Two-dimensional gas chromatography (GC×GC) and GC×GC–time-of-flight (TOF)-mass spectrometry (MS) have received increasing attention as powerful techniques for separation and analysis of extremely complex mixtures. Over the past 20 years, solid-phase microextraction (SPME) has become a staple sample preparation technique for a large variety of GC analyses. With a few additional considerations, SPME is combined readily with GC×GC or GC×GC–TOF-MS to provide easily automated and solvent-free sample preparation. This combination provides up to six dimensions of separation power in analytical method development that can be used to provide the required selectivity or can cause unwanted selectivity through sample losses or slow kinetics. In this article, the combination of SPME with GC×GC and GC×GC–TOF-MS is discussed using mixtures of solvents as example analytes. Optimization of the injection process is emphasized, as extractions remain generally the same as in traditional SPME–GC. To ensure good peak shapes, the injection liner, carrier gas flow rate and column temperatures must be optimized carefully. The differences in carrier gas flow optimization between GC×GC with flame ionization detection and GC×GC–TOF-MS also are discussed.

Since the development of comprehensive two-dimensional gas chromatography (GC×GC) by Phillips and coworkers in 1990, and most notably in the past five years, GC×GC and multidimensional GC by heartcutting have become increasingly popular among analysts in a variety of disciplines.

  A number of recent reviews describe advances in GC×GC that demonstrate its high selectivity and ability to separate extremely complex mixtures, so these are not repeated here.

  GC×GC is moving rapidly from the realm of being a research technique for hard-core chromatographers and into use by the broader community. GC×GC coupled with time-of-flight-mass spectrometry (GC×GC–TOF-MS) provides perhaps the ultimate in chromatographic separation and identification power, combining the separation power of multidimensional GC with the sensitive detection and identification capability of electron impact TOF-MS. A recent article demonstrating GC×GC–TOF-MS in athletic doping control illustrates the separation, detection and quantification power of GC×GC–TOF-MS.

  Further selectivity and sensitivity may be achieved by adding a concentrating and selective extraction technique, such as solid-phase microextraction (SPME), to GC×GC analysis. In this article, we discuss the principles, advantages and challenges in combining SPME with GC×GC and with GC×GC–TOF-MS. While the same extractions can be used with both traditional and multidimensional GC, there are additional injection considerations that result from the two-column combination in multidimensional separations and the additional selectivity afforded by the two column system can also require rethinking of sampling procedures and extraction chemistry.  

SPME was developed about 20 years ago as a solvent-free alternative to traditional extraction methods for the analysis of volatile organic contaminants in water.

  Headspace SPME subsequently became the method of choice for volatile compounds such as solvents.

  Because of its ease of use and automation, SPME has become one of the most popular sample preparation methods for GC. Being a solvent-free extraction and injection technique, SPME has unique properties and capabilities as an injection method.

  Most notably, there is no interference in the chromatogram from the presence of the large solvent peak found in traditional liquid injection chromatograms. There is also none of the beneficial band focusing due to solvent effects, as there is no traditional solvent. The only band-focusing mechanism available is cold trapping at the head of the analytical column. A complete discussion of the beneficial solvent effects, and how to avoid deleterious effects involved in traditional liquid injections, is provided in the comprehensive text on injection by K. Grob,

  which is a must-read for any chromatographer seeking to optimize or troubleshoot GC injections. Lacking solvent effects, SPME–GC injections must be optimized carefully, depending upon analyte volatility and instrumental conditions with an eye toward minimizing width of the injected band. 

Effective injection using SPME requires careful consideration of the desorption conditions within the inlet. Generally, desorption should be carried out as rapidly as possible, followed by thermal refocusing of the desorbed analytes at the head of the analytical column. The most important variables are: the diameter of the glass sleeve, purge time, flow rate of the carrier gas, inlet temperature, initial column temperature, stationary phase and film thickness of the capillary column.  

For lower vapour pressure analytes such as drugs or hydrocarbons larger than about C

, SPME injection conditions are usually very straightforward. Very often, a traditional straight-bore splitless glass sleeve of about 2 mm inside diameter, with no packing or glass wool, will suffice. Purge time and flow rate must be set sufficiently to fully desorb the analytes and transfer them during the splitless period to the column. This can be studied simply by varying purge time and flow rate (one at a time) and by maximizing the detector response versus the purge time or flow rate. Inlet temperature is set sufficient to volatilize the analytes but not higher than the maximum temperature for the fibre coating being used. Initial column temperature should be set low enough to focus the analytes at the column head. For less-volatile compounds, this is quite straightforward, as demonstrated in our previous work.

  The fibre is often left in the inlet for a time following the splitless period, with the purge flowing, to remove any remaining analyte or interferences and avoid carryover to the next sample. 

Figure 1, which was published originally about 12 years ago,

  demonstrates several aspects of traditional SPME–GC injections. It shows separation of several alkanes using a temperature-programmed separation on a nonpolar column following SPME. The SPME injection was performed using a large 4 mm inside diameter glass sleeve in the inlet. Generally, this wide diameter sleeve would never be used for SPME or splitless injections. This one remained in the GC system by accident during an SPME training exercise. The early eluted compounds show significant band broadening as they are transferred from the fibre to the inlet during an entire 60 s splitless period. The initial temperature of the temperature programme is not sufficiently low and the stationary phase film is not sufficiently thick to effectively retain and focus these analytes. As the molecules get larger, the peaks become sharper, finally becoming equivalently sharp as the same compound injected using a traditional fast liquid injection. These lower vapour pressure compounds are very effectively focused. This illustrates the need for much more careful SPME injection optimization for more volatile analytes, yet little optimization for less volatile analytes, assuming that they can be desorbed in the first place.  

With no solvent effects or thermal focusing, volatile compounds are much more difficult to focus at the column head, so rapid desorption and transfer to the column is a must. First, a small diameter, 0.75 mm, glass sleeve designed for SPME should be used. This will facilitate rapid desorption, but beware that the small diameter of the glass sleeve can limit the volumetric flow rate in the inlet, causing potential difficulty in using wider (0.32 mm or more) columns or high split ratio following desorption and possibly causing flow-related errors with some electronic flow controllers. The initial column temperature should be set as low as possible. In our laboratory, we attempt to optimize methods for initial column temperatures of 35 °C or higher to avoid the inconvenience of using a cryogenic oven while maintaining reasonable turnaround time between temperature programmes. A thicker stationary phase film or careful matching of the stationary phase and analyte polarities (to enhance analyte retention) will allow higher initial column temperature. However, for analysis of highly volatile compounds, a cryogenic oven and a thick stationary phase film might be needed. 

The ease of extraction and automation in SPME and the high resolving power of GC×GC and GC×GC–TOF-MS make these a natural combination. The first report of SPME–GC×GC in the literature by Perrera and colleagues

  occurred in 2002 for the analysis of volatile emissions from wounded plants. Recent applications have involved the analysis of pesticide residues in tea, identification and fingerprinting in cachaca (a distilled beverage) production, honey volatiles and bumblebee gland secretions.

  In each of these applications, headspace SPME was used under varying conditions. In the bumblebee example, for instance, a 100 mm polydimethylsiloxane (PDMS) fibre was exposed in the presence of a flying male bumblebee inside a glass container. In this article, as in many on SPME–GC, the extraction and GC conditions are described quite thoroughly, while the SPME injection conditions are missing necessary details such as inlet liner dimensions, inlet pressure during desorption and desorption temperature, often making the work difficult to reproduce without significant additional optimization. 

The experimental work described in this article illustrates the special considerations involved in the use of SPME with GC×GC and GC×GC–TOF-MS using the analysis of common solvents as examples. Solvents were chosen because, although these mixtures can be separated by traditional single dimension GC, they are familiar to nearly all gas chromatographers, providing a common point of reference. The full resolving power of GC×GC is demonstrated in the references cited herein. 

All chemicals and reagents were purchased from Sigma-Aldrich (St Louis, Missouri, USA), were of the highest purity available, and were used without further purification. Water was 18.2 MΩ Milli-Q (Millipore, Milford, Massachusetts, USA) obtained in our laboratory. SPME fibres were obtained from Sigma Aldrich Supelco (Bellefonte, Pennsylvania, USA) and were conditioned according to the manufacturer's instructions. 

The GC×GC–flame ionization detection (FID) system was obtained from LECO (St Joseph, Michigan, USA) and consisted of a model 6890 gas chromatograph (Agilent Technologies, Wilmington, Delaware, USA) modified by the addition of the secondary oven, thermal liquid nitrogen modulator and the associated pneumatics. Instrument control and data acquisition were performed using ChromaTOF software (LECO). For GC×GC–TOF-MS, a Pegasus 4D time-of-flight mass spectrometer and the associated control software (LECO) were added to the original GC×GC system. The first dimension column was a 60 m × 0.32 mm, 1.8 μm 

 DB-624 (J&W/Agilent, Wilmington, Delaware, USA) and the second dimension column was 1.5 m × 0.25 mm, 0.25 μm 

 Rtx-WAX (Restek, Bellefonte, Pennsylvania, USA).  

For GC×GC–FID work, the first dimension column was 30 m × 0.25 mm, 1 μm 

 Rtx-5 (Restek). The second dimension column was 1.1 m × 0.25 mm, 0.5 μm 

 Rtx-WAX (Restek). The first dimension column was temperature programmed with a 35 °C hold for 10 min and ramped at 10 °C/min to 220 °C.  

The carrier gas was helium (AGL, Wayne, New Jersey, USA), the inlet temperature 250 °C and the transfer line temperature 240 °C. The mass spectrometer settings were 70 eV electron impact at 200 spectra/s with mass range 30–300 Da. Glass inlet sleeves were obtained from Restek.  

Modulation was performed using a two-stage modulator with cryogenically cooled liquid nitrogen for the cold jets and heated nitrogen gas for the hot jets. The modulator was operated to provide a 2 s modulation period and programmed to maintain the same temperature as the secondary oven. The modulator gave a 0.4 s hot pulse and a 1.6 s cooling time between stages. For the FID work, the modulation period was 4 s. 

Solvent mixtures were prepared at approximately 1.00 ppm each in water by first preparing 100 ppm each in methanol and diluting 1:10 with water. Mixtures included Class 1, 2 and 3 solvents for pharmaceutical residual solvent analysis as described in the 

) General Chapter <467> Residual Solvents

  or as described in the text accompanying each figure. 

The combination of SPME with GC×GC and GC×GC–TOF-MS provides numerous possibilities for obtaining selectivity in complex separation problems. If the steps in a chromatographic method are each considered as an opportunity to separate analytes of interest from the matrix and interferences, up to six dimensions of separation — sampling, extraction, injection, first dimension separation, second dimension separation and detection — may be obtained. In each dimension, both the kinetics (rate) and thermodynamics (partitioning) of the process should be considered, along with that step's effects on the later steps in the process. For example, the enhanced separation power of GC×GC and TOF-MS might allow earlier steps such as sampling, extraction and injection to be simplified. However, poor sampling, extraction, or injection can have magnified negative effects on later GC×GC separation and detection. If sampling or extraction are ineffective or not properly performed, even the most sophisticated chromatographic and detection systems might not produce required results. The following discussion illustrates this idea for the case of SPME injection of common solvents. The literature references provide further examples of the resolving power of GC×GC. 

Figure 2 is a portion of a GC×GC–TOF-MS total ion chromatogram of a 100 ppm solvent standard, injected by traditional split injection with a split ratio of 50:1. The main plot, typical of GC×GC data, is shown as a contour plot (looking down from above onto the chromatogram surface) with the brighter red colour showing larger peaks. This is a very rapid injection process that should produce narrow peaks in the first dimension and does, in this case, with many of the peaks fully contained in a single 2 s modulation period. In the second dimension, the peaks are very sharp, ranging from about 80–150 ms in width at baseline. For SPME injections to be considered effective and to not sacrifice the resolution advantages in GC×GC, peak width and shape performance should be similar to this traditional fast injection. The benefits of both GC×GC and GC×GC–TOF-MS are seen in this plot. The two dimensional system provides complete separation of all analytes; note the separation of peak 1 from the interfering peak and the separation of peaks 12, 13 and 14, which would have been coeluted in a traditional one-dimensional separation. The addition of a TOF-MS detector allows rapid identification of each peak: at 200 spectra/s, each 2 s modulation period includes about 400 mass spectra.  

A small portion of an unoptimized SPME–GC×GC–FID chromatogram of common solvents is shown in Figure 3, showing a three-dimensional plot of one area of a larger chromatogram. The extraction was by headspace of a 100 ppm standard of several solvents using a carboxen-PDMS fibre, which is selective to small polar molecules. This chromatogram illustrates the two challenges in developing methods for volatile compounds by SPME–GC×GC: tailing or poor peak shape in both dimensions. Note the L-shaped appearance of the peaks. Tailing or additional peak broadening in the first dimension is caused by the slow injection process common in SPME injections. In the second dimension, tailing can be caused by slow modulation of the eluted first dimension peak into the second column or by multiple analyte interactions with the second dimension column, common in many separations on polar columns. Therefore, successful SPME injections in GC×GC require careful optimization of both the first and second dimension column flows and geometry. This separation is roughly based upon vapour pressure in the first dimension and on polarity or hydrogen bonding character in the second. Flow optimization in GC×GC–FID can be compromised by the necessity of high column head pressure, leading to peak broadening in both dimensions as illustrated in the classical van Deemter equation.

A portion of an unoptimized SPME–GC×GC–TOF-MS chromatogram of a solvent mixture is shown in Figure 4. The extraction was performed by direct immersion of a 100 mm polydimethylsiloxane (PDMS) fibre into 20 mL of 100 ppm solution of ICH class 1, 2 and 3 solvents in water. This is a nonpolar fibre that is by far the most popular fibre coating in use today. It is considered by many to be nonselective, but this example shows that it is, in fact quite selective. When compared to the simple liquid injection in Figure 2, several of the polar compounds are either missing or at much lower recovery, including ethyl acetate, 2-butanol, 2-methyl-1-propanol and 1,4-dioxane. Some possible unwanted selectivity from the sampling procedure may also have affected this analysis — two less-polar compounds are also missing: 2,2-dimethyl propane and 1,2-dimethoxy ethane, possibly due to evaporation from the polar aqueous solution while awaiting analysis.  

Interestingly, SPME clearly provides an additional channel of selectivity that can either complement the selectivity offered by multidimensional GC or can degrade it. This injection was performed using a 2 mm inside diameter glass sleeve, generally used for splitless liquid injections and SPME injections of less volatile analytes. Clearly, the peaks are broadened significantly and are tailing in both dimensions. The significant first-dimension broadening is due to the slow transfer of analyte from the fibre through the inlet sleeve and onto the column, with no corresponding focusing mechanism. Note that the later peaks are less broadened, as expected: these compounds are somewhat focused by the low initial column temperature. Additional broadening also is seen in the second dimension, most likely a result of both the extreme first-dimension band broadening and ineffective focusing of the modulated peaks in the second column. Also, with the high vacuum provided by the TOF-MS as detector, the second-dimension retention time was shortened to 2 s from 4 s, as seen Figure 3 and in our previous work, with no loss of resolution.

The total ion chromatogram shown in Figure 5 was optimized by using a 0.75 mm inside diameter inlet liner in place of the 2 mm liner and by increasing the column flow rate during injection to minimize the extracolumn volume in the inlet and the related peak broadening. SPME desorption occurs with the inlet in splitless mode. Although desorption from the fibre coating is quite rapid in a heated inlet, the desorbed analytes must still traverse the glass sleeve, which is often slow.

  This also demonstrated unwanted selectivity caused by a problem with the sampling procedure, as the vial containing the analytes was allowed to sit in the autosampler tray over a weekend. This clearly allowed time for several of the hydrocarbons to evaporate from the aqueous solution, reducing or eliminating them entirely from the analysis. Unfortunately, highly selective and sensitive detection might not make these compounds reappear, and even if it did, reproducible quanification would be problematic. For volatile analytes, the narrow diameter glass sleeve combined with a higher flow rate generated peaks almost as narrow as those seen in the liquid injection. Earlier eluted peaks have similar widths and shapes to the later peaks. The solvent peaks, all of which are again present, now show acceptable peak shape and little band broadening versus a traditional liquid injection.  

One challenge in both traditional liquid and SPME injections with GC×GC is that a second injection onto the second-dimension column is performed during the modulation process. On our system, this is accomplished by a two stage modulator using cold and hot jets of nitrogen to alternatively freeze narrow analyte bands at the head of the second column and then to rapidly desorb them into the column. To accomplish this, the timing sequence of the cold and hot jets must be carefully optimized. Several authors have reviewed these method development steps.

  Additionally, in GC×GC–TOF-MS, the carrier gas velocity in the second-dimension column, which is usually very short (1–3 m), is quite high, often reaching 200 cm/s or higher. The second-dimension separation, therefore, is a very fast separation on a short column. Seemingly minor problems with injection bandwidth will be significantly magnified in this situation. 

 As seen in Figures 3–5, in GC×GC–TOF-MS, the hold-up time (

) in the second dimension is quite short, in this data, about 0.4 s. For the 1.5 m × 0.25 mm column employed, this implies an average linear gas velocity of about 350 cm/s and a volumetric carrier gas flow rate of about 11 mL/min under a near vacuum as the outlet of the 1.5 m column is at about 10

  Torr. This flow is very far to the right, in the mass transfer region of a typical van Deemter plot, and furthermore, it is so high that the second dimension temperature should be optimized carefully to maximize retention. In our work, the second-dimension temperature was lowered as much as practical with this instrumentation: to 5 °C above the first dimension. This leads potentially to the band broadening and tailing seen in the second dimension chromatograms. The maximum retention factor (

) seen in this data is about 3, with many of them less than 1. Also, for the 1.5 m second-dimension column, there are only about 3000–4000 theoretical plates. If resolution is considered a function of retention factor, selectivity and theoretical plates and retention factor and theoretical plates are low, then selectivity must be kept high to achieve resolution. This implies careful study and optimization of the stationary phase choice and lowering the second-dimension temperature to enhance retention.  

SPME combined with GC×GC or GC×GC–TOF-MS provides a straightforward, yet powerful separation system. However, as the separation power increases so does the need for careful optimization and understanding of each step. For SPME, both the extraction and injection conditions are important in obtaining effective separations. The critical injection parameters are glass sleeve diameter, inlet flow rate, and band focusing at the column head. If focusing is strong (low column temperature, thick film, high retention) then inlet liner conditions are not as critical, however if focusing is weak (higher column temperature, thin film, low retention), then inlet liner conditions should be optimized carefully to provide the fastest sample transfer possible using a narrow glass sleeve and high flow rate. In this work, it is clearly seen that even the highly sophisticated columns and detection afforded in GC×GC–TOF-MS are not capable of mitigating a poor job optimizing the previous sampling and injection. However, the separating power of SPME–GC×GC–TOF-MS is perhaps unrivaled, offering up to six dimensions of separation for complex samples. 

The authors gratefully acknowledge the contribution of technical and instrumentation support from LECO Corporation, columns from Restek, and SPME fibres from Supelco. J.S. and M.D. acknowledge financial support from the Clare Booth Luce Foundation. N.S. acknowledges financial support from Sanofi-Aventis for the Centre for Academic Industry Partnership. 

Nicholas H. Snow, Jerianne Sinex and Mandelle Danser,

 Seton Hall University, South Orange, New Jersey, USA. 

, 438–454, 540–553, 726–741, 821–840 (2006). 

Solid Phase Micro Extraction Theory and Practice

7. J.J. Langenfeld, S.B. Hawthorne and D.J. Miller, 

Split and Splitless Injection in Capillary Gas Chromatography

10. P.M.M. Perrera, P.J. Marriott and I.E. Galbally, 

Articles

Regular Article

In this article, the combination of SPME with GCxGC and GCxGC–TOF-MS is discussed using mixtures of solvents as example analytes

Multiple Dimensions of Separations: SPME with GCxGC and GCxGC–TOF-MS

Green chemistry is an overarching philosophy of chemistry defined by a set of 12 principles introduced by Anastas and Warner.

  The principles can be applied to organic chemistry, inorganic chemistry, biochemistry, physical chemistry and analytical chemistry. In all disciplines, the focus is on minimizing the risks and maximizing the efficiency of any chemical process. Green chemistry seeks to reduce and, ideally, eliminate pollution at its source to protect human health.  

In the last ten years, green chemistry has gained momentum: many universities created green-chemistry curricula; symposia were organized on green chemistry and green engineering all over the world; and journals and websites dedicated to this topic were launched.  

Moreover, this momentum was not only occuring in academia but also in industrial and pharmaceutical environments (e.g., the Pfizer Analytical Green Chemistry Network).

The relationship between green chemistry and analytical chemistry is two-fold. On the one hand, analytical chemistry is by definition 'green' as it can measure the greenness of a chemical product or technology, the environmental pollution of a chemical process, the consequences of chemicals on health and so on. On the other hand, analytical chemistry is also a target for green chemistry (alongside other areas of chemistry) because several of its principles apply to analytical chemistry (e.g., the use of hazardous solvents in chromatographic analysis and in sample preparation methods).  

Important in the discussion of green analytical chemistry, a term coined by J. Namiesnik at the end of the nineties,

  is its definition. At present, the definition of L.H. Lawrence

  is the most comprehensive one: "the use of analytical chemistry techniques and methodologies that reduce or eliminate solvents, reagents, preservatives and other chemicals that are hazardous to human health or the environment and that may also enable faster and more energy-efficient analyses without compromising performance criteria." The latter prerequisite is often the bottleneck in translating existing methods to green methods.  

We also have to state that 'thinking green' in analytical chemistry is not always straightforward. We often receive remarks during presentations on green chromatography that what is considered green is actually not green and vice versa. Consider the following examples: 

. Aqueous waste streams are more difficult to clean than organic waste streams. 

Replacing acetonitrile with biodegradable ethanol

. Some sources indicate that acetonitrile, ranked by the EPA as a hazardous solvent, produces NO

 by incineration (jointly reponsible for acid rain) while other sources claim that NO

 is not formed. Note, however, that the production of LC-grade acetonitrile, a by-product of the synthesis of acrylonitrile needs much more energy than the production of pure ethanol. 

. Highly appreciated in green chromatography (sub- and supercritical fluid chromatography) but carbon dioxide is also linked to global warming. 

 We do not want to go into these controversial discussions in this series of articles and would simply prefer to describe approaches that are generally accepted as less harmful to the environment.  

Part 1 of the series discusses reducing solvent usage or switching to benign solvents in liquid chromatography (LC). Part 2 will mainly concentrate on gas chromatography (GC) and supercritical fluid chromatography (SFC), both, by definition, greener than LC. Part 3 will present and discuss the features of green sample pretreatment techniques, that are often much more productive and cost-effective than classical enrichment/fractionation methods.  

Reducing the Internal Diameter of LC Columns

In the last year, because of the shortage and high price of acetonitrile, we have been overloaded with documentation, leaflets, e-mails and so on from the different instrument manufacturers and column suppliers on how to reduce the consumption of acetonitrile. The emphasis here was on practical and economical reasons, rather than on ecological ones. 

Acetonitrile is by far the most used solvent in LC because of its reputed unique characteristics such as dissolving a wide range of solutes, low acidity, minimal chemical reactivity, low UV cut-off and MS compatibility. Reducing acetonitrile consumption is by far the first option in greening LC but the principles in doing so also apply to other organic solvents and additives. As an example, replacing the additive trifluoro-acetic acid (TFA) which is cytotoxic, corrosive and persistent in the environment with formic acid which has low toxicity and decomposes to CO

One analytical LC instrument, equipped with a conventional LC column (15 to 25 cm in length, 4.6 mm i.d. packed with 5 μm particles) operated at the commonly used flow rate of 1 mL/min, generates, upon continuous operation, nearly 1.5 L solvent each day which is approximatley 500 L solvent in a year, of which typically 50% is organic solvent, in most cases acetonitrile. This is enormous and strategies to reduce the consumption of LC solvents are being actively pursued.  

Relevant column-related parameters towards a lower solvent consumption include reduction of the internal diameter and, using shorter column lengths and smaller particles. 

An obvious way to reduce solvent consumption without losing efficiency is to reduce the flow rate. Longstanding theory shows that, at least in LC, the efficiency (

) of the column is independent of the internal diameter (Equation 1). This is in contrast with gas chromatography where the separation is improved by reducing the internal diameter. In LC, 

 is dependent upon the length of the column (

The maximum plate number is obtained at the optimal velocity (u

) and, when the internal diameter of the column is reduced, the flow rate has to be down-scaled by a factor (F) according to Equation 2 to work at u

Upon switching from a 4.6 mm i.d. column to an identical 2.1 mm i.d. column, F equals 4.8. Knowing that a 4.6 mm column is typically operated at 1 mL/min, the 2.1 mm column should be operated at 0.21 mL/min. A major advantage that evidently arises is the reduced solvent consumption with a factor of 4.8 without compromising separation. Switching from a 4.6 mm i.d. to a 2.1 mm column is no problem on state-of-the-art LC instrumentation, although analysis times can increase slightly due to the gradient delay caused by the volume of both the pump and injector. Reduction of the internal diameter is often accompanied with an increase in sensitivity. This is a direct consequence of the reduced dilution of the solutes in the mobile phase and the appearance of more concentrated bands at the detector. This sensitivity gain is typically experienced with UV, fluorescence and electrospray ionization mass spectrometry (ESI-MS), which are concentration sensitive. The increase in sensitivity for UV and fluorescence is equal to the down-scaling factor F. Examples of extreme down scaling are shown in Figure 1.  

A tryptic digest of 10 proteins was analysed on (a) a 5 cm × 0.2 mm i.d. PS-DVB monolithic capillary column operated at 2.5 μL/min (total mobile phase volume 55 μL) and on (b) a 1.2 m × 0.1 mm i.d. packed with 5 μm ODS particles and operated at 450 nL/min (total mobile phase volume 135 μL).

  The mobile phase consisted of acetonitrile, water, TFA. Detailed experimental conditions can be found in reference 6. Note the long analysis time on the second column. For most applications and definitely in a QA/QC environment such analysis times are unacceptable. In the omics field, however, analysis time and thus solvent consumption can be related to the obtained information in a certain time interval. The second column, because of its high peak capacity, enabled the identification of more proteins. The more information obtained in a given time, the greener the method is. 

Note that the green character of these miniaturizations is only valid when micro- and nanolitre pumps are used. High volume pumps operated in the split-flow mode are not green in this respect (see further). Capillary and nano-LC are typically used in the bio-field (e.g., proteomics) because in addition to high efficiency, very high sensitivity is obtained for small sample sizes due to the concentration-sensitive nature of nanospray-ESI-MS. Other examples of extreme down-scaling in LC can be found in the literature (e.g., Smith et al.

Capillary- and nano-LC have further developed into chip-based microfluidic systems in which injection, sample concentration, separation and electrospray-needle are integrated. 

A classification of LC techniques according to the internal diameter and the impact on the flow rate, and, therefore, solvent consumption and sensitivity is presented in Table 1.  

Table 1: Relation i.d., solvent consumption and sensitivity at uopt 

A reduction of the solvent consumption by column miniaturization requires instrument adjustments. Extra-column volumes must be minimized, accordingly to control pre- and post-column band broadening. These include the injector and detector cell volume and the volumes of all connections between the injector and detector. In principle, the down-scaling factor applies here as well. Down-scaling from a 4.6 mm column to a 2.1 mm column can readily be achieved without major instrumental adaptations. 

However, one has to realize that a gradient delay is to be expected. Further down-scaling requires a more thorough rewiring. While a 20 cm tubing with an internal diameter of 0.17 mm and a volume of 4.54 μL is ideally suited to connect a 4.6 mm column to a detector, it will induce peak broadening when using a 1 mm column. When working in gradient mode, pre-column dead volumes are less of an issue because one can generally benefit from on-column focusing due to the weak-eluting power of the initial mobile phase conditions. In isocratic mode, pre-column volumes should be carefully evaluated as well.   

Note that at present, 1 mm i.d. columns are seldom used. As well as instrument requirements, 1 mm i.d. columns commonly exhibit lower plate numbers compared with similar columns in terms of length and particle size. Apparently, packing 1 mm i.d. columns is more difficult and challenging than larger i.d. columns and Equation 1 is not respected. 

Apart from the extra-column volumes, the pumps themselves should be adapted. There is not a single pump available covering the flow rate range of nL/min (nano-LC) till mL/min (conventional-LC). Standard LC pumps successfully work from 50 μL/min to 5 mL/min. While 4.6 mm, 2.1 mm and to a lesser extent 1 mm columns can be operated using the same pump, sub-mm columns require a different pump or the use of flow splitting. Instrument vendors opt for both approaches. While a number of vendors cover the mid μL/min–mid nL/min range making use of splitters, others deliver these flows without splitting. As mentioned before, the former can no longer be considered as green chromatography given the fact that the majority of the mobile phase is diverted to the waste.  

Another option to decrease solvent consumption is by increasing the 'chromatographic productivity'. This is generally achieved by reducing the particle size in combination with shortening the column length. From Equation 1 it can be derived that a column with 5 μm particles and 15 cm in length should give approximately 15000 theoretical plates. When using sub-2 μm particles, a column length of 5 cm is sufficient to obtain the same number of plates. As the column length is reduced by a factor of three, analysis time is, in the first instance, reduced by the same factor. However, efficiency versus mobile phase velocity curves for sub-2 μm columns are flat and high flow rates can be applied without losing efficiency. 

Choosing sub-2 μm particles comes with instrumental adaptations. A major downside of smaller particles is the column-pressure increase. When the particle size is halved, pressure goes up by a factor of four. In the example discussed before, switching from a 15 cm × 2.1 mm i.d. × 5 μm 

 column to a 5 cm × 2.1 mm i.d. × 1.8 μm 

 column operated at a flow rate three times higher results in approximately an eight-fold increase in back-pressure. Instrumentation has to be capable of operating at higher pressures. A variety of ultra-high pressure LC (UHPLC) systems are currently on the market and are gradually replacing more conventional LC instrumentation. 

The interplay of particle size and pressure on chromatographic productivity is best illustrated by means of kinetic plots. We refer the reader to some recent publications for detailed information.

  Typical kinetic plots are shown in Figure 2.  

In the plots speed of analysis for conventional 5 μm and 1.8 μm particles for a 400 bar instrument are compared with 1.8 μm particles on a 1200 bar instrument. The x-scale represents the log 

 and the y-scale the analysis time. Above the plots, we arbitrary selected separations for which 10000 plates (high throughput), 25000 plates (high productivity) and 100000 plates (high resolution) are needed. For the high throughput separations, relative analysis times will be 10 (5 μm at 400 bar), 2.5 (1.8 μm at 400 bar) and 1.6 (1.8 μm at 1200 bar). This means that the same analysis is six times faster on the 1.8 μm (1200 bar) than on the 5 μm (400 bar) column. A factor of 5 is noted for 25000 plates and a factor of 4 for 100000 plates. Note that 100000 plates can never be reached with 1.8 μm particles on a 400 bar instrument. Obviously high plate numbers means long analysis times and thus huge amounts of solvent.  

High throughput analysis for the determination of antioxidants in vegetable oils (European Parliament and Council Directive No. 95/2/EC — 1995) is illustrated in Figure 3.  

All antioxidants included in the EC directive are separated in less than 2 min on a C18, 5 cm × 2.1 mm i.d., 1.8 μm 

 column operated at 1070 bar and 45 °C (see further). Total solvent consumption was 3.8 mL (flow 1.9 mL/min). The same analysis on a 15 cm × 4.6 mm i.d., 5 μm 

 particles was 18 min with 18 mL solvent consumption.

It is important to note — and this is often misleading in leaflets by instrument and column manufacturers when they describe solvent savings exceeding 90% — this result is a combination of reduced particle size and reduced internal diameter.   

Another point of discussion is the speed of analysis! Speed of analysis is often confused with solvent saving. Figure 4 shows the optimized analysis of an active pharmaceutical ingredient (metoclopramide) and its impurities on a sub-2 μm particle column. Because u

 spans a broad range (efficiency versus mobile phase velocity curve is flat), the mobile phase flow can be increased from 0.22 to 0.66 mL/min to speed up the analysis (a to b). However, in both analyses the solvent consumption is approximately 3 mL. Note that at high pressure (1020 bar) selectivity changes occur because of frictional heating

  and the temperature had to be decreased to obtain the same separation (c). Nevertheless, according to the definition of green analytical chemistry by L.H. Lawrence "more energy efficient" can be an argument here.  

Temperature is a powerful variable in LC to tune speed, selectivity, efficiency and detectability.

  By operating at higher temperature, the viscosity of the mobile phase decreases and diffusion in the mobile phase becomes faster. The consequences are illustrated in Figure 5 showing the kinetic plots for the 1.8 μm column installed on a 1200 bar LC system operated at 30 °C and 80 °C. Analysis times are further reduced with a factor of about 2.  

If speed is not important and the analysis time at ambient temperature is acceptable, the amount of organic modifier in the mobile phase can be reduced significantly and, in some cases, be eliminated completely resulting in pure aqueous mobile phases.

  The eluotropic strength of water in reversed-phase LC increases drastically as a function of temperature and at 200 °C is close to that of acetonitrile at ambient temperature.  

In Figure 6, the influence of the temperature and flow rate on the separation of seven phenylurea pesticides on a 5 cm × 2.1 mm i.d., 1.8 μm 

 column is shown. At 40 °C and a flow rate of 0.35 mL/min (a), all compounds elute within 11.5 min with the mobile phase acetonitrile/water (30:70). At 80 °C (b), the analysis time is reduced to about 5 min (less than half mobile phase consumption) while the selectivity for compounds 4 and 5 is significantly enhanced. Elevated temperature also allows the use of higher flow rates without losing efficiency.

  The flow rate was increased to 0.85 mL/min (c) leading to an analysis time of 2 min (about 1/5 of the initial analysis time). In the latter case the solvent consumption is the same as at 0.35 mL/min and 80 °C.  

Part one focusses on ways of reducing and replacing solvents commonly used as mobile phases

Green Chromatography (Part 1): Introduction and Liquid Chromatography

As I write this column, I have just returned from the Pittsburgh Conference in Orlando, Florida. One of the things that I enjoy about Pittcon is doing booth duty, when I get to meet some of you loyal readers of "LC Troubleshooting". Sometimes the conversations turn to specific problems that you are having with your liquid chromatography (LC) system or separation. Often these can be a mind-stretching conversation with give-and-take that is not as convenient in an e-mail exchange. However, some of these conversations make me realize that sometimes we all get too focused on the details of the method without backing up and determining how they fit into the big picture. In this month's column, let's look at a couple of examples of this.    

In theory, at least, a chromatographic peak should be Gaussian in shape, with no fronting or tailing. Nearly every method, however, has peaks that exhibit some degree of peak tailing, where the back edge of the peak does not reach the baseline as quickly as the front edge rises from it. We measure peak tailing as either the asymmetry factor A

 or the tailing factor, TF. These are calculated as indicated below with reference to Figure 1: 

where A and B are the peak half-widths, measured at 10% of the peak height for the asymmetry factor and 5% of the peak height for the tailing factor. In past years, methods were plagued with tailing peaks, especially when basic compounds were analysed on the older, low-purity, type-A silica columns. These columns had a high population of acidic silanol groups responsible for tailing. Today's newer, high-purity, type-B silica columns are much less prone to tailing. In fact, sometimes tailing is so small that we begin to notice peak fronting. 

One conversation related to peak fronting of 0.8, as measured with the asymmetry factor. The person was very concerned about the source of the fronting and how to correct it. It is easy to get off on a conversation about the sources of fronting peaks, which, in general, are rare today with reversed-phase methods. Peak fronting is most commonly attributed to a gross column failure that we sometimes refer to as bed collapse. Some deterioration of the packing material takes place and the particles inside the column shift, creating a void in the column. This will cause all the peaks in the chromatogram to front, and will not be corrected by column reversal or flushing. The lady had replaced the column with a new column twice and the fronting persisted, so it is unlikely that column collapse was the problem. Another possible problem source is insufficient buffer in the mobile phase. Also, in the past I have seen references to peak fronting in ion-pairing separations being corrected with a change in temperature, but these methods used type-A columns; I have not seen this on type-B columns, so a temperature change may no longer be effective.  

As the conversation became more involved, one of my colleagues, who was listening, waved the yellow caution flag. "Wait a minute," he said. "How much fronting are you seeing?" Well, a little simple math says that an asymmetry factor of 0.8 is equivalent to the same peak distortion as a peak tail of 1.25. The release specifications that many column manufacturers use in their quality testing process indicate that a column with 0.9 < As < 1.2 is acceptable. In other words, a brand new column might exhibit a little fronting or tailing. If the method we were discussing had an asymmetry factor of 1.25, we wouldn't be having this conversation, would we? As my daughter used to say, "Don't sweat the petty stuff (...and don't pet the sweaty stuff)." This is not a problem that is worth investigating. 

Another Pittcon attendee dropped by to discuss a problem he was having with a method for the analysis of a drug in serum. When he calculated recovery of the drug from spiked samples, he found 102% recovery. Having low recovery, for example, 98%, is easy to explain, but he was concerned about having too much recovery — how is it possible to recover more drug than you put in? We must remember that errors in most laboratory processes, including sample preparation and chromatography, are distributed evenly about the mean. For example, it is just as likely that a pipette will deliver 0.5% more than the nominal value as it is to make the same error on the low side. As a result, the overall error of the method should be distributed about the mean value. However, with most sample preparation processes, we lose sample along the way, so the average recovery is <100%. For example, if the average recovery were 96 ±2%, we would never see >100% recovery, so when we do see a method with >100% recovery, we might be surprised. But there is nothing abnormal about such values. 

As the conversation went along, questions centred on the source of the error. It turns out that the method recovery was measured by comparing extracted serum samples with an aqueous reference standard. While this is a reasonable technique to make a gross check of overall extraction efficiency, it is not appropriate for method calibration. With bioanalytical methods (drugs in biological matrices), the regulatory guidelines call for a matrix-based standard curve. This means that the current method should use blank serum as the matrix and spike it at the appropriate concentrations to generate the calibration curve. This provides some internal correction for some of the variables that might be beyond the control of the user. For example, ion enhancement or ion suppression with mass spectrometric (MS) detectors can be a problem with serum- or plasma-based methods. By using a matrix-based standard curve, in which the calibrators are treated the same as the samples, it is much like solving simultaneous equations in algebra — the constant factors drop out.  

But up comes the yellow flag again! Why are we having this conversation? The regulatory guidelines for methods like this allow for precision and accuracy of ±15% at all concentrations above the lower limit of quantification (LLOQ) and ±20% at the LLOQ. A 2% error, as in the present case, is insignificant relative to the allowable variability. There are other fish to fry. 

 I am reminded of a story told to me by a colleague in a laboratory I used to manage. He had worked for a major pharmaceutical company that, like most pharma companies, was very interested in improving their processes. As part of the data-gathering process, when each new LC method was completed, the time taken to develop the method was added to a database. After a sufficient amount of data was gathered, it was possible to calculate the average method development time for an LC method. All was well and good until the next method was developed and it took longer than the average to complete. The staff was chastised for poor performance because the laboratory manager expected all methods to be developed in less than the average time. This reminds me of Garrison Keillor's mythical town of Lake Wobegon that he describes on his National Public Radio broadcasts. The tag line at the end of his Lake Wobegon news always ends with "...and all of the children are above average." If the laboratory had sufficient data to determine a statistically significant average, it is unreasonable to expect all methods to be developed in less than the average time — duh! Now if the average represented all LC methods developed in the pharmaceutical industry, one company's goal of developing methods in less than the average time might be reasonable. Or in a continuous-improvement environment, it might be reasonable to expect a target development time to be less than one standard deviation above the mean. But all methods less than the average time? What are they smoking? 

Another place where we can get distracted from the big picture is related to trying to reduce analysis costs. My guess is that I get this question in at least half of the LC classes that I teach: "How can I extend the life of my column?" Whenever we are looking at trying to improve a method, we need to consider the cost. How much does the current status cost? How much can I save with the desired change? How much will it cost me to get there?  

Unfortunately, many laboratories consider the purchase of an LC column to be a capital expenditure. Yes, it is expensive, but in terms of the overall cost of analysis, it should be considered a consumable item. Do a few calculations and you'll convince yourself (and hopefully your boss). When I was managing a contract analytical laboratory, we were often asked for a quote for budget purposes. For a typical LC method with ultraviolet (UV) detection, we used a number of $50/sample for this purpose. If I pay $500 for a column and only get 500 samples through it before it fails, the cost is $1/sample for the column. This is 2% of the overall cost of the method in the present example. A 500-injection lifetime is pretty short for most methods, so you might be prompted to spend some time trying to increase the column lifetime. Let's say that you do some experimentation and find that by instigating a special cleaning procedure, you can extend the column lifetime to 1000 injections. Well, you've just cut your column costs in half. This sounds pretty good until you consider the overall savings. You've reduced the column burden on the method from 2% to 1%. Is it worth the trouble? Instead, it might have been more appropriate to focus on a more expensive part of the process — maybe it is report generation or sample tracking. There are enough things to take up our time in the laboratory without creating new ways to spend time that have little return on the overall value of the process. 

So, what's the common thread with these stories? It is very easy to get focused on one specific aspect of an LC method and get distracted from the overall goal of the method.  

In the first case, why is peak asymmetry of 0.8 a concern? If it is because we're not used to seeing fronting peaks in most LC methods, we might be worrying about nothing. If it is because we're concerned about losing resolution between that peak and a small peak that is eluted just in front of it, then the concern might be more valid. Should we focus on peak fronting or on adjusting the relative peak positions? 

In the second example, recovery of 102% turned out to be unimportant in the context of a bioanalytical method with allowable precision and accuracy of ±15–20%. But if the method were a pharmaceutical content uniformity assay, in which ±2% is the allowable variation, 102% recovery is a real concern. 

Continuous improvement, including the reduction of method development time, is an admirable goal. But is it reasonable to instantly expect all methods to be better than average? Setting more achievable intermediate goals for method improvement would be more reasonable, more likely to succeed, and certainly better accepted by the method development staff. 

These examples bring to mind one of my favorite authors when I was managing a laboratory: Eliyahu Goldratt (

). In what some people refer to as a BFO (blinding flash of the obvious), Goldratt introduced me to the concept of the bottleneck. You can spend all the time you want trying to improve a process, but if it does not affect the rate-limiting step, your efforts will be of little help. Instead, if you can improve just this one step, the whole process will be improved. This principle applies very easily to the LC laboratory. Don't spend too much time investigating insignificant aspects of a method — focus on what will really make a difference. 

 is vice president of LC Resources, Walnut Creek, Califorina, USA; and a member of 

 editorial advisory board. Direct correspondence about this column to "LC Troubleshooting", 

, Poplar House, Park West, Sealand Road, Chester CH1 4RN, UK, or e-mail Alasdair Matheson, the editor, at 

LC Troubleshooting

Look at the big picture. It may not be appropriate to obsess on the details

Too Much Scrutiny

Green chromatography has changed from a buzzword to reality. Last year, interest in green liquid chromatography increased dramatically due to the acetonitrile shortage — but more as a way to save money than the environment!  

 in 2010 a team from the Research Institute for Chromatography (RIC) will review green chromatography in practice in an exclusive three-part series.  A major part of the discussion will focus on use of hazardous solvents in chromatography and sample preparation and describe some green alternatives. In both areas considerable progress has been made to reduce solvent use, switch to more benign solvents, and/or eliminate organic solvents.  

Several of the 12 principles of green chemistry

  directly apply to analytical chemistry: (1) prevent waste, (5) use safer solvents, (6) increase energy efficiency, (8) reduce derivatives, (10) design for degradation and (11) real-time analysis for pollution prevention.  

An important point when discussing green analytical chemistry — a term coined by J. Namiesnik — is its definition.

  L.H. Lawrence defined it more specifically as "the use of analytical chemistry techniques and methodologies that reduce or eliminate solvents, reagents, preservatives and other chemicals that are hazardous to human health or the environment and that may also enable faster and more energy-efficient analyses without compromising performance criteria."

  The latter prerequisite has often been the bottleneck in translating existing methods to greener methods.  

The first part of the series in this month's issue will discuss reducing solvent use by miniaturization, switching to benign solvents and eliminating organic solvents in liquid chromatography. Ultra high pressure LC (UHPLC) using short narrow-bore columns packed with sub-2 μm particles is currently the most straightforward way to reduce solvent consumption. Method translation from conventional LC to UHPLC is rather simple and solvent saving can be as high as 90%. Further miniaturization to capillary or chip format resulting in μL to nL/min flow rates is another option if validation criteria can be met. Switching to more benign solvents is much more complicated — but possible.  

Part 2 of the series in September will focus on gas chromatography (GC) and supercritical fluid chromatography (SFC). GC is by definition much greener than LC (although dwindling supplies of helium and its associated increasing cost should be taken into consideration) and several applications presently performed by LC, for example, PAH analysis and some pharmaceutical analyses, can be perfectly performed by GC. By far the most widely advertised "green chromatography method" is SFC due to its sparse use of solvents. Is this correct? Do we not need high concentrations of organic modifier to keep the solutes in solution? SFC will be critically evaluated in the framework of green chromatography and generic green methods will be presented.  

The series will conclude in December and will concentrate on green sample pretreatment techniques, that are also often much more productive and cost-effective than classical enrichment/fractionation methods. The new methods in which miniaturization and/or solventless operation are key factors have aided in designing integrated analytical systems, providing high-throughput, unattended operation and exceptional performance. 

Pat Sandra is director of the Research Institute for Chromatography (RIC), Kortrijk, Belgium, professor in Separation Science at the Ghent University, Belgium and director of the Pfizer Analytical Research Centre-UGent, Belgium.

, Oxford University Press, New York (1998) 

www.epa.gov/greenchemistry/pubs/principles.html

Pat Sandra and his team will discuss how green chromatography works in practice

Green Light for Green Chromatography?

The International Symposium on Chromatography (the ISC Symposium)

 is one of the top international congresses in separation science. With the first symposium held in London in 1956, this conference is the oldest series of scientific meetings devoted to the field of chromatographic science. It is an international forum for scientific discussion about all chromatographic methods, along with combined techniques, such as multidimensional chromatography coupled to mass spectrometry or NMR, automation and miniaturization. The symposium is held biannually in European countries.  

The 28th International Symposium on Chromatography (ISC 2010)

 will take place in Valencia (Spain) at the Faculty of Pharmacy in the campus of Burjassot of the University of Valencia, from 

 and will consist of invited lectures by scientists at the forefront of the field, oral contributions organized in parallel sessions and poster presentations. Workshops, seminars and short courses given by experts in this field will also be a part of the Symposium programme and will offer an excellent opportunity to learn about most recent scientific and technological developments in separation science. Methods and applications to be presented at 

include: fundamentals on chromatography; gas chromatography; liquid chromatography; multidimensional chromatography; hyphenated techniques; fast separations; column technology; electrodriven separations; supercritical fluid chromatography; enantioseparations; new detection methods; nanotechnology; industrial processes and product analysis; environment; speciation analysis; food quality and safety; clinics and pharmaceutics; life sciences/bioanalysis; forensics; and polymers.    

Workshop-seminars of instrument manufacturers and an extensive exhibition of instrumentation, accessories and supplies will run in parallel to the scientific programme and will display the latest innovations in the field of instrumentation, column technology, sample preparation, software tools and related subjects. Important dates to consider are 

: The deadline for oral and poster contributions; 

: Deadline for last minute posters and early bird registration. 

For more information please visit the event website at 

			Hynes Convention Center & Sheraton Boston Hotel, Boston, USA  

16th International Symposium on Separation Science

Conference Halls of the National Research Council (CNR), Rome, Italy  

SFC 2010 — 4th International Conference on Packed Column SFC

			Radisson Blu Royal Viking Hotel, Stockholm, Sweden  

Send your event details to Doug Kitson at 

Departments

The International Symposium on Chromatography (the ISC Symposium) is one of the top international congresses in separation science. With the first symposium held in London in 1956, this conference is the oldest series of scientific meetings devoted to the field of chromatographic science.

Event News

Monolith pioneer, Frantisek Svec, brings readers up to date on the latest developments in monoliths for use in chromatography. Breakthroughs in the organic monoliths include phases for small molecule separations, nanoparticle-modified surfaces, new approaches to polymeric monoliths and design of monolithic layers. Less common inorganic monoliths are highlighted. Finally, commercial sources for monolith technology are covered for those who would like to purchase rather than synthesize their columns.

Monoliths are separation media in a format that can be compared to a single large 'particle' that does not contain interparticular voids. As a result, all the mobile phase must flow through the stationary phase. Monolithic columns are about two decades old. Although a few approaches to monolithic stationary phases had been suggested in the past, they were, for the most part, unsuccessful. Thus, serious research into monolithic formats started in the late 1980s and early 1990s. The focused work of several research groups led to a family of useful monoliths comprising compressed hydrophilic gels, macroporous polymer discs, columns, tubes and silica rods.  

At the beginning, most of these new developments were not accepted quickly by the chromatography community as demonstrated by the publication difficulties encountered during the first wave of manuscripts submitted by early workers such as Stellan Hjertén, Nobuo Tanaka and ourselves. Fortunately, these and other authors were persistent in their research endeavours, the science was interesting and the audience preinformed about monolith advantages through presentations at conferences. Today, both organic and inorganic monolithic separation media have been generally accepted as an alternative type of chromatographic stationary phases enabling rapid separations of a wide spectrum of analytes. The number of publications concerning monolithic technologies grows steadily and monolithic products are available commercially. A very detailed account of these materials can be found in a monograph

  describing the war chromatographers were waging against diffusion of analytes in the stagnant mobile phase within pores of standard packings and the new weapons they invented and used including sub-2 µm particles, core-shell packings, high temperature and monoliths. These new column technologies are being continuously perfected and the chromatographic separations are getting more efficient and faster. However, here I will focus on monoliths. In contrast to diffusion, which is the typical driving force for mass transfer within the pores of particulate stationary phases during chromatographic processes in packed columns, convective flow through the pores of monoliths enables a substantial increase in the speed of the separation all at lower pressure drop than experienced by packed columns of similar efficiency. 

The latest advances in monolithic column technology traditionally have been included in 

 before the HPLC symposium held in the US.

  Typically, the focus of these contributions was on porous polymer-based monolith, treating formats such as discs, columns, capillaries, tubes, chips and so forth, each separately. In contrast, this contribution will focus on a few selected leading-edge developments in the field and is also expanded to include monoliths formed from inorganic materials. 

Organic Monoliths for Separation of Small Molecules

Porous polymer monoliths proved to be an excellent separation medium for large molecules such as peptides, proteins, nucleic acids and synthetic polymers. However, they failed to match the efficiency of silica-based monoliths in the separations of small molecules. This deficiency has been attributed to the lack of mesopores and low surface area and recently led to a series of studies aiming at the preparation of organic polymer-based monoliths capable of separating even the small molecules in an isocratic mode. For example, Bonn's group in Innsbruck, Austria used the route that we suggested in 1995

  and stopped polymerization of 1,2-bis(4-vinylphenyl)ethane in its early stage to obtain monoliths with large surface area.

  Jandera's group in Pardubice, Czech Republic, carefully optimized the polymerization conditions to create monoliths containing a larger volume of mesopores.

  However, it has always proven difficult to prepare polymer monoliths in a single-step that possess both large through-pores, enabling flow at low back pressure, as well as a multiplicity of small pores. We recently introduced monolithic poly(styrene-

-divinylbenzene) precursor capillary columns that have been prepared using standard polymerization techniques that lead to monoliths with only large pores. However, hypercrosslinking in the next step then affords a monolith containing an array of small pores. This hypercrosslinked monolithic column exhibited a surface area of up to 600 m

 /g, more than one order of magnitude larger than that of typical polymer-based columns. This monolithic column contained a significant proportion of newly formed mesopores, which enabled quite a good separation in isocratic mobile phase mode and also proved useful for the separations in size-exclusion mode.

  Figure 1 shows the fast isocratic separation of benzene derivatives featuring high efficiency on one of these columns.      

Modification of Surface Chemistry with Nanoparticles

While most of the methods controlling the surface chemistry of porous polymer monoliths described so far rely on copolymerization of functional monomers, chemical modification of reactive groups of the monolith, or grafted chains formed from functional monomers, a new technique has been introduced recently to the monolithic arena: functionalization with latex nanoparticles.

  Its roots stem from an old technology used by Dionex Corporation (Sunnyvale, California, USA) for the preparation of column packings for ion chromatography.  

Gold nanoparticles represent another type. Recently, we used them to functionalize the pore surface in porous polymer monoliths.

  The procedure is simple: First, poly(glycidyl methacrylate-

-ethylene dimethacrylate) monolith reacts with cysteamine to afford thiol functionalities and then the nanoparticles are formed 

Monolith pioneer, Frantisek Svec, brings readers up to date on the latest developments in monoliths for use in chromatography

LCGC Europe-05-01-2010

Multiple Dimensions of Separations: SPME with GCxGC and GCxGC–TOF-MS

Green Chromatography (Part 1): Introduction and Liquid Chromatography

Too Much Scrutiny

Green Light for Green Chromatography?

Event News

New Developments in the Field of Monoliths for Chromatography