CHROMacademy is a chromatography e-learning programme that provides continuing profesional development for chromatographers to make them more effective in the work place. There are over 3000 pages of e-learning on HPLC, GC, MS and Sample Preperation techniques to improve practical knowledge for the absolute novice (

) to the expert who needs a refresher in a particular topic (

Advanced Topics in LC-MS Spectral Interpretation

, for example). The site can be also used for induction training or programmed learning activites and there are tutorials and assessments built into all learning modules to check progress. The first official webinar: 

Essential Guide 1: Split /Splitless Injection for Capillary GC

 attracted 568 visitors. The recorded session is available via the CHROMmunity at 

 (under the WEBCASTS Tab). Other Webcasts include 

Essential Guide 2:  Mobile Phase Optimization Strategies for Reversed Phase HPLC; Essential Guide 3: Understanding and Optimizing Detectors for Capillary GC

Mobile Phase Optimization Strategies for Reversed Phase HPLC

Waters has launched an on-line community featuring resources for users of the company's Empower software and NuGenesis scientific data management system (SDMS) as well as access to experts and shared user experiences. The site provides easy access to the latest information about software, targeted support materials and exclusive content only available to the community, according to the company, To date some of the most popular discussion topics among members include data sharing between colleagues around the world, Window 7 support for Empower and SDMS and validation requirements for chromatography data systems. 

Shimadzu Scientific Instruments has partnered with Ceres Nanosciences and Nonlinear Dynamics to release the Nanotrap Biomarker Discovery Platform, which the company claims gives researchers the ability to enrich, preserve and screen low-level biomarkers directly from complex biological samples. Nanotraps are core shell hydrogel particles with porous interiors and size exclusion shells. Progenesis MALDI, the software component of the platform, is reported to generate high-quality proteomics data to define biomarkers. It uses the Progenesis approach and technology to improve the reliability and reproducibility of results which, according to the company, adds speed, objectivity and statistics to biomarker discovery. 

DataApex has announced enhancements for Clarity Chromatography Software available in the updated version 2.8. According to the company, the added optional extension for natural gas analysis (NGA) calculations greatly enhances use in the petrochemical industry. The NGA extension performs the compression factor, mean molecular weight, density, Wobbe index and other calculations using different norms. The new Launch Manager utility allows the system to start with predefined profiles with different configurations and user preferences to facilitate switching instrument configurations. The OQ Validation Wizard guides the user through the operational qualification of the software that can now validate digital detectors. 

Alpha MOS has developed a new database for aroma and chemical characterization by gas chromatography analysis. The AroChemBase library uses the Kovats Index as a reference method and is reported to include 2900 compounds of which 1400 have sensory attributes, with chemical information (Name, formula, CAS number, molecular weight, Retention Index) available for several columns. According to the company, when used with a two-column GC-based electronic nose or GC system, it allows a dual-column identification and ranking. The database can be enriched with user data and it is also possible to extract tailored sub-libraries.  

Bio-Rad has released a fully integrated software and database package to provide multiple tools, including database building and management, search, analysis, structure drawing and reporting within a single user interface. According to the company, the KnowItAll Informatics System 8.2 includes a series of new report templates for more efficient output and improved design and a function to preview chromatograms and spectra to be used for searching, interpretation or database creation before the file is actually opened. The informatics system is reported to offer a fully integrated environment with flexible and expandable software and database solutions for chromatography, MS, cheminformatics; chemometrics, metabolomics and more. 

Labtronics has announced the release of Nexxis iLAB 5.0, a total lab automation system. This system integrates ELN, SDMS and instrument integration into a single package that is ready to go out of the box, according to the company. The company claims that it enhances the value of an existing LIMS by providing extra functionality. It controls the analyst step-by-step to prevent mistakes, enforces training and prevents an analyst from doing work if training has expired, according to the company. Expiration dates on chemicals and the calibration on instruments are current and enforced. 

Evaporators and concentration systems website

Genevac has announced an online resource that the company claims brings together the latest technological innovations, applications advances and links to references that detail the current scope of evaporative sample preparation techniques used in clinical and diagnostic testing. Visitors to the website can learn how the company's evaporators and concentration systems are used by clinical biochemists for a number of testing methods, including vitamin D analysis, vitamin K analysis, drugs of abuse testing and neonatal screening. To enable scientists to understand the detail of the various evaporative and concentration techniques used in these applications, further background information can be found in the technology section. 

Send any digital developments in chromatography to Doug Kitson at 

Departments

Digital Update

The tools for analytical resolution of enantiomers have evolved in recent years with advances in chromatographic techniques. As in any analytical method, the separation of enantiomeric pairs should achieve a rapid and complete resolution of the two chromatographic peaks and must also be reproducible and robust. Ideally it should also separate sample impurities, reach a low LOD/LOQ (limit of detection/limit of quantification) and show an appropriate elution order. Moreover, the ideal chromatographic conditions should ensure the stability of the sample during the analysis in addition to the compatibility of the sample medium with the mobile phase and the column. 

Such a list of conditions for the resolution of challenging samples requires decisions concerning both the chiral stationary phases and the chromatographic modes employed. Our goal to find the best chromatographic method must be tempered with the use of a minimum number of chiral stationary phases (CSPs), thus, an efficient screening strategy is essential.  

The resolution of racemic mixtures by chromatography has reached maturity in the last decade.

  Although most racemates and diastereomeric mixtures can be separated at analytical level, the challenge is to achieve complete resolution of the components while reaching high-speed, preserving efficiency and with limited screening efforts. 

Resolution of enantiomers by chromatography is predominantly performed by using chiral stationary phases.  A number of chiral selectors are currently commercially available, such as proteins, polysaccharides, antibiotics, brush-type molecules, ionic exchangers, crown ethers, cyclodextrins and multiple polymers.

  One of the most widely used group of CSPs are the silica-based polysaccharide-derived chiral supports. They can operate in different modes and, therefore, a preliminary decision tree to devise the screening strategy is substantial when a new sample is received (see Figure 1).  

The starting point is to decide which technology is to be used with a specific type of selector. Both liquid chromatography (LC) and supercritical fluid chromatography (SFC) are powerful tools that have been used successfully. The decision, therefore, is typically based on equipment availability and/or suitability for the scale and type of molecule. Although SFC lacked sensitivity in its early development, recent advances have resulted in the efficient use of SFC with mass spectrometry (MS) detectors. 

In LC method development, one may use organic solvent mixtures or water-compatible mobile phases. Normal phase applications (NP) have historically been more widely used for the separation of enantiomers. However, the reversed phase (RP) separations, together with polar organic modes, should be seriously considered when samples are in aqueous media to exploit the LC–MS compatibility of these mobile phase systems. 

The present article aims to cover the practical approaches applied in our laboratories to screen analytical samples. Based on extensive experimental work, we will focus on the results achieved with a range of 3 and 5 μm silica-based phases, containing amylose and cellulose derivatives as chiral selectors in a coated or immobilized fashion. Strategies for efficient HPLC method development with these CSPs in normal phase, polar mode and reversed phase conditions, as well as SFC, will be addressed. The different modes will be reviewed with the preferred primary screening, as well as the potential alternatives for higher peak efficiency (switch to smaller particles), fast analysis and unique selectivities with new selectors. 

The Chiralpak and Chiralcel analytical columns [Daicel Chemical Industries Ltd (Tokyo, Japan)] were used in this study. The 5 μm columns were 4.6 × 150 mm for RP-based separations and 4.6 × 250 mm for NP and polar eluents. The analytical columns based on 3 μm silica gels were in two formats: 4.6 × 150 mm and 4.6 × 50 mm. 

The mobile phases for liquid chromatography were prepared from HPLC grade solvents. Diethylamine (DEA), trifluoroacetic acid (TFA), formic acid (HCOOH) and ammonium bicarbonate (NH

) were used as the mobile phase additives for the resolution of non-neutral molecules. 

The chromatographic tests were performed on two separate Agilent LC systems. One was used for columns packed with 3 μm CSPs. This system consisted of the following modules: Agilent 1100 Series QuatPump (Massy, France) with vacuum degasser, Agilent 1100 Series well plate autosampler, Agilent 1200 Series thermostated column compartment, Agilent 1100 Series diode array detector B with a high pressure micro flow cell (1.7 μL), and 0.12 mm i.d. flow capillaries. 

The second system was mainly used for parallel testing of the 5 μm packed columns. Its configuration was similar to the first one except for the following modules: Jet Stream column oven (VWR, France), Agilent 1100 Series diode array detector B with its standard flow cell (16 μL) and the original 0.17 mm i.d. flow capillaries. 

Method Development in Organic Mobile Phases

Given the increasing number of chiral compounds in drug development and other domains, efficient and automated HPLC screening using columns packed with various CSPs has been a challenging subject and discussed by several authors.

  The strategies to achieve enantiomer separations are quite variable from one laboratory to another depending on the type of substances in screening, the equipment available and the R&D background. However, the motivation for the development of screening approaches remains common. We all search for the easiest, the most successful and reliable way to analyse a series of molecules with diverse structures and chemical properties in a reasonable time frame. This is the standpoint for chiral compound screening. 

The standard screening protocols adopted by companies and research groups worldwide use either three or four analytical columns in the primary screen. Two main options, already discussed in a series of publications,

  include the three immobilized phases of the newest generation (Chiralpak IA, IB and IC) or alternatively the four traditional coated columns (Chiralpak AD-H and AS-H, Chiralcel OD-H and OJ-H). Such configurations with polysaccharide-derived columns, tested on a randomly composed sample pool and with four solvent mixtures, could afford complete enantiomer resolution for over 90% of the racemates examined and enantiorecognition above 95%. 

After further investigation it was concluded that the small group of solvents and their mixtures could lead to good selectivity values for most of the compounds tested and could be considered as a primary set of mobile phases for screening with these CSPs. The primary solvent configuration for both screening systems is presented in Figure 2. In practice, these four-component mobile phase sets are normally set up using a quaternary HPLC screening system.  

It is well known that the nature of the mobile phase has major effects on the enantioselective performance of a CSP. The proposed screening systems were selected to maximize the complementary behaviour among the CSPs of the set and also between solvents on the same column. It is important to note that the elution order changes in certain cases due to the different selectors or mobile phases. This phenomenon can often be observed not only for the elution order, but also for the separation of certain sample impurities. Such systematic screening can be a tool to choose the most suitable CSP/solvent combination for the researcher's specific aims. 

The polysaccharide-type CSPs seem to be most frequently used with organic eluents (alkane mixtures or the polar solvents discussed above). However, resolution of enantiomers on the polysaccharide columns by aqueous eluents has a history almost as long as their applications with organic mobile phases.

  The choice of RP mode has often been related to the need for direct injection of the samples of biological extracts, but also due to low retentivity of the compounds under NP conditions, low solubility of polar and ionic samples in organic solvents and lack of appropriate enantioselectivity in organic mobile phases. An additional and increasingly important reason to prefer RP methods is their suitability for LC–MS applications. 

Method development in RP mode on polysaccharide-derived CSPs has been discussed in several articles

  and some new applications have been recently developed.

  Nevertheless, only coated supports were considered in most of these reports and LC–MS compatibility was only covered partially, if at all. 

As a logical sequence of the recent investigations with organic solvents in HPLC and SFC,

  an intensive study on the chromatographic behaviour of the immobilized CSPs towards various RP mobile phase systems was undertaken in our laboratories. This has allowed the development of simple and straightforward strategies for efficient method development.

The mobile phase selection should take into account the nature of the molecules to be analysed. Under this primary guideline, a 'decision tree' can be defined to start the method development (Figure 3). The selected mobile phases do not really differ from the proposals for achiral RP screening.  

The organic modifier plays an important role in regulating retention and modulating enantioselectivity. The first choices of modifiers to be combined with water are acetonitrile (ACN) and methanol (MeOH), as in achiral RPLC. In the case of the immobilized supports, other solvents, such as tetrahydrofuran (THF), can be used. For complex separations it may be profitable to investigate the use of these other solvents with the immobilized columns in combination with ACN- and MeOH-containing mobile phases to modulate recognition. 

As for the aqueous solution, the most common and effective pH value for the resolution of acidic molecules is in the range 2.0–2.5 in which the ionization of most organic acids will be suppressed. Formic acid is a good choice where LC–MS compatibility is required, while other acids (TFA, acetic acid, H

Based on the same reasoning, basic aqueous solutions should normally be in use to suppress the ionization of basic solutes. Where RP methods suitable for MS detection for basic compounds are being developed, the ammonium bicarbonate solution adjusted to pH 9 is an excellent option, although some other buffers may also be useful in terms of enantiorecognition. 

SFC applications in the separation of enantiomers are a growing field and deserve a dedicated article for themselves. Experiments with polysaccharide-derived CSPs show that excellent success rates can be achieved by using alcohol co-solvents.

  In particular, ethanol, methanol and isopropanol are very good initial co-solvent choices. Acetonitrile seems to be less successful, but it may be an option for a secondary screening. 

Immobilized CSPs are compatible with a much wider range of solvents than the coated phases. The use of such 'extended range' solvents was investigated for SFC screening in the same way as LC separations. Initially these solvents were used to enhance the sample solubility, to solve difficult separations or to process the compounds that are unstable in alcohols. Subsequently, we have found that these solvents can also offer exceptional enantioselectivity profiles. THF, methyl-tert-butyl ether (MtBE), dichloromethane (DCM) or ethyl acetate, for example, can be used as the CO

 modifier. In some cases, pure DCM, MtBE and ethyl acetate may not be strong enough to elute certain compounds. In these cases, the addition of small percentages of an alcohol mixed with the co-solvent is advised. 

As already mentioned, the success rate is already very high with the alcohol modifiers. Therefore, it has become established practice to use either the three immobilized columns (Chiralpak IA, IB and IC) — or alternatively the four coated ones (Chiralpak AD-H and AS-H, Chiralcel OD-H and OJ-H) — in the column set for screening with the alcohol modifiers as first strategy. Then the solvent range can be broadened on the immobilized columns if no satisfactory separation is achieved or sample solubility/stability becomes an issue.  

SFC and LC are complementary techniques and comparable success rates are achieved with the same set of columns. Nevertheless, recognition patterns are sometimes different and lead to different retentive and selectivity profiles. These two factors plus the likely sample solubility issues will determine the choice of one technique or the other for separation of a given compound. 

The use of polysaccharide-based CSPs of 3 μm particle size, in combination with the screening strategies outlined above, allows high-speed development of methods for the rapid and highly efficient separation of enantiomers.

Newly developed 3 μm supports show certain advantages for analytical purposes. Their performance and potential in fast routine chiral analysis, compared with the already existing 5 μm supports, are based on their higher column efficiency and more favourable mass transfer kinetics. These benefits on enantiomer resolution associated to the small particles are important for difficult separations and on gain of analysis time. 

Short, 4.6 mm i.d. columns packed with these 3 μm CSPs afford rapid and high-efficiency resolution of enantiomers when used in conventional modern HPLC instruments. Very high-speed analysis is possible by optimization of flow rate and temperature. Some examples of such fast analyses at high flow rate are depicted in Figure 4, with proven column packing stability over the whole HPLC pressure range.  

Although most separations can be achieved with a reduced number of columns and solvents, there remains a need for unique selectivities, which allow the resolution of certain challenging isomers or their impurities. Our research effort over the last decade has been devoted to the development and selection of a reduced series of selectors showing enantiomeric recognition complementary to the primary column sets used for screening. The new CSPs exhibiting the outstanding complementary characteristics are cellulose and amylose derivatives bearing methyl- and chloro-substituted phenyl groups, instead of the dimethyl- or dichloro- present in the standard systems (see Figure 5 for structural features). These columns were initially developed and used in our labs solely for preparative purposes but following their successful use for nearly a decade, they were introduced in 3 μm and 5 μm formats for LC and SFC separations. As shown in Figure 6, their compound-specific recognition can be helpful to resolve samples with multiple components, such as the two metolachlor enantiomeric pairs, in a single run.  

The current portfolio of columns available for the separation of enantiomers offers a powerful technology platform for the resolution of most enantiomers at analytical level. The selection of a limited series of CSPs, together with the rational choice of mobile phases and technologies, leads to new screening strategies that are compatible with time constraints and with the need for high success rates.  

It is our conclusion that an adequate compromise between resolution needs and development time can be found with an optimal screening setting. However, the current status is expected to evolve to address new challenges in the resolution of specific samples. This will result in the development of novel phases and technologies and we will have, in turn, to evolve new screening strategies to match these new needs. 

 received her PhD degree in Pharmacy from the University of Barcelona (Spain). After two years postdoctoral work in the University of Vienna (Austria) under the supervision of Professor Wolfgang Lindner, she joined Chiral Technologies Europe (Strasbourg, France). She is now manager of technical operations in the same company. Her main interests include the applications of chiral selectors for the resolution of enantiomers at analytical and preparative scale. 

Regular Article

A relatively small set of columns can be used on a routine basis in different modes to meet modern analytical needs.

Finding the Best Separation for Enantiomeric Mixtures

A new concept for the extraction of solid samples has been developed. The popular pressurized liquid extraction (PLE) technique has been modified so that the extraction cell can be divided, which greatly increases the flexibility and versatility of this technique. The system uses simple adaptors to couple commercially available extraction cells, which may be loaded with either sample or adsorbent(s). These segmented extraction cells make it possible to selectively retain analytes or matrix to achieve simultaneous extraction and clean-up or fractionation. The ultimate aim is to perform simultaneous extraction and clean-up and obtain a purified extract that is ready for instrumental analysis, all in line with current trends in analytical chemistry to automate and streamline the analytical procedures to reduce the analysis costs, solvent consumption, manual labour and increase the quality of analysis. Selected modes of operations are presented and a few recent environmental applications of modular PLE are reviewed.

Pressurized liquid extraction (PLE) was introduced in the mid-90s as an alternative to tedious classic extraction methods such as Soxhlet extraction or sonication for solid samples.

  It involves extraction with solvents at high temperature and pressure, which is highly beneficial for the extraction efficiency. The elevated temperature and pressure leads to significantly higher capacity of the extraction solvent to dissolve the target analytes, improves the rate of mass transport and effectiveness of sample wetting and matrix penetration, which improves the desorption of analytes from active sites on, and within, the sample particles. Instruments for PLE consists of a stainless-steel extraction cell, which is filled with sample and kept at the desired pressure and temperature with the aid of a pump for solvent delivery, a flow restrictor or on/off-valve, electronically controlled heaters; and one or more vials for extract collection. PLE may be conducted in two ways:  

, where the solvent is continuously pumped through the extraction cell. 

, where the extraction cell is filled with solvent, pressurized for a specified time and then drained to the collection vial. It is also possible to combine the two or to perform multiple extraction cycles. Dynamic PLE is suitable for rapidly equilibrating (easily extracted) samples and, conversely, static PLE is suitable for slowly equilibrating samples. As PLE is primarily used for samples that are difficult to extract, the static PLE approach is more widely used. The majority of PLE applications (about 75%) have been performed in the static extraction mode. 

Such systems can reach temperatures up to 200 °C and pressures up to 200 bars and can accommodate cells of various volumes, [i.e., 1, 5, 11, 22, or 33 mL (ASE 200) and 34, 66 or 100 mL (ASE 300)]. Both systems are produced by Dionex (Sunnyvale, California, USA) 

Since its introduction a decade ago, PLE has grown rapidly in popularity, as indicated by an almost exponential increase in the number of scientific publications and conference presentations (Figure 1, left). It has also been adopted by many commercial laboratories and more or less completely replaced Soxhlet and other traditional extraction techniques. 

Over time, there has been a shift in major application areas. Initially, PLE was almost exclusively used for solid environmental samples, primarily soils and sediments, later it was also adopted by researchers working with biotic samples and food control laboratories and, recently, it has gained increasing popularity amongst natural product chemists and laboratories working with product control (Figure 1, right). 

Generally, the PLE extracts are characterized, or analysed for target (mainly organic) analytes, by various chromatographic techniques, primarily gas chromatography (GC) (75–80%) and liquid chromatography (LC) (10–15%), but also ion chromatography (IC) (ca. 8%) and capillary electrophoresis (EC) (ca. 2%); with mass spectrometry (MS) as the most frequent detection technique. The main areas have been summarized in some recent reviews: abiotic environmental,

PLE is very efficient, as mentioned earlier, and generally gives equal to or better recoveries than Soxhlet.

  However, the exhaustive nature of PLE is both its largest assets and one of its main drawbacks. Although PLE allows the sample to be (particle) filtered (Figure 2, left), through its flow-through design and sometimes produces extracts that can be analysed directly using highly selective techniques such as liquid chromatography-tandem mass spectrometry (LC–MS–MS), its low selectivity more often leads to extracts rich in co-extracted material, which has to be removed prior to instrumental analysis.

  This process is tedious and costly, severely hampers the sample throughput, negatively influences the quality of analyses and frequently involves the use of large volumes of harmful organic solvents. Alternative procedures are, therefore, needed. 

Some authors have used a pre-extraction (Pre-PLE, Figure 2) with a weak solvent to remove some interfering (less strongly sorbed) compounds before the extraction of the compounds of interest.

  The extract complexity may also be reduced by using a sequential extraction procedure with solvents of increasing solvent strength.

  Such procedures may also be used to assess the strength of matrix–analyte interactions.

The selectivity of the extraction or leaching process may be further enhanced through the addition of a matrix retainer to the extraction cell. Use of alumina as a fat retainer was already suggested in 1996 in a Dionex application note.

  Since then, many other adsorbents have been used for the same purpose, for example, Florisil, silica gel and diol- and cyanopropyl-silica.

  For chemically inert compounds such as PCBs and dioxins our group has recently shown than even better results may be obtained using sulphuric-acid-impregnated silica.

  Matrix retainers have also been used for abiotic samples. Peterson and Richter used an ion-exchange resin to retain unwanted ionic interferences during extraction of perchlorate from vegetation samples,

  Ong et al. used silica gel to retain humic matter and other polar co-extractants during PLE of polycyclic aromatic hydrocarbons (PAHs) from soil

  and Kim et al. used a silica/cupper mixture to simultaneously remove polar compounds and sulphur when extracting PAHs from sediment.

  In the closely related matrix solid-phase dispersion (MSPD) technique the matrix retainer is mixed with the sample prior to the PLE extraction.

  Both the matrix retainer and MSPD technique can be adjusted to retain particular matrix constituents by choosing an appropriate retainer or dispersion material and by carefully tuning the solvent composition and PLE parameters.  

There are also a few recent applications in which the concept of PLE with in-line sample purification has been taken one step further, by using the adsorbent bed not only for matrix removal but also for analyte fractionation. Lundstedt et al. used 2% deactivated silica-gel -filled extraction cells (Figure 3, left) to separate parent PAHs from carbonyl-containing PAH oxidation products (oxy-PAHs).

  The former were extracted with cyclohexane/dichloromethane (9:1) at 120 °C and the latter with cyclohexane/dichloromethane (1:3) at 150 °C. By using a similar approach Poersschmann et al. were able to fractionate lipids into two fractions containing (1) neutral lipids (acylglycines, sterol esters and long-chain free fatty acids and (2) polar phospholipids.

  In both cases, analyte classes of different polarity were separated through adsorption chromatography processes. 

Another mode of separation, shape selective separation on activated carbon, has proven to be very useful. By using carbon-filled extraction cells halogenated aromatic compounds, for example, PCBs and dioxins, can be fractionated according to planarity (Figure 3, right). Because the PCBs are less planar than the dioxins they can be extracted with apolar or moderately polar solvents or solvent mixtures, pass the carbon trap, and be recovered in the first fraction together with the lipids. The planar dioxins, on the other hand, binds tightly to carbon and can only be released by backward elution with an aromatic solvent such as toluene. Depending on the amount of carbon, the PLE settings, and the solvent composition the dioxin-like non-ortho PCB may be directed to the first or second fraction. Following miniaturized multilayer silica clean-up the toluene fraction is ready for non-ortho PCBs and/or dioxins analysis by bioassays,

The applications described above serves as illustrative examples of what can be accomplished using 

 purification techniques. However, although the streamlined approaches significantly reduces the analysis time, manual labour and solvent volumes, compared with the traditional Soxhlet extraction and multi-step open column clean up, there are also limitations of the 

 clean-up and fraction techniques. These primarily stem from the intrinsic ambiguity in exhaustively extracting analytes from complex samples, efficiently trapping analytes or matrix on an adsorbent and, in the case of analyte trapping, quantitatively releasing analytes without co-extracting large quantities of matrix material. This equation cannot be solved using current commercial hardware for PLE, which houses sample and matrix retainer or adsorbent in the same compartment.  

A flexible modular approach for PLE with 

 clean-up has been recently developed, in which extraction cell segments may be added or removed as needed to overcome the previously discussed problems and ensure optimum extraction efficiency and selectivity. 

A modular system using in-house manufactured adaptors to couple existing ASE 200 extraction cells (Dionex Sunnyvale, California, USA) was recently developed (Figure 4).

  The adaptors consist of two cylindrical stainless steel guides, with threading identical to the original PLE end-caps, and a PEEK disc with a stainless steel frit pressed into its centre. The guides primarily align the extraction cell segments and keep the PEEK disc in place, but are not tightened hard. Instead, it is the compression unit of the ASE system that presses the extraction cell segments into the PEEK disc and creates the seal. These adaptors allow two or three cells to be serially connected. All combinations of 1, 5, 11 and 22 mL cells, except two- or three-22 mL cells, are possible. The same company produce a system with a similar design (ASE 350) and it should be possible to manufacture larger adaptors to couple its larger cells (34, 66 and 100 mL) to increase the sample capacity. 

Due to the cell and/or heater oven designs of other PLE systems on the market it is currently difficult to transfer this modular approach to ther instrument platforms. Fluid management systems (FMS) do, however, offer a similar solution in which PLE is coupled through an evaporative interface to a multi-column system for subsequent (at-line) clean-up. This system is primarily intended for dioxin analysis 

The separation of the extraction cells into two or three separate compartments greatly enhances the flexibility and versatility of the PLE with the 

 purification concept. Modules with sample, matrix retainer and adsorbents or ion-exchange resins may be added or removed as needed to obtain the desired separation, much like the solid-phase extraction (SPE) cartridges that are frequently used for extraction and purification of aqueous samples; or for purification of extracts of solid samples. The combinations are almost endless, but a few are described below and in Figure 5. 

The 'Trap and Release' mode (Figure 5, top left) is very similar to the procedure described in Figure 3 and the corresponding text, with one major difference, the sample modules can be removed in Modular PLE prior to elution of the trapped material. The 'Class Separation' mode (Figure 5, top right) is closely related to Trap and Release, but involves sequential extraction of the trap. The final extraction of the most tightly bound analytes is done in the reverse direction, after turning the trap. For samples of moderate complexity it may also be possible to perform extraction and purification based on a 'Dual Mode Clean-up' (Figure 5, lower left), by loading a two-module extraction cell with sample and an adsorbent, then extracting the sample, trapping the desired analytes on the adsorbent (adsorbent A), removing the sample module, and back-extract the trapped material through a second adsorbent (adsorbent B). More complex samples may be handled using a three-module 'Matrix Retainer and Trap' set-up (Figure 5, lower right), in which the compartments are packed with sample, matrix retainer and adsorbent, respectively. The samples are extracted and the extracted material passes through a matrix retainer and adsorbent which, once again, is used to trap selected analytes. The sample and matrix retainer modules are then removed and the trap is sequentially extracted. 

Spinnel et al. combined a user-friendly sampling technique based on gas adsorption on polyurethane foam plugs (PUFP) and aerosol particle removal using glass fibre filters with Modular PLE for cost-efficient extraction and sample preparation of polychlorinated dibenzo-

-dioxins and dibenzofurans ('dioxins' or PCDD/Fs) in flue gasses from municipal solid waste incinerators.

  The procedure utilized the 'Trap and Release' mode (Figure 5) with the sampling material packed in the upstream cell, followed by an activated carbon-filled cell. The dioxins were extracted from the PUFP and the filter using three cycles of 

-heptane/acetone (static time 5 min; temperature 150 °C; pressure 2000 psi). The upper cell and the coupling assembly were removed and the cell with the carbon trap was capped. The trap was inverted and extracted with three cycles of toluene (static time 7 min; temperature 175 °C) to back-elute the dioxins.  

The procedure offers clear advantages as compared with normal PLE and selective PLE with 

  Because the carbon adsorbent is very selective to planar compounds the bulk of matrix is passing through the carbon trap during extraction. Furthermore, the coupled cell assemblies enable the removal of the matrix before back-flushing the carbon trap, thereby preventing extraction of unwanted matrix constituents during the exhaustive toluene extraction of the trap. 

Excellent agreement was obtained between Soxhlet extraction and multi-step clean-up and Modular PLE followed by a miniaturized open column clean-up (Figure 6). For mono- to tetra-CDD/F the results of the two extraction techniques were statistically equivalent while Modular PLE provided superior extraction of highly chlorinated congeners. This analytical strategy was proposed to be used to improve national and international dioxin emission inventories. 

In a subsequent study, Spinnel et al. further developed the Modular PLE approach for dioxin analysis.

  A matrix retainer module filled with sulphuric acid impregnated silica gel was introduced between the sample, in this case salmon (Salmon salar) muscle mixed with sodium sulphate and the carbon trap. This 'Matrix Retainer and Trap' procedure (Figure 5, lower left) provided extracts clean enough to be analysed by gas chromatography–high resolution mass spectrometry analysis (GC–HRMS) after solvent evaporation to an appropriate final volume. 

The results obtained were in good agreement with those of a Soxhlet reference method. A slightly higher total dioxin equivalency concentration was obtained for Modular PLE (89.3 ± 8.3 pg/g fresh weight) than for Soxhlet extraction (81.3 ± 7.8 pg/g fresh weight), which indicate an improved extraction efficiency.  

The congener profiles of the major (indicator) PCBs (no. 25, 52, 101, 138, 153 and 180), as well as dioxin-like mono-ortho PCBs (no. 105, 114, 118, 123, 156, 157, 167 and 189), non-ortho PCBs (no. 77, 81, 126 and 169) and 2,3,7,8-substituted PCDD/Fs also agreed well between the methods (Figure 7), although with the expected positive bias for the modular PLE results. Careful inspection of the profiles revealed that the highly chlorinated PCDD/Fs were particularly well extracted using Modular PLE. 

Complementary Use of Modular PLE and Hyphenated Chromatographic Systems

Application of Modular PLE within the major PLE application areas, (i.e. abiotic environmental, food, feed and biota, and natural products) may sometimes be challenging. For example, in combining extraction and in-line purification it will be difficult to separate less retained compounds, often non-polar compounds without functional groups. Thus, the first fraction obtained in 'Class Separation' procedures (Figure 5, top right) may be complex. In such cases, the recently developed comprehensive two-dimensional chromatographic techniques, for example, comprehensive two-dimensional gas chromatography with time-of-flight mass spectrometric detection (GC×GC–MS), may be used to obtain the necessary analyte resolution.

  If so, the lack of selectivity during extraction and clean-up could be made up for by the exceptional resolving power of the comprehensive chromatographic techniques. 

Although this paper only illustrates a small range of the possibilities of modular PLE its advantages are clear. Simultaneous extraction and clean-up result in reductions in analysis costs, solvent consumption, manual labour and improved quality of analysis. The future of modular PLE depends both on its acceptance by the analytical chemistry community and on the development and commercialization of dedicated cell assemblies and/or modular PLE instrumentation. 

Interested readers may find inspiration and food for thought in existing SPE procedures related to their field because the same basic principles for analyte trapping, matrix removal and target fractionation apply for both techniques. In terms of hardware development there are several routes worth exploring, including (but not limited to): dedicated modular PLE systems; improved adaptors for the coupling of existing cells; the design of cells that can be directly connected; and the development of sample and adsorbent modules that can be sequentially loaded into exciting high-aspect ratio cells. Finally, the current rapid development of selective solid-phase materials, such as molecular imprinted polymers (MIPs) and immuno-sorbents, may result in new exciting (analyte and matrix) trapping materials for modular 

Selected modes of operations are presented and a few recent environmental applications of modular PLE are reviewed.

A Modular Approach to Pressurized Liquid Extraction with in-cell Clean-up

Ron Majors, editor of "Column Watch" and "Sample Prep Perspectives," has been with 

 for over 26 years. Currently a senior scientist with Agilent Technologies, Wilmington, Delaware, Ron is known industry-wide as one of the premier chromatography experts in the field. He is also a member of 

John Dolan has been writing "LC Troubleshooting" for 

 for over 25 years. One of the industry's most respected professionals, John is currently a principal instructor for LC Resources, Walnut Creek, California He is also a member of 

For decades, reversed-phase (RP) chromatography has dominated HPLC applications. Almost as soon as bonded reversed phases became available in the early 1970s, this mode surged ahead as the most popular. Through the years, the technique has become more efficient, columns more stable, and new types of phases beyond the traditional C8 and C18 give different selectivity that is required for more difficult separations. Here, we focus on the characterization of reversed-phase columns with particular emphasis on the hydrophobic-subtraction model. A well-attended Pittcon 2010 session brought together many experts to update our knowledge in this area and help liquid chromatographers make more educated decisions on phase selection and usage.

  over 95% of all respondents used reversed-phase (RP) chromatography in their daily work. The popularity of this technique is undoubtedly due to its versatility. Reversed-phase chromatography can separate nonpolar, polar, ionizable and ionic compounds, sometimes at the same time. For mobile phases, reversed-phase chromatography uses water (often buffered) and a water-miscible organic solvent; both are relatively plentiful in most parts of the world in HPLC grade purity.  

At Pittcon 2010, Dr Lloyd Snyder of LC Resources (Walnut Creek, California, USA) and Professor Peter Carr of the University of Minnesota (Minneapolis, Minnesota, USA) organized a full-day symposium on RP column selectivity with an emphasis on the hydrophobic-subtraction model.

  The two sessions brought together academic and industrial people who are expanding on and using this model to tackle the subject of column selectivity in this most popular mode.  

Brief History of RP Chromatography Chemistries

The technique of high performance liquid chromatography (HPLC) had its beginnings in the late 1960s. In the beginning of HPLC, there was no reversed-phase chromatography as we know it today.  

 Fatty Acids by Reversed-Phase Partition Chromatography

  which outlined the basics of liquid–liquid chromatography with a nonpolar stationary phase and a polar mobile phase, lay dormant for a couple of decades while workers in the field pursued other chromatographic approaches such as paper, adsorption and thin-layer chromatography (TLC). The problem for liquid–liquid partitioning reversed-phase chromatography in those early days was first homogeneously coating, then keeping the coated nonpolar stationary phase on the surface of the polar base material while an aqueous–organic solvent mixture was running over its surface. 

This liquid–liquid partition mechanism was never very successful in TLC and also proved unsuccessful in the early days of LC. However, the development of a hydrocarbon polymer-coated Zipax (called Zipax HCP) material by Kirkland and DeStefano

  gave a more stable surface than the coated liquid phases. However, as admitted by Kirkland coworker Reed Williams with the Dupont Liquid Chromatography business at the time, "With the liquid- and polymer-coated packings, one dared not change the mobile phase very much for fear of stripping the stationary phase from the packing."

  The early brush-type bonded silicate ester phases (Si–O–CH

  suffered from some of the same problems not because the phase would mechanically strip off of the packing, but because the chemical bond could be easily hydrolysed and was thermally unstable.  

The answer to the dilemma came in the successful development of chemically bonded silicone (siloxane) phases (Si–O–Si–C) based upon the work of Aue and Hastings.

  The first commercial, chemically bonded phase was Permaphase, developed by Kirkland and coworkers.11 Rather than being coated, Permaphase was a chemically bonded polymeric phase applied to the pellicular support Zipax. Compared with liquid–liquid partitioning phases and polymer-coated phases, with this polymeric-bonded phase, column life was much longer, stronger mobile phases could be used and gradient elution could help solve the 'general elution problem'. However, due to slow solute diffusion in the polymeric layer, column efficiency was not as great as anticipated. The solution, now in popular use today, was the development of the nonpolar, monomeric bonded siloxane phase.

  Here a monochloro- or monoalkyoxy-silane containing a strongly hydrophobic hydrocarbon moiety, such as octadecylsilane (ODS, C18), was reacted with the surface silanol to produce a stable, higher efficiency bonded phase. Monomeric phases are, by far, the most widely used reversed-phase packing today and the basis for most theoretical and practical models of retention, efficiency and selectivity. 

Gas chromatography (GC) had been plagued by the proliferation of many stationary phases. Unlike GC, with only the stationary phase and temperature as major variables, liquid chromatographers initially felt that the ability to manipulate the mobile phase would cut down the number of stationary phases to only a few. In the preface of the 1974 book on chemically bonded phases, editor Eli Grushka, then at the Chemistry Department of the State University of New York at Buffalo, stated, "We do not wish to encourage proliferation of many bonded phases, each suited for a particular separation."

  He went on to state, "Further work should be directed, perhaps, at a better understanding of bonded phase characteristics, thus developing a relatively small number of systems that could be utilized for multiseparation purposes." Nevertheless, to date, there are well over 1000 reversed-phase columns alone

  and the list keeps growing. Thus, a number of workers

  have tried to come up with a simple scheme to understand selectivity in reversed-phase chromatography. The hydrophobic-subtraction model

  appears to be gaining favour as an approach that can be fairly universal in its application. The remainder of this instalment of "Column Watch" will be devoted to the papers presented at Pittcon 2010 on this model as well as other approaches to reversed-phase chromatography selectivity. 

The Hydrophobic-Subtraction (H-S) Model of Reversed-Phase Column Selectivity

Many different procedures have been used to describe the selectivity of reversed-phase columns (for example, references 15–20). The H-S model differs from all of these in being derived experimentally, without initial assumptions about the various interactions between solutes and the column, yet results in a quantitative (and presumably fundamental) description of each column–solute interaction. Several thousand measurements of retention factors 

 for 150 different solutes and about 100 type-B alkylsilica columns (C

EB for a reference solute (ethylbenzene), and terms i–v describe contributions to retention from five solute-column interactions: (i) hydrophobic, (ii) steric interaction, (iii) hydrogen-bonding of basic solutes to (acidic) silanols, (iv) hydrogen bonding of acidic solutes to basic groups within the stationary phase and (v) cation-exchange between a cationic solute and ionized silanols –SiO

. The Greek letters (η', σ' and so forth) refer to properties of the solute, while capital letters describe properties of the column that can be measured by means of Equation 1: column hydrophobicity 

, steric resistance to penetration by the solute 

 (a measure of silanol 'acidity' or ionization). These five parameters can be easily measured

  and used to characterize different columns in terms of selectivity. Except for 

, which varies with mobile-phase pH, each parameter can be assumed constant for different mobile phases or temperatures. Two additional interactions are present for cyano (π–π, dipole–dipole) and phenyl (π–π) columns, but are of limited significance for most samples. 

Two columns can be compared in selectivity by means of a function 

 can be regarded as the distance between columns 1 and 2 in a five-dimensional space defined by the five column parameters. Two columns with a small value of 

 will be similar in terms of selectivity, while large values of 

 indicate two columns of quite different selectivity. The empirical factors (12.5, 100, etc.) recognize differences in the relative contributions of 

Henry agreed with Neue that large groups of solutes should be studied by LC–MS to survey a wide range in phase properties. He also made a plea for manufacturers to provide more information on the chemistry of their phases.

Because of the fundamental nature of the H-S model, it seems suited to a wide range of applications of column selectivity, some of which were reported for the first time at Pittcon 2010:  

Selection of similar columns, as back ups for routine assays.

Selection of dissimilar columns, for a change in separation during method development, the development of orthogonal separations to ensure that no 'new' peaks are present and 2D separation.

Avoidance of columns that are more likely to result in tailing peaks (usually for bases, but sometimes for acids).

Development of new stationary phases of unique selectivity.

A better understanding of stationary phase degradation during routine use.

'Slow' column equilibration for ionized solutes.

Deeper insights into the nature of reversed-phase retention and column selectivity, as well as the factors that determine column selectivity.

Quantitative predictions of separation for different columns, with the possibility of choosing a specific, 'best' column for a given sample.

Let's take a closer look of some of these applications using the H-S model. 

Reversed-phase HPLC is widely used in the pharmaceutical industry. Most pharmaceutical methods require a back up column. The H-S model can be used to find a column that is 'equivalent' based upon the testing parameters mentioned previously. M. Marques of the 

) reviewed both the history of column classification by the 

 and two current procedures for the measurement of column selectivity.

 approach, developed in cooperation with the National Institute of Standards and Technology, is based upon the retention times of five test compounds: uracil, toluene, ethylbenzene, quinizarin and amitriptyline. The H-S model comprises the second 

 as the Product Quality Research Institute procedure (the PQRI provided support for the evaluation of the H-S model). The 

) allows the user to compare the selectivity of two columns by either procedure. There are 120 C18 columns in the 

 database and 480 reversed-phase columns of all kinds in the PQRI database (which continues to be updated). Two similar, widely promoted methods for comparing column selectivity were also noted: the Advanced Chemistry Development (ACD, Toronto, Ontario, Canada) ACD Labs Column Selector and Katholieke Universiteit (Leuven, Belgium) Column Classification System. Over the past 18 months, the column equivalency section of the USP website has been visited 1400–1900 times per month. It had been hoped that users would offer comments on which of the two 

 procedures they preferred, but so far, little feedback from the public has been received.  

A presentation by A. Schellinger of Abbott Laboratories (North Chicago, Illinois, USA)

  was based entirely upon the hydrophobic-subtraction model. The thrust of the work was to compare the selectivities of various groups of phases chosen based upon their H-S parameters, their 

 values (Equation 2) and their positions in the H-S triangle of Zhang and Carr

  for some 25 drugs containing multiple basic sites.

  The point was to test the concept "Will phases chosen to be similar based upon the H-S model actually be similar based upon their selectivities as established by a set of basic drugs?" Four groupings of reversed phases were chosen:  

Phases that are internally similar but quite different from the preceding phases.

Phases chosen to be different from one another and all other phases tested. 

By and large there was a good correlation between the standard error (S.E.) values of the drug-based comparison of phases to the S.E.-based upon a comparison using test compounds from the 16 solutes used by Snyder and Dolan

  to establish the phase parameters. The chief deviation was a Nova Pak cyano phase (Waters, Milford, Massachusetts, USA). Schellinger also discussed the use of gradient elution to compare phases. The standard conditions used to measure the H-S model parameters are isocratic (50:50 acetonitrile–water phosphate buffers at pH 2.8 or 7.0). It was shown that gradient conditions give very similar column classification if one bases selectivity on the scatter in plots of retention time vs retention time not log 

 as is done in the original isocratic Snyder–Dolan method.

The use of the H-S model can be quite helpful in column and bonded phase development from the commercial side. F. Dorman (Restek, Bellefonte, Pennsylvania, USA)

  reviewed the use of the H-S model in his laboratory for the purpose of comparing different columns in terms of selectivity. In one study, measurements of the column-selectivity parameters 

 and so forth were made for column packings for which either the bonding process or starting silica was changed. As expected, a change of silica was found to have the largest effect, affecting mainly values of silanol acidity 

 at low pH. A comparison of selectivity among various Restek columns were investigated next, with a sample of the 16 test-compounds that are used to measure values of 

 and so forth. Each of the five sample-column interactions of Equation 1 are exhibited strongly by two or more of these test-compounds, so resulting chromatograms for each column provide a visible display of changes in each interaction (as well as corresponding changes in 

, etc.). Thus, the Restek IBD column was found to have greater hydrogen-bond basicity 

, so that phenols and acidic solutes were retained most strongly. Similarly, the Aq-C18 and PFP-propyl columns had the most acidic silanols (largest 

) and preferentially retained protonated basic solutes. Finally, Equation 2 was used to rank different columns in terms of selectivity. 

In his use of the H-S model at Grace Davison Discovery Sciences (Carnforth, Lancashire, UK), I. Chappell

  began by describing how column selectivity (as measured by values of 

 and so forth) changes with bonding level (ligand concentration) for extended-polar-selectivity phases and concluded that this enables the design of phases with selectivity matched to the application. He then considered the manufacture of packings for large-scale preparative separations, where a selectivity similar to that obtained with more costly spherical packings is required for these low-cost irregular silicas. The question he considered was "How do we measure and control selectivity on transfer between silica types?" By varying the bonding level, it was possible to adjust for differences in the base silica, resulting in the same column-selectivity parameters for different bonded phases. His final conclusion was "The Snyder/Dolan/Carr approach to measuring reversed-phase selectivity is a powerful tool in media design and control." 

We focus on the characterization of reversed-phase columns with particular emphasis on the hydrophobic-subtraction model

LCGC Europe-06-01-2010