A 785 (1997) 49. [21] D.J. Miller, S.B. Hawthorne, Anal. Chem. 69 (1997) 623. [22] M. Siskin, A.R. Katritzky, Chem. Rev. 101 (2001) 825. [23] A.R. Katritzky, D.A. Nichols, M. Siskin, R. Murugan, M. Balasubramanian, Chem. Rev. 101 (2001) 837. [24] H. Weing¨artner, E.U. Franck, Angew. Chem. 44 (2005) 2672. [25] T.S. Kephart, P.K. Dasgupta, Talanta 56 (2002) 977. [26] T.M. Pawlowski, C.F. Poole, Anal. Commun. 26 (1999) 71. [27] T. Kondo, Y. Yang, Anal. Chim. Acta 494 (2003) 157. [28] J.W. Coym, J.G. Dorsey, J. Chromatogr. A 1035 (2004) 23. [29] O. Chienthavorn, R.M. Smith, Chromatographia 50 (1999) 485. [30] T. Teutenberg, O. Lerch, H.-J. G¨otze, P. Zinn, Anal. Chem. 73 (2001) 3896. [31] A.M. Edge, S. Shillingford, C. Smith, R. Payne, I.D. Wilson, J. Chromatogr. A 1132 (2006) 206. [32] L. Spearman, R.M. Smith, S. Dube, J. Chromatogr. A 1060 (2004) 147. [33] N.M. Djordjevic, P.W.J. Fowler, F. Houdiere, J. Micocol. Sep. 11 (1999) 403. [34] B.A. Jones, J. Liq. Chromatogr. 27 (2004) 1331. [35] T. Teutenberg, H.-J. Goetze, J. Tuerk, J. Ploeger, T.K. Kiffmeyer, K.G. Schmidt, W.gr. Kohorst, H. Rohe, H.-D. Jansen, H. Weber, J. Chromatogr. A 1114 (2006) 89. [36] F. Harvey Doyle, R.M. Smith, J.K. Roberts, P.A. James, 29th International Symposium on High Performance Liquid Phase Separations and Related Techniques 26–30 June, Stockholm, 2005. [37] M.O. Fogwill, K.B. Thurbide, J. Chromatogr. A 1139 (2007) 199. [38] E. Loeser, P. Drumm, J. Sep. Sci. 29 (2006) 2847. [39] R. Tajuddin, R.M. Smith, Analyst 127 (2002) 883. [40] M. Kivilompolo, K.E. Vainikka, K. Hartonen, T. Hy¨otyl¨ainen, M.L. Riekkola, 11th International Symposium on Supercritical Fluid Chro-

[41] [42] [43] [44] [45] [46] [47]

[48] [49] [50] [51] [52] [53] [54] [55] [56] [57] [58] [59] [60] [61] [62] [63] [64] [65] [66] [67] [68]

[69]

[70]

[71] [72] [73] [74] [75] [76] [77] [78] [79] [80] [81] [82] [83]

matography, Extraction and Processing, Pittsburgh, August 2004, poster C-14. L.J. Lamm, Y. Yang, Anal. Chem. 75 (2003) 2237. J.D. Thompson, J.S. Brown, P.W. Carr, Anal. Chem. 73 (2001) 3340. S.M. Fields, C.Q. Ye, D.D. Zhang, B.R. Branch, X.J. Zhang, N. Okafo, J. Chromatogr. A 913 (2001) 197. D. Guillarme, S. Heinisch, J.L. Rocca, J. Chromatogr. A 1052 (2004) 39. R.G. Wolcott, J.W. Dolan, L.R. Snyder, S.R. Bakalyar, M.A. Arnold, J.A. Nichols, J. Chromatogr. A 869 (2000) 211. G. Mayr, T. Welsch, J. Chromatogr. A 845 (1999) 155. M.G. Tebrake, R.M. Smith, S.A.C. Wren, I.D. Wilson, 25th International Symposium on High Performance Liquid Phase Separations and Related Techniques, HPLC’ 2001, 17–22 June, Maastricht, 2001. M.M. Sanagi, H.H. See, J. Liq. Chromatogr. 28 (2005) 3065. F. Gritti, G. Guiochon, Anal. Chem. 78 (2007) 4642. Y. Yang, M. Belghazi, A. Lagadec, D.J. Miller, S.B. Hawthorne, J. Chromatogr. A 810 (1988) 149. H.A. Claessens, M.A. van Straten, J. Chromatogr. A 1060 (2004) 23. I.D. Wilson, Chromatographia 52 (2000) S-28. S.J. Marin, B.A. Jones, W.D. Felix, J. Clark, J. Chromatogr. A 1030 (2004) 255. P. He, Y. Yang, J. Chromatogr. A 989 (2003) 55. Y. Yang, L.J. Lamm, P. He, T. Kondo, J. Chromatogr. Sci. 40 (2002) 107. T. Teutenberg, J. Tuerk, M. Holzhauer, S. Giegold, J. Sep. Sci. 30 (2007) 1101. N. Nagae, T. Enami, S. Doshi, LC–GC N. Am. 20 (2002) 964; LC–GC Eur. 16 (2003) 418. R.D. Morrison, J.W. Dolan, LC–GC Eur. 13 (2000) 720. R. E. Majors, M. Przybyciel, LC–GC N. Am. 20 (2002) 584; LC–GC Eur. 15 (2002) 780. W. Kiridena, C.F. Poole, W.W. Koziol, Chromatographia 57 (2003) 703. T.H. Walter, P. Iraneta, M. Capparella, J. Chromatogr. A 1075 (2005) 177. T. Enami, N. Nagae, Bunseki Kagaku 53 (2004) 1309. S.W. Pettersson, B.S. Persson, M. Nystr¨om, J. Chromatogr. B 803 (2004) 159. Shimadzu News 1 (2004) 8. J.A. Lippert, T.M. Johnson, J.B. Lloyd, J.P. Smith, B.T. Johnson, J. Furlow, A. Procter, S.J. Marin, J. Sep. Sci. 30 (2007) 1141. T.E. Young, S.T. Ecker, R.E. Synovec, N.T. Hawley, J.P. Lomber, C.M. Wai, Talanta 45 (1998) 1189. J.E. O’Gara, K.D. Wyndham, J. Liq. Chromatogr 29 (2006) 1025. Y.F. Cheng, T.H. Walter, Z. Lu, P. Iraneta, B.A. Alden, C. Gendreau, U.D. Neue, J.M. Grassi, J.L. Carmody, J.E. O’Gara, R.P. Fisk, LC–GC N. Am. 18 (2000) 1162. K.D. Wyndham, J.E. O’Gara, T.H. Walter, K.H. Glose, N.L. Lawrence, B.A. Alden, G.S. Izzo, C.J. Hudalla, P.C. Iraneta, Anal. Chem. 75 (2003) 6781. Y. Liu, N. Grinberg, K.C. Thompson, R.M. Wenslow, U.D. Neue, D. Morrison, T.H. Walter, J.E. O’Gara, K.D. Wyndham, Anal. Chim. Acta 554 (2005) 144. L.A. Al-Khateeb, R.M. Smith, manuscript in preparation. J. Nawrocki, C. Dunlap, A. McCormick, P.W. Carr, J. Chromatogr. A 1028 (2004) 1. J. Nawrocki, C. Dunlap, J. Li, J. Zhao, C.V. McNeff, A. McCormick, P.W. Carr, J. Chromatogr. A 1028 (2004) 31. J. Nawrocki, M.P. Rigney, A. McCormick, P.W. Carr, J. Chromatogr. A 657 (1993) 229. J. Li, Y. Hu, P.W. Carr, Anal. Chem. 69 (1997) 3884. N. Wu, Q. Tang, J.A. Lippert, M.L. Lee, J. Microcol. Sep. 13 (2001) 41. B. Yan, J. Zhao, J.S. Brown, J. Blackwell, P.W. Carr, Anal. Chem. 72 (2000) 1253. J.H. Knox, P. Ross, Adv. Chromatogr. 37 (1997) 73. J.H. Knox, P. Ross, Adv. Chromatogr. 37 (1997) 121. E. Forgacs, J. Chromatogr. A 975 (2002) 229. R. Tajuddin, R.M. Smith, J. Chromatogr. A 1084 (2005) 194. E. Young, R.M. Smith., manuscript in preparation. D. Guillarme, S. Heinisch, Sep. Purif. Rev. 34 (2005) 181.

R.M. Smith / J. Chromatogr. A 1184 (2008) 441–455 [84] T. Yarita, R. Nakajima, K. Shimada, S. Kinugasa, M. Shibukawa, Anal. Sci. 21 (2005) 1001. [85] O. Chienthavorn, R.M. Smith, S. Saha, I.D. Wilson, B. Wright, S.D. Taylor, E.M. Lenz, J. Pharm, Biomed. Anal. 36 (2004) 477. [86] R.M. Smith, O. Chienthavorn, I.D. Wilson, B. Wright, S.D. Taylor, Anal. Chem. 71 (1999) 4493. [87] C.A. Bruckner, S.T. Ecker, R.E. Synovec, Anal. Chem. 69 (1997) 3465. [88] B.A. Ingelse, H.G. Janssen, C.A. Cramers, J. High Resolut. Chromatogr. 21 (1998) 613. [89] Y. Yang, A.D. Jones, J.A. Mathis, F.A. Francis, J. Chromatogr. A 942 (2001) 231. [90] Y. Yang, T. Kondo, T.J. Kennedy, J. Chromatogr. Sci. 43 (2005) 518. [91] J. Finell, J. Tanner, H. Vettenranta, K. Hartonen, M.L. Riekkola, 11th International Symposium on Supercritical Fluid Chromatography, Extraction and Processing, Pittsburgh, August 2004, poster B-09. [92] R. Nakajima, T. Yarita, M. Shibukawa, Bunseki Kagaku 52 (2003) 305. [93] T. Yarita, R. Nakajima, S. Otsuka, T. Ihara, A. Takatsu, M. Shibukawa, J. Chromatogr. A 976 (2002) 387. [94] D. Guillarme, S. Heinisch, J.Y. Gauvrit, P. Lanteri, J.L. Rocca, J. Chromatogr. A 1078 (2005) 22. [95] S. Shen, H. Lee, J. McCaffrey, N. Yee, C. Senanayake, N. Grinberg, J. Clark, J. Liq. Chromatogr. 29 (2006) 2823. [96] E.W.J. Hooijschuur, C.E. Kientz, U.A. Th Brinkman, J. High Resolut. Chromatogr. 23 (2000) 309. [97] J.R. Bone, R.M. Smith, B.L. Sharp, Patent Application WO 0218939 A3 (2000).

455

[98] R.M. Smith, E. Young, J.R. Bone, B.L. Sharp, manuscript in preparation. [99] K. Albert (Ed.), On-line LC-NMR and Related Techniques, Wiley, 2002. [100] J.L. Wolfender, K. Ndjoko, K. Hostettmann, Phytochem. Anal. 12 (2001) 2. [101] R.M. Smith, O. Chienthavorn, I.D. Wilson, B. Wright, Anal. Commun. 35 (1998) 261. [102] O. Chienthavorn, R.M. Smith, I.D. Wilson, B. Wright, E.M. Lenz, Phytochem. Anal. 16 (2005) 217. [103] S. Saha, R.M. Smith, E. Lenz, I.D. Wilson, J. Chromatogr. A 991 (2003) 143. [104] R.M. Smith, O. Chienthavorn, S. Saha, I.D. Wilson, B. Wright, S.D. Taylor, J. Chromatogr. A 886 (2000) 289. [105] D. Louden, A. Handley, S. Taylor, I. Sinclair, E. Lenz, I.D. Wilson, Analyst 126 (2001) 1625. [106] D. Louden, A. Handley, R. Lafont, S. Taylor, I. Sinclair, E. Lenz, T. Orton, I.D. Wilson, Anal. Chem. 74 (2002) 288. [107] L. Pereira, S. Aspey, H. Ritchie, J. Sep. Sci. 30 (2007) 1115. [108] T. Andersson, K. Hartonen, T. Hy¨otyl¨ainen, M.L. Riekkola, Analyst 128 (2003) 150. [109] J.D. Thompson, P.W. Carr, Anal. Chem. 74 (2002) 1017. [110] J.R. Bone, R.M. Smith, Anal. Commun. 36 (1999) 375. [111] T. Yarita, T. Nakajima, M. Shibukawa, Anal. Sci. 19 (2003) 269; reprinted in AIST Bull. Metrol. 2 (2004) 555. [112] Y. Yang, A.D. Jones, C.D. Eaton, Anal. Chem. 71 (1999) 3808. [113] M.M. Sanagi, H.H. See, W.A. Wan Ibrahim, A.A. Naim, J. Chromatogr. A 1059 (2004) 95.