Coupling liquid chromatography (LC) and mass spectrometry (MS) is always a 'work in progress' with the final goal of providing the most sophisticated detector capable of identifying and quantifying organic and inorganic compounds at trace level. Well-established LC–MS interfaces and several novel ones are constantly being developed and different operating strategies have been adopted to overcome the apparent difficulties of coupling the two techniques. 

LC–MS is complex because LC operates with a high-pressure liquid phase, while the MS analyser must operate under high-vacuum gas phase conditions and this appears, in principle, an absolute limitation. The LC mobile phase has a variable composition and high-resolution separations often rely on the use of modifiers that are not always welcome in the MS instrumentation. Physicochemical properties of the analytes can vary dramatically and this influences instrument response, or requires different settings in the interface and in the mass spectrometer.  

Despite these limitations, the use of LC–MS has grown in many areas of research due to technical advances in both techniques. 

The key of this success is represented by the development of the atmospheric pressure ionization (API) interfaces, among which electrospray ionization (ESI) is the most widely used for medium to highly polar compounds because of its high sensitivity and wide molecular weight (MW) range. Interfacing LC and ESI has produced remarkable advances in biochemical analysis, allowing the mass determination of high-molecular-weight, non-volatile biological molecules directly from the liquid phase. This approach is now an invaluable tool in proteomics and has opened many doors in this and other areas of research, including pharmaceutical analysis, environmental analysis, food analysis, bioanalysis and metabolomics. 

Atmospheric pressure chemical ionization (APCI) comes into play for small–medium MW and less polar analytes, while the more recently developed atmospheric pressure photo ionization (APPI) and atmospheric pressure laser ionization (APLI) are used for non-polar compounds that are poorly ionized by the other two techniques. However, the ionization of non-polar compounds is still limited in many cases. The ionization is 'soft' in all cases, it takes place through low-energy chemical processes that lead to a protonated or deprotonated molecular ion (with or without adducts), with a scarce or absent fragmentation. Therefore, only a partial information is obtained and MS–MS, MS

, or high resolution accurate mass MS are required for analyte characterization. The attempt to create reference MS–MS library spectra is a difficult task, still to be fulfilled, because of the number of factors that influence the CID (collision ion dissociation) process into an MS–MS instrumentation.  

Many authors report that quantitative analysis in ESI and, to a lesser extent, in APCI, is affected by the presence of interferences from the matrix that induce ion suppression or enhancement.

  Matrix effects (ME) are responsible for unreliable quantitative data, even within a series of replicates, scarce reproducibility, linearity and accuracy of the overall method. ME are highly dependent on the sample nature and the unpredictable response that they induce can be detected, even among different lots of the same sample, or with the same method. A possible explanation relies on the assumption that the analyte and the co-eluted matrix can compete in the liquid phase for the available charges and for the access to the droplet surface for gas-phase emission.

  During method development each step should be optimized to reduce ME. Sample clean-up procedures, more efficient chromatographic separations and novel mass spectrometric approaches can play a significant role, together with appropriate data analysis. Nevertheless, soft ionization techniques are not very well suited for the analysis of true unknown compounds, such as in gas chromatography–mass spectrometry (GC–MS), in which 70 eV electron ionization (EI) allows the collection of library spectra for sample identification. Therefore, the demand for new LC–MS instrumentation is strongly increasing in many applications. 

A niche of LC–MS instrument development involves the use of electron ionization. From a historical standpoint, it is worth mentioning that EI pioneered the LC–MS development but, despite an initial success, mainly offered by the particle beam (PB) interface,

  it was rapidly outpaced by the development of the soft ionization techniques. They opened the way to the analysis of molecules of biological interest, attracting most researchers' attention. Consequently, the efforts to develop a more efficient EI-based interface were cast off, although a considerable number of small–medium molecular weight substances can provide very good EI spectra. The operating principle is simple and it is based on the gas-phase ionization of the sample, disregarding its chemical background, as long as it can withstand the typical EI source conditions (high vacuum and temperature). The absence of ion–molecule or ion–ion reactions reduces the effect of the mobile phase in the ionization process reducing ME, avoiding any post-column adjustments.  

Similarly in GC–MS, the spectrum obtained is very informative; its high reproducibility allows an easy comparison with the spectra from commercially available electronic resources (such as NIST or Wiley). It can also benefit the algorithms developed by the National Institute of Standards and Technology (NIST) and Automated MS Deconvolution and Identification System (AMDIS), which extract the analytes' mass spectra in complex chromatographic mixtures in the presence of several overlapping peaks. The identification is automatic, legally defensible and provides structural information that can help to identify unknown compounds.

However, we cannot understate the fact that the operating conditions of EI and LC are quite dissimilar and this might explain some of the limitations observed in the past. Poor sensitivity, signal instability, reduced linearity and disappointing optimization were everyday frustrations, especially when working in reverse-phase conditions. Various authors attempted to efficiently generate EI spectra from a liquid effluent. Kientz et al.

  were able to generate EI spectra based on an eluent-jet formation by means of inductive heating of the micro-LC effluent and momentum separation in a jet separator; Dijkstra et al.

  improved that technique with a particular emphasis on its use under chemical ionization (CI)–MS conditions. A considerable effort in the exploitation of EI in LC–MS has been done by Amirav et al., who explored the potential of supersonic molecular beams (SMB-MS) to produce intense molecular ions in EI spectra from an LC effluent. The authors called this approach Cold EI to emphasize that the SBM serves as a medium for EI of vibrationally cold molecules.

  A spray of liquid is formed at high pressure, then the sample particles are vaporized prior to sample expansion, as isolated molecules, from a supersonic nozzle. 

A supersonic molecular beam, containing vibrationally cold sample molecules, is then collimated and proceed axially through an EI ion-source to obtain Cold EI mass. The spectra provide an enhanced molecular ion, but also library-searchable fragments for improved confidence level in the identification. Our research group has been working for more than a decade and through several designs to develop an up-to-date LC–MS based on EI. Keeping in mind the operating principles of PB, we significantly improved sensitivity for any mobile-phase composition by drastically reducing the mobile-phase intake.

  This first approach could be considered as a miniaturization of the PB interface, therefore, we called it microparticle beam and it was followed by its evolution, called capillary EI (CapEI, commercialized by Waters, Milford, Massachusetts, USA, in 1999). Both devices used microscale flow rates (1–5 μL/min) and they were successfully tested in the analysis of several classes of compounds under different reverse-phase HPLC conditions.

  In particular, for the first time, a significantly improved tolerance for nonvolatile buffers was observed in mass spectrometry.

  Taking advantage of the extremely low flow rate typical of the nano-HPLC columns, we conceived a completely new interface, particularly simple in the design. The effluent from the column is directly introduced into the ion source at nano-scale flow rates (< 500 nL/min) with the advantage to further reduce the adverse effects of mobile-phase vapours. We called it Direct-EI because the nano-column is directly coupled to the MS, with the same simplicity of a capillary GC column, without any intermediate apparatus. 

One of the most challenging efforts in LC–MS is to eliminate the solvent vapours before they enter the high vacuum region. In ESI interface, despite the fact that the liquid phase plays a fundamental role in the ionization process, it must be removed prior to entering the analyser, because its instantaneous vaporization is sufficient to raise the pressure to inacceptable values. This operation should be performed very carefully to avoid sample losses and interferences with the ionization process, with the aim to generate a solvent-free ion beam focused into the high vacuum analyser region. In EI this issue must be addressed at an early stage, in fact the typical ion source conditions are high vacuum (approx. 10

 torr) and high temperature, therefore, only a very limited liquid intake should be allowed into the ion source. The development of nanoscale LC columns has allowed the separation of complex mixtures at nanolitres per minute flow rate. This is a sufficiently low flow rate to be introduced into a modified EI ion source, without breaking the vacuum and compromising the correct functioning. Keeping in mind these considerations, we developed the Direct-EI interface, in which the entire interfacing process takes place in the small volume of the ion source, with a simplicity comparable to a GC–MS system. The liquid phase enters the interface via a capillary connecting tubing. 

As illustrated in Figure 1, an aerosol is formed in high vacuum conditions, followed by a quick droplet desolvation and final vaporization of the solute, upon contact on a target surface prior to the ionization. The tip of the nebulizer is extended into the ion source so that the spray expansion is completely contained inside the volume. The eluate must reach the ion source as liquid phase and to prevent a premature in-tube solvent evaporation the nebulizer and the connecting tubing must be thermally insulated from the source heat. This is very important to obtain a good quality aerosol and to avoid solute precipitation that might clog the tip. The solute conversion to the gas phase is promoted by the source heat. 

The temperature is kept between 200 °C and 350 °C and has a double function: to compensate for the latent heat of vaporization during the droplet desolvation and to convert the solute into the gas phase upon contact on the source surface. The transition from liquid to aerosol is very rapid and the mix of vapours is homogeneously distributed in the ion volume. The flow rate introduced should not exceed 0.75 μL/min for most reverse mobile phase compositions, mainly imposed by the column backpressure. It normally generates less than a mL/min of vapour, which is within the pumping capacity of most MS systems, and it is roughly the same of the carrier gas (helium) introduced by a conventional GC capillary column. At this flow rate we did not report any adverse effects on the MS hardware (filaments, lenses, insulations, etc.). Conversely, the lowest flow rate capable of generating a fine and homogeneous spray is approximately 100 nL/min. The design of the interface allows the fast removal of the solvent, improving the signal-to-noise (S/N) ratio and the sensitivity, and eliminating chemical ionization interferences. Solvent signals can be detected in the low mass range of the mass spectrum recorded, but they can easily be subtracted, preserving the intrinsic quality of the EI spectrum. 

Another advantage offered by the nano-columns is that the reduced flow rate allows the introduction of a steady stream of non-volatile buffers, as mobile-phase modifiers, into the ion source. Our previous EI based interfaces, and now the Direct-EI, demonstrate that the combination of reduced flow rate and EI enhances the tolerance towards chemically modified mobile phases.

  Many chromatographic separations benefit from the presence of non-volatile buffers in the mobile phase. In ESI, non-volatile species cause salt depositions on the metal surfaces and could completely block ion transmission. In addition, if the anion and the cation pair too strongly with the analyte, then the analyte ions might be prevented from carrying the excess charge on the droplet surface; as a result, the ESI response may be very low. For this reason volatile buffers are used instead. In EI the analyte is ionized in the gas-phase independently and the signal is not influenced by the presence of pre-formed ions in the form of salts or strong acids and bases. Routine cleaning is enough to get rid of the very limited salt deposition into the ion source. For the same reasons a wider selection of solvents can be used as well. 

We already mentioned that quantification procedures based on ESI and APCI can be severely affected by ME. In Direct-EI, as opposed to API techniques, the gas-phase ionization process is a physical-based odd electrons one and involves a high-energy content. Fragmentation is spontaneous, pathways are highly reproducible and independent from the type of instrument. Co-eluted components are ionized independently from each other, so that signal intensity is invariably related with the concentration of each analyte. In ESI the ionization takes place in the liquid phase, so that any co-eluted substance from the matrix can interfere with the available charges inducing either ion suppression or enhancement of the signal.  

This interface was tested on an Agilent 5475 MSD (Agilent Technologies Inc., Santa Clara, California, USA), and compared to our previous designs, it shows the following improvements: (a) LODs to the low-picogramme level for most substances in SIM; (b) higher spectral quality with full spectrum searching capability using electronic libraries; (c) no chemical ionization (CI) signals in the mass spectrum with highly accurate reproduction of isotope ion clusters; (d) full compatibility with nonvolatile buffers and other salts in the mobile phase; (e) absence of ME in quantitative applications; (f) extended range of linear response; (g) good intra and interday signal stability.  

Direct-EI offers a valid and reliable alternative to other LC–MS interfaces for a large number of small–medium molecular weight molecules, with the only condition to withstand a gas phase ionization. The diagram in Figure 2 demonstrates how 101 compounds, spanning from n-alkanes to sugars, with very different polarity and molecular weight can be successfully ionized with Direct-EI. This interface was tested in many fields of applications (environmental, pharmaceutical and biological) under different analytical conditions, at trace-level concentrations and with various matrices.

  In many aspects, Direct-EI can bridge the gap between GC–MS and LC–MS because we found that many compounds that are chemically sensitive or difficult to vaporize by GC–MS can take advantage of the nano-flow nebulization and give stable and intense signals (i.e., fatty acids, phenoxyacid pesticides). Other analytes that give a poor response, or do not ionize at all with ESI can be successfully analysed with Direct-EI (i.e., organochlorine pesticides, diethylene glycol). 

Multiresidue analysis of substances with different polarities can be highly frustrating. In fact, the protocols for low-polar, thermostabile analytes determination are traditionally based on gas chromatography (GC), mainly coupled to detection techniques such as electron capture detection and mass spectrometry.

  Conversely, LC–MS represents the method of choice for high-polar, poorly volatile compounds,

  but falls short when low polar compounds are present as well. Twelve OCPs together with seven phenoxyacid pesticides in water samples, with different physicochemical properties, were selected and analysed in a single step with Direct-EI-MS, after off-line SPE sample clean-up. Thanks to the gas-phase ionization, no ME were reported, regardless of the complexity of the original matrix. In Figure 3 another application of environmental interest regarded the analysis of organophosphorus pesticides and endocrine disrupting compounds (EDC) in water samples. 

A similar example regards the analysis of fragrances and other perfumery compounds. Figure 4 shows a scan profile of 36 substances at a concentration of 1 mg/mL, almost completely separated by the column. Some of the analytes were not GC amenable, but gave an uneven response with ESI. For all of them it was possible to record interpretable EI mass spectra as well as for three interferences present in the original mixture. 

To further test the performance of Direct-EI, and to assess its behaviour in terms of ME, we selected ibuprofen and phenacetin as model compounds and human plasma as complex matrix. Plasma samples were cleaned-up with two different procedures: liquid–liquid extraction and solid-phase extraction and ME was evaluated using the post-extraction addition method. The blank extracts were spiked with a given amount of a standard of the analytes and analysed in parallel with LC–ESI-MS–MS and LC-Direct-EI-MS. The effect of matrix composition on the response of the two interfaces for the target analytes is summarized in Table 1. In ESI-MS–MS analysis the presence of the matrix induced a consistent signal suppression for ibuprofen, whereas we observed signal enhancement for phenacetin. This trend was reported in all extracts with a drastic effect on both method precision and accuracy, regardless of the clean-up procedure used for sample preparation. On the contrary, the response with Direct-EI was not influenced by the presence of the matrix, under any circumstance. These results demonstrate that the method based on LC–ESI-MS–MS cannot be validated in the present form; therefore, strategies to address ME are mandatory. 

Table 1: Relative ME evaluation in human plasma extracts with LCâESI-MSâMS and LC-Direct-EI-MS.			

Another application involves the profiling of free fatty acids (FFA) in plasma samples.

  Preliminary results with LC-Direct-EI-MS analysis have shown several advantages over the current protocols that entail GC–MS and LC–API-MS methods. First of all, the sample preparation is faster because it does not require the compounds' derivatization. The response of the target compounds is not influenced by ME, and reproducible, high-quality library-matchable spectra are obtained. On the other hand, although sensitivity is satisfactory for the quantification in human plasma, it is not as high as that of GC–MS and LC–API-MS. Our future efforts will be dedicated to increasing sensitivity. 

The Direct-EI represents a valid alternative in LC–MS for the analysis of small–medium molecular weight compounds in a wide range of polarities, with a sufficient thermal stability. A large number of applications can benefit from this approach that fills the gap between GC–MS and API-based LC–MS. Our experiments demonstrate that Direct-EI is a robust and stable platform, where a "direct", simple approach combines with high operational flexibility and a large extent of information with an affordable instrumentation. Its performance is scarcely influenced by compound structure and mobile phase composition. In addition, the reduced flow rate allows the introduction of non-volatile modifiers and to limit ME thanks to the gas-phase ionization. Future efforts will be addressed to increase sensitivity, in which Direct-EI is still less competitive with ESI in terms of very low detection limits for specific analytes.  

The authors would like to thank Agilent Technologies for their support with this research. 

 graduated at the University of Urbino, Italy, in 1983. From 1987 to 1989 she was appointed as a post-doctoral associate at the Massachusetts Institute of Technology, Cambridge, Massachusetts, USA, where she expanded her experience in LC, LC–MS and MS–MS. From 1988 she has been assistant professor at the University of Urbino, where she teaches Analytical Chemistry. Her research focuses on new advances in micro- and nano-HPLC columns, and on the development of LC–MS techniques in wide range of applications. 

 graduated at the University of Urbino, Italy in February 1992. In October 1996 he received his PhD in Chemical Sciences at the University of Camerino. His current position is Assistant Professor of Analytical Chemistry University of Urbino. His principal field of interest is LC–MS coupling, but also has experience in method development in complex biological and environmental matrices. 

 graduated at the University of Urbino in 2000, and she earned her PhD in Chemical and Pharmaceutical Sciences at the same University in 2004. Since 2004 she has been involved in the LC-Direct-EI-MS project as a postdoctoral associate. Her scientific activity is focused on the following topics: development of analytical methods for sampling, purification and pre-concentration of analytes in complex matrices of environmental, biological and pharmaceutical interest; development and application of LC–MS technique for environmental and biological samples. 

 is currently a professor of Analytical Chemistry at the University of Urbino. He has extensive experience in the field of MS, LC and LC–MS. He is the director of several projects dealing with instrument development and design on LC–MS with some of the major instrument manufacturers and actively collaborates with the pharmaceutical industry with innovative LC–MS applications. He has published more than 100 scientific articles in leading analytical chemistry and mass spectrometry journals. 

1. B.K. Matuszewsky, M.L. Constanzer and C.M. Chavez-Eng., 

4. W.M.A Niessen, P. Manini and R. Andreoli, 

, A. Cappiello, Ed., 193–210 (Elsevier, Amsterdam, The Netherlands 2007). 

Articles

Regular Article

The operating principles of the Direct-Electron Ionization interface, which is gaining popularity in many LC–MS applications

Towards a Universal Detector for Small Molecule Applications: Direct-EI in LC–MS

Standards are as important in chromatography as columns, mobile phases, injectors and detectors.

 There's hardly an analytical laboratory to be found that does not stress the need for the certification of all components involved in an analysis. Quite remarkably though, standards are not always thought of as being an essential part of a certified analysis because many analysts prepare their own standards, while the other attributes are almost always purchased ready-made from a certified supplier. And while instrumental errors are often indicated by the instruments themselves and small deviations from the settings of, for example, temperature and pressure do not in most cases affect the results in a serious way, the user cannot equally easily recognize problems with the standards. 

Problems with Manufacturer Instrument Certification

Before we look at the chromatography standards themselves, a small detour along the certification of the instruments may help to gain some insight into the pitfalls regarding certified analysis. All of the GC and HPLC manufacturers are ISO 9000 certified. Unfortunately, an ISO 9000 certificate does not at all guarantee that the instrument is suitable for the job that the analyst has in mind, although it does provide more certainty that any additional devices that are purchased will be of equal quality — or will have the same deviation. 

To cite the manager of a large industrial analytical laboratory: "There is no obstacle to acquire an ISO 9000 certificate for producing life vests made from concrete."  

The ISO Guide 17025 was designed for that reason.

 It states the criteria that the analyser meets and how this was tested. Still, this does not guarantee that the instrument will provide the correct answer to the analysis. A well-known example concerns the certification of the total organohalogen analyser (EOX, TOX). In this instrument, the sample is burnt to convert all organic halogen to yield HX, which is then automatically washed, dried and quantified via coulometric titration. One certified instrument that is currently on the market sometimes measures a mere 30% of the correct value, for example, for high-boiling PCB's, while at the same time other instruments, including a non-certified one, do provide reliable values for high boiling compounds that are difficult to burn completely. Why does the certificate not mean very much in this case? It is stated that the certified EOX-analyser has been tested using Aldrin (in accordance with the Dutch NEN 5735 Guide

). It so happens that Aldrin yields a result of 100% — even in the worst available analyser and is, therefore, not an adequate measure for the quality of an EOX-device. And so this was a poor choice on the part of the NEN-committee concerned. (The building sector, however, is pleased with this choice as it appreciates analysis reports that state low EOX-values in soil samples). 

Another example of a certified analysis that gives a false sense of security concerns the test procedure that resulted in the heparin scandal. The Chinese manufacturer of this globally distributed anticoagulant illegally mixed this drug with over sulphated Chondroitin sulphate (OSCS) a much cheaper anticoagulant, but one with serious side effects. This was not recognized because of the prescribed test procedure.

Both examples demonstrate that it is more important to do business with a 

and competent manufacturer than to overrate the value of a certificate that states that the product concerned passed the final inspection during the manufacturing or production. It remains a fine thing to doubt the reliability of a certified product, be it an instrument or a standard.  

Standards for Certified Reference Materials (CRMs)

It is clear for these reasons that ISO Guide 9000 does not suffice for the manufacturing of CRMs (certified reference materials, more commonly described as standards and test mixtures). It is for that reason that additional Guides such as the ISO Guide 17025 referred to above and ISO Guides 31–35 have been set up.

 These ISO Guides require that a comparative chromatographic analysis be conducted as a means to inspect the preparation. And that is not all. The corresponding certificate also provides information regarding the shelf life of the standard. The producer must exclude the possibility of a reaction with dissolved air during storage or a reaction between the components themselves. This increases the reliability of the product. 

Of course, a professional manufacturer of a mix-standard would not combine any components of which one can expect an inter-reaction based on theoretical grounds. Despite this, it is important to always check the deterioration of the composition in practice. ISO Guide 35 describes how the CRM producer comes to an expiration date for the standard via initial and on-going stability studies. 

If an ISO Guide 31 certificate states that the shelf life of a product is three years, then one can conclude that an equal standard was made at least three years earlier and that it was recently subjected to a chromatographic analysis that showed that the composition had not changed. 

Just how easy it is to handle CRMs in the wrong way is evident from the following case:  

A 9000-2005 certified lab purchases a certified standard with 15 components, which is manufactured in series and is, therefore, much less expensive than a custom-made standard. However, a few months later the client actually needed a standard with one additional component and with the same (one year) shelf life. The client, therefore, bought a certified standard solution containing only this sixteenth component and then prepared by himself a new 16-component standard by mixing both original standards. The new standard is then filled in multiple ampoules, intended as a supply for the next year to come. He properly describes these actions in his management report and demonstrates such during the next audit.  

Then the following happens: the accreditation inspector rejects the newly mixed standard because the remaining shelf life of the purchased 15-component CRM is less than the intended shelf life (one year) of the self-prepared mixture of the certified standards. The client asks the supplier for an updated certificate showing a longer shelf life for the 15-component mixture. This is a fine example of handling certified standards in the wrong way. The inspector should have said that the certified shelf life of any standard only applies up until the date when the ampoule is opened. If the composition is altered by him at a later stage, then he must store part of the newly mixed products to ascertain later whether the composition of the standards remains as expected in the course of the period in which it is used. It makes no sense to establish the expiration date of a custom-made standard until the product is manufactured. In other words, one cannot claim that the shelf life of a custom-made standard is one year until a period of one year has indeed passed and the appropriate checks have been made. When these (chromatographic) checks are successfully repeated after another year, the standard can be assigned a two-year shelf life, etc. 

ISO Guide 34 extensively describes the stringent requirements that apply to a reference material producer. He is to store part of the manufactured product and inspect the quality after a certain time. This provides insight into the shelf life. In addition, the producer can then even re-certify previously supplied standards and, for example, specify a longer shelf life than initially specified on the original certificates.  

Sometimes a supplier cannot guarantee the shelf life of a standard right away, because the standard was custom-made and had not been produced before.  

The certificate supplied with the standard concerned stated "on-going stability programme" under the heading "shelf life". Some of these clients informed the supplier that this was not satisfactory to the inspector of the RvA (Dutch Accreditation Council) who demanded that the certificate state an exact shelf life. It appears that the inspector concerned was not aware of the fact that a standard that has been manufactured according to ISO Guide 34

) for the first time is to be certified in this way. In view of the Accreditation Council's powerful position, the client has no idea how to cope with its demands. It would be to the credit of the Accreditation Council if it were to provide its staff with more adequate instructions for this situation.   

Taking all of the above into consideration, one cannot but conclude that it is much safer to purchase standards from specialist suppliers, rather than produce the standards on one's own accord. The specialist sells the CRMs in large quantities, often worldwide. Should the product contain an error as a result of the delivery or for some other reason, then there will be a more than considerable chance that someone somewhere will discover the error. ISO Guide 33 even states that the CRM will be less expensive than compiling a standard on one's own, even if a CRM is very expensive — if one does the calculations right! 

Employing and interpreting a certificate is not an easy thing to do. The subject should find a place in the chemistry curriculum at university and for QC-training for newly employed staff should pay ample attention to this issue. 

was one of the first scientists in The Netherlands to research gas chromatography for food analysis in the 1950s. From 1980 until 2008 he was the owner and managing director of a wholesaler of standards and other GC/LC supplies. He was frequently contacted by customers because they encountered problems with accreditation inspectors when using custom-made standards. He is currently an independent consultant at Rugolution. E-mail: 

is principal at McDowall Consulting, Bromley, Kent, UK. He is also a member of the 

1. R. Boqué, A. Maroto and Y. Vander Heyden, 

2. ISO/IEC Guide 17025 ( 2005):  General Requirements for the Competence of Testing and Calibrating Laboratories. ISO, International Organization for Standardization. 

 Quantitative determination of the content of extractable organo halogen compounds (EOX)

CE of Contaminated Heparin, CASSS CE Pharm

Reference materials — Contents of certificates and labels

Calibration in analytical chemistry and use of certified reference materials.

General requirements for the competence of reference material producers.

Reference materials, general and statistical principles for certification.

Questions of Quality

There is more to using and interpreting certificates for chromatography standards than meets the eye

Certificates for Chromatography Standards: Sense and Nonsense

This mini-review of a new sample preparation technique called "electromembrane extraction" demonstrates how the combination of an electroextraction with hollow-fibre liquid-phase microextraction can lead to a very selective, rapid sample preparation method for the extraction of charged substances from complex matrices such as plasma, breast milk and urine. Several important parameters for successful extraction are presented and application examples of various analyte–matrix combinations are tabulated.

A lot of effort has been devoted recently to the miniaturization of existing liquid–liquid extraction methods to make them more versatile and powerful. Reducing the use of hazardous organic solvents due to environmental and cost concerns, time reduction, ease of automation, possible on-line coupling, high-throughput capability and the small amounts of matrix available are major incentives that have motivated scientists working towards miniaturization. In bioanalysis, which in this context is defined as the analysis of small drug molecules and their metabolites in biological samples, the complexity of the matrices requires selective and specific sample preparation methods to isolate the analytes of interest.  

Macromolecules, salts, cellular material, fat or lipids in biological matrices such as urine, plasma, whole blood and breast milk can disturb the separation and data analysis steps. In addition, the analytes of interest can often exist in low concentrations (pg/mL–μg/mL). Therefore, a sample preparation method with a high degree of selectivity and enrichment is crucial for a successful analysis. 

With these considerations kept in mind, a totally new approach to sample preparation was proposed in 2006.

 The innovative concept, called electromembrane extraction (EME), combined the technical setup for hollow-fibre liquid-phase microextraction (HF-LPME)

 with known principles for electroextraction.

 This combination offers a highly selective sample preparation method using simple equipment and gains a high degree of enrichment within a short period of time.  

The EME method extracts charged substances from a small sample volume through a thin membrane of organic solvent immobilized in the wall of a hollow fibre and into a receiver solution inside the lumen of the hollow fibre. This extraction process is forced by an applied potential difference across the membrane and this combination of well-known liquid–liquid extraction processes with electrokinetic migration yields a rapid and selective sample preparation method for ionic substances. EME has shown to be compatible with a wide range of biological matrices — for example, plasma, whole blood, urine and breast milk — preparing clean extracts in a short period of time with simple and inexpensive equipment.  

This instalment of "Sample Preparation Perspectives" is a mini-review of the EME technique and shows the investigation of several parameters that affect recovery of charged analytes. In addition, a number of application examples will illustrate its potential for the successful extraction of drugs from complex matrices. 

The technical set-up for the equipment used in EME is based upon earlier experience with HF-LPME and is shown in Figure 1(a). The hollow fibre used is made of porous polypropylene, which is compatible with a broad range of organic solvents. The thickness of the wall of the hollow fibre is 200 μm, with a pore size of 0.2 μm and an internal diameter of 1.2 mm. 

Figure 1: Diagrams of (a) EME set-up and (b) principle, with pethidine as the model substance.			

A piece of the hollow fibre is cut to a length of 25 mm and mechanically closed at the lower end with a pair of pincers. The upper end is sealed to the end of a 22 mm pipette tip by heating. To make the supported liquid membrane (SLM), the fibre is dipped in an organic solvent for 5 s to fill the pores in the walls and the excess of organic solvent gently removed with a medical wipe. 

The fibre connected to the pipette tip is guided through a punched hole in the sample compartment cap as illustrated in Figure 1(a). The pipette tip works as a mechanical support for a 0.5 mm-thick platinum wire placed inside the lumen of the hollow fibre. Another platinum wire is introduced directly into the donor phase through the sample compartment cap. When coupled to a power supply, these inert wires act as electrodes, thus creating an electrical field across the SLM. In this way, the equipment makes a closed electrical circuit, where the SLM functions as a resistor.  

The volume of the sample varies between 150 μL and 500 μL, depending upon the sample compartment size. However, the compartment is never filled more than half full because of the need for convection space. The sample is shaken on a platform shaker during the extraction to increase the physical movement of the analytes in the bulk donor phase and to reduce the thickness of the stagnant layer at the interface between the donor phase and the SLM. The acceptor phase volume is set to 25 μL and is introduced into the lumen of the hollow fibre by a microsyringe. When the predetermined extraction period is finished, 20 μL of the acceptor phase is collected by the microsyringe and transferred to a vial for analysis in a capillary electrophoresis (CE) instrument

 or by high performance liquid chromatography (HPLC).

 The major driving force in EME is the potential difference across the SLM, in which charged analytes are drawn from the donor phase, across the interface between the donor phase and the organic membrane, through the organic membrane, across the interface between the organic membrane and acceptor phase and toward the electrode of opposite charge in the acceptor phase. The principle of the extraction is shown in Figure 1(b). To ensure full ionization of the model analytes, the pH in both the donor phase and the acceptor phase should be well controlled. In the extraction of basic analytes from water, the pH in the aqueous donor phase was adjusted to 2.0 with 10 mM hydrochloric acid.

 The acceptor phase also consisted of 10 mM hydrochloric acid, which increased the solubility of the analytes in the aqueous solution compared with the SLM. In the case of acid extractions, the analytes were dissolved in 10 mM sodium hydroxide to a final pH of 12.0, which was also the pH in the acceptor phase.

 The properties of the organic solvent impregnated in the SLM are important for a functional EME system. Based upon earlier experiences with HF-LPME, the nitro compound 2-nitrophenyl octyl ether (NPOE) was tested as the organic solvent for extraction of basic drugs with different polarities. An electropherogram representing the extract after EME of five hydrophobic basic drugs with NPOE as the SLM is shown in Figure 2. Hydrophilic model analytes (log P < 1.0) were unable to penetrate the interface between the donor phase and the SLM with pure NPOE. The high polarity of these analytes with low partition into the SLM seemed to counteract the influence of the electrical field. 

Figure 2: Electropherogram obtained from extraction of five basic analytes (NPOE as the SLM).			

To increase the solubility of the analytes in the organic solvent, di-(2-ethyl-hexyl) phosphate (DEHP) was added to the SLM as an ion-pair reagent.

 The results in Table 1 illustrate the importance of the nature of the organic solvent. The addition of 10% DEHP to the membrane increased the recoveries of the hydrophilic drugs tested from zero up to 22–54%, while the recoveries of the more hydrophobic analytes tested were reduced dramatically with DEHP presented in the SLM. Also, the hydrophobic drugs ion-paired with DEHP, and were efficiently transported into the organic membrane. However, these ion pairs were highly hydrophobic, and the strong interaction partly prevented them from liberating to the acceptor phase. The addition of 10% tris-(2-ethylhexyl) phosphate (TEHP) to NPOE altered the situation, as shown in Table 1. Despite a similar structure to DEHP, TEHP did not function as an ion-pair reagent, as it does not contain any ion-pairing seats. The hydrophilic drugs were not extracted by the addition of TEHP to the SLM. However, the recoveries of the hydrophobic drugs increased with TEHP in the SLM, probably caused by alteration of the polarity in the SLM. A combination SLM consisting of 10% DEHP and 10% TEHP in NPOE led to extraction of almost all the basic drugs tested, showing that development of a membrane that permitted electrokinetic transport of drug substances in a very large log P window was possible. 

Table 1: The influence of organic solvent on recovery.			

Proper organic solvents for extraction of acids have been examined.

 The aromatic nitro compounds were found to be inefficient for the acidic model analytes and aliphatic alcohols with different chain length were tested based upon experiences with HF-LPME.

 The alcohols 1-octanol and 1-heptanol were found to be suitable solvents because of their insolubility in water and easily impregnate the fibre. In addition, the alcohols showed an appropriate electrical resistance to the applied voltage — that is, the current was low enough to avoid bubble formation and excessive electrolysis but still sufficiently high for the system to maintain the properties of a closed circuit. Recoveries between 8% (hexobarbital) and 100% (fenoprofen) were obtained with 1-heptanol as the SLM (Table 2). 

Table 2: List of substances and matrices tested in EME			

The effect of different organic solvents was strongly dependent upon the potential difference applied across the SLM. The optimal potential difference with NPOE as the SLM was 300 V, which showed recoveries between 16% (pethidine) and 78% (methadone) for the nonpolar model analytes.

 To reduce the applied potential difference, other organic solvents were tested.

 Different SLMs were made with different nitro compounds including ethyl nitrobenzene (ENB) and 1-isopropyl-4-nitrobenzene (IPNB). It was shown that 10 V was the potential difference that showed the highest recoveries, which were in the ranges of 54–80% (ENB) and 59–93% (IPNB). The results show that the chemical nature of the organic solvent in combination with a proper potential difference can be used to design very selective extractions of different analyte classes. 

Other parameters that were found to influence the recoveries in EME included the convection speed, the ion balance between the donor and acceptor phase and the temperature.

 Because of the successful extractions with only 10 V as the driving power, a simple 9 V battery was tested as the only power supply.

 This replacement of the power supply did not influence the recoveries of the drugs tested, making the idea of a portable sample preparation device possible. 

 The original idea for EME was to employ the same set-up that was used for HF-LPME. The only technical difference between these two systems was the electrical circuit introduced in the EME set-up. The platinum electrodes coupled to a power supply created a potential difference across the SLM, hence, changing the extraction mechanism from passive diffusion to electrokinetic migration. A systematic study of the differences between the two methods was performed regarding the extraction mechanisms, extraction times, recoveries and selectivity.

 Overall, the introduction of another transport force into the EME system made a significant change in the extraction time compared with the existing HF-LPME model. Generally, the most time-consuming step in bioanalysis is the sample preparation step. Therefore, the extraction speed is of great importance in development of a new extraction method. The time to reach a steady-state level is determined by different parameters in the driving force of the extraction. The only differences between the two systems were the pH in the donor phase, which was 2.0 in the EME system and 12.0 in the HF-LPME system, and the voltage applied across the SLM in the EME system.  

The recoveries as a function of the extraction times were examined and the extractions were completed when a steady state level was reached. The graphs in Figure 3 compare the results from an EME extraction with an HF-LPME extraction of the same compound, haloperidol. The time-saving effect obtained from the voltage application in EME was significant, with a reduction in the equilibrium time from about 60 min with passive diffusion as the driving force to 5–10 min with an electrical field as the major driving force. The speed advantage EME offered compared with HF-LPME was most remarkable using the smallest sample compartment tested (300 μL, 31 mm × 4 mm i.d.). In the small compartment, the short electrode distance created a relatively high electrical field strength, resulting in a stronger driving force and hence increased extraction speed. 

Figure 3: Comparison of extraction speed in EME and HF-LPME.			

 A number of biological matrices have been tested in combination with EME. The matrices covered a wide range of complexity from urine, to diluted and untreated plasma, to whole blood, to breast milk. We will now look at some of these examples. 

 Extractions of basic drugs from both diluted plasma

 have been tested. These experiments showed that the extraction recoveries were almost independent of the dilution and the pH in the plasma. In other words, extraction from plasma samples without any pretreatment was possible. 

The time to reach a steady-state level from untreated plasma was somewhat extended compared to extraction from phosphate buffer (pH 7.4), as exemplified with nortriptyline in Figure 4. The slow extraction kinetics in plasma was probably caused by two factors: the increased viscosity of plasma samples and drug interactions with plasma proteins. To check whether the viscosity was a deciding factor or not, the plasma sample was diluted with pure water in the ratio 1:1 in a subsequent experiment. The results are shown in Figure 4. It can be seen that lowering the viscosity of the plasma samples did not increase the extraction speed significantly. The steady-state recoveries were comparable in phosphate buffer as well as untreated and diluted plasma. 

Figure 4: Recoveries of nortriptyline as a function of extraction time using phosphate buffer (pH 7.4), untreated plasma, and plasma:water (1:1) as sample matrix.			

 Experiments with EME from whole blood samples have been performed.

 The recoveries obtained from extraction from untreated whole blood spiked with some basic drugs were in the range 19–51%. They were comparable to EME from untreated plasma, even though whole blood is a more viscous and complex matrix than plasma. In some subsequent experiments, whole blood samples were diluted with pure water in the ratio 1:1 or 1:3 to test if a less viscous matrix would increase the recoveries. For the majority of the model analytes tested, the dilution step led to improved recoveries. 

 EME of diluted urine samples has been performed.

 The recoveries obtained after 5 min EME from diluted urine were in the range of 37–50%, which were significantly lower than EME from pure water samples under otherwise identical conditions. To test the effect of variety in the water and salt content, urine samples from different people and collected at different times were spiked with the basic model analytes and subjected to EME. The results showed no significant difference between the recoveries. 

 The recoveries of the drugs tested were in the range 39–55% from breast milk and the resulting electropherograms showed very few interferences. Extractions from samples collected from two different women and from the start and end of lactation showed no significant differences in the recoveries.  

The repeatability for the extraction from human plasma was tested at two different concentration levels. The repeatability of the extractions was 5–17% RSD.

 Other EME applications have been performed simultaneously to the works briefly described in this article. EME of peptides,

 have shown the potential of the technique. Table 2 gives a comprehensive overview of the substances and matrices that are exposed to EME. The usage of electrical potential difference in sample preparation is also described by Arrigan and his group.

 Their research focuses on the electrochemical properties of the interface between two immiscible electrolyte solutions (ITIES), which can be used in separation and detection of ionic molecules from biological matrices.  

A new idea about an electrical field as the extraction driving force combined with existing hollow-fibre extraction technology is described briefly. It is shown that the technique offers rapid and selective isolation and enrichment of ionic substances. The extraction recoveries are strongly dependent upon the nature of the organic solvent. This dependence makes EME a highly selective extraction method that uses the nature of the organic solvent and the applied potential to control the extractions. 

The time-saving effect by using an electrical field as the driving force is demonstrated during a comparison between EME and liquid-phase microextraction. The potential of EME is shown during extraction from different biological matrices. Extractions from diluted urine, diluted and untreated plasma, diluted and untreated whole blood and diluted breast milk are noticed, showing promising results. Normally, existing liquid–liquid extraction methods require pH adjustment of the matrix before extraction of ionic analytes. In that way, EME offers possible advantages, where ionic substances can be extracted directly from untreated biological samples without disturbing the chemistry in the sample. 

The high extraction speed and the simple equipment needed makes EME a promising tool for field sampling, either from blood samples at the bedside in a hospital or in environmental analysis. The EME experiments performed with a common battery as the only power supply were promising in this respect.  

 is with the Department of Pharmaceutical Chemistry, School of Pharmacy, University of Oslo, Oslo, Norway. She obtained her PhD about Electromembrane Extraction in October 2009 and is continuing her research regarding microextraction and membrane technology in Oslo. 

"Sample Preparation Perspectives" editor 

 is a senior chemist, Columns and Supplies Divison, Life Sciences and Chemical Analysis Group, Agilent Technologies, Wilmington, Delaware, USA and is a memebr of the Editorial Advisory Board of 

. Direct correspondence about this column to 

, Poplar House, Park West, Sealand Road, Chester CH1 4RN, UK. E-mail: 

1. S. Pedersen-Bjergaard and K.E. Rasmussen, 

2. S. Pedersen-Bjergaard and K.E. Rasmussen, 

3. S. Pedersen-Bjergaard and K.E. Rasmussen, 

Sample Prep Perspectives

How the combination of an electroextraction with hollow-fibre liquid-phase microextraction can lead to a selective, rapid method

Electromembrane Extraction: The use of Electrical Potential for Isolation of Charged Substances from Biological Matrices

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). The next Webcast is:

 Essential Guide 2:  Mobile Phase Optimization Strategies for Reversed Phase HPLC. 

Lablogic Systems has released Version 4.0.5 of its Laura radiochromatography software, which the company claims brings together more than 150 improvements and innovations from the last twelve months. These changes include the optimization of the Active Counting Mode facility, which is reported to make it more reproducible with better resolution, sensitivity and control. Also included are interfaces for the iScan TLC radiopharmaceutical scanner and Waters Alliance HPLC system and dual-absorbance detectors. Other products receiving new or enhanced support include the Agilent Binary-SL pump, Canberra MCA UniSpec detector, Gilson autosampler, Gilson FC204 fraction collector, LabLogic's own SoFie stop-flow system, the Mini-Scan TLC scanner, Shimadzu HPLC system, Shimadzu autosamplers and Xcalibur MS software. 

DataApex has released the update of its chromatography software. Clarity version 2.7 brings new control drivers covering Hitachi LaChrom Elite HPLC system (L-2100/2130 pump, L-2200 autosampler, L-2300 Column Oven, L-2400 UV Detector) and the HTA autosampler HT800L. According to the company, the software can now control more than 250 instruments. The new version enhances several functionalities that bring users more comfort during installation and configuration. The update is available for current users free-of-charge. The demonstration version and the complete list of controlled instruments can be downloaded from the company's website.			

Phenomenex has announced a new web-based tool to optimize customer HPLC methods for use with the company's Kinetex core-shell columns. The benefits to be gained by switching from fully-porous particle columns are also calculated. With the calculator, researchers input their method running conditions and details regarding current chromatographic results. According to the company, in less than one minute, the calculator delivers an optimized method with the recommended column and optimal run conditions for the user's system, whether it's HPLC or UHPLC. The calculator also provides expected backpressures, efficiency gains, solvent reduction, time savings and resolution improvements. The calculator can process details of both isocratic and gradient methods.

Departments

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 (What is Chromatography?) to the expert who needs a refresher in a particular topic (Advanced Topics in LC–MS Spectral Interpretation, for example).

Digital Update

When did you start investigating diamond as a stationary phase — and why?

Our group started investigating diamond as a potential stationary phase for chromatography in 2005, following a discussion about the possibility of fractionating such abrasive microparticles based on size. I was surprised by the amount of different types of synthetic diamonds that have been manufactured by large-scale industrial production and their easy availability. 

Importantly, at this time, Prof. Georgii Lisichkin and Dr Inna Kulakova from the Laboratory of Organic Catalysis of Lomonosov Moscow State University started their project on chemical modification of diamond surfaces. At the beginning our research was motivated by a simple curiosity to test a new exotic material as our group had previous experience of using non-traditional adsorbents in HPLC, including micro-flakes of copper, hyper-crosslinked polystyrene and porous glassy carbon monoliths. 

What unique properties do diamond stationary phases possess and what obstacles could this help them overcome compared with traditional phases?

Every chemist knows about the chemical inertness and hardness of diamond. Less is known about other intriguing properties of diamonds, such as an extremely low thermal expansion coefficient and a very high thermal conductivity (see Table 1). For example, the thermal conductivity of diamond is almost 150 times higher than stainless steel SS316 and thermal expansion is 240 times lower. The combination of these two superior properties with an excellent chemical stability in various aggressive liquids potentially makes diamond an ideal stationary phase for HPLC at elevated temperatures. 

Table 1: Physicochemical properties of some materials (as nonporous, 25 Â°C)			

The most popular carbonaceous stationary phase in HPLC is porous graphitic carbon (PGC), which was developed more than 20 years ago and later produced under the trade name Hypercarb. The unique selectivity of PGC towards polar organic compounds is mainly due to the so-called "polar retention effect on graphite" or PREG.

				  This effect is attributed to the orbital overlap between conductivity electrons in PGC and a lone pair of electrons or π-system in analytes. Diamond, another allotropic form of carbon in sp

  configuration, has significantly higher resistivity than graphite. So, one can expect some difference between adsorption properties of conducting graphite and non-conducting diamond stationary phases. However, doping of the diamond with boron atoms dramatically improves the electrical conductivity, so, potentially, diamond can be a useful alternative because of its favourable mechanical stability. There are also some indications in the literature showing interesting selectivity of diamond to various types of biomolecules.

  Taking into account the excellent biocompatibility of diamonds this could be an interesting area of investigation. 

Would the cost of diamond make such stationary phases too expensive for routine analysis?

Actually, the first application of diamonds was in HPLC in 1973 and was performed with the relatively big chromatographic column: 25 cm length and 3 mm i.d. packed with 10 micron microparticles of natural diamond.

  A simple calculation using actual and dead volumes of a chromatographic column packed with nonporous particles, with a density of 3.51 g/cm

  for diamond from Table 1, shows that the column content was equal to roughly 20 carats! Actually, natural diamonds are not suitable as a stationary phase because they are not chemically pure and may have different structural defects in the particles, so at present researchers use only synthetic diamonds for the research. 

Obviously, there are currently no commercially available diamond phases used in chromatography or in solid-phase extraction (SPE). But, there is large-scale production of various microparticulated diamonds for the preparation of abrasive materials and polishing compositions, so the actual cost of these technical grade diamonds is at the same level, or less, than the same-sized bare silica particles for HPLC.  

In our research, we use microdisperse sintered detonation nanodiamonds (MSDN). The name of this material reflects the preparation steps: dynamic synthesis of detonation nanodiamonds by blasting explosives in oxygen-deficient conditions, followed by purification and isolation of the nanodiamonds fraction from the reaction soot, sintering of the aggregates of size 25–35 micron and, finally, crushing and fractionation of size. The current cost of MSND is approximately 10 USD per gramme. 

What is the most significant breakthrough you have made in this research?

There are two main obstacles to the wide introduction of diamond-based stationary phases for chromatography. Very poor separation efficiency was always one of them. However, we have achieved a maximum separation efficiency of 43600 theoretical plates per metre for the separation of substituted benzenes on a 150 × 4.0 mm i.d. column packed with 4–5 micron MSDN particles in normal-phase chromatography mode (Figure 1). It is not a lot when compared with the efficiency of current HPLC columns, but it is almost 20–30 times higher than values reported previously for diamond-based stationary phases. 

Another problem is the lack of data on the adsorption properties of detonation nanodiamond and their surface chemistry, so it is difficult to identify the areas of possible applications of MSND. Because of harsh conditions during synthesis and purification, more than 20 different functional groups can be simultaneously identified on the surface of MSND. The surface chemistry of nanodiamond can also be different from different producers. Recently we finished an important study on the characterization of the adsorption properties of MSND by mapping the retention of alkylbenzenes and comparing with other common adsorbents, including silica, alumina and PGC (Figure 2). The ion-exchange properties of MSND and its ability to retain polar organic substances have also been investigated. Thus we, probably, removed these obstacles and this achievement will now hopefully open up this area further. 

The other important aspect of our results is the demonstration of the applicability of standard size stainless-steel columns with an internal diameter of 4 mm, packed with diamond for chromatographic separations at 160–180 °C (Figure 3) and relatively high flow rate of 1.2 mL/min. 

Surprisingly, we did not notice any chromatographic peaks distortions and achieved a 5-fold improvement in separation efficiency compared with separations performed at 60 °C. Obviously, it illustrates the excellent heat dissipation within the relatively bulky diamond column bed. 

Which applications could they be useful in?

In terms of exploiting separation selectivity, bare MSNDs are very versatile and can be used in several different chromatographic modes, such as ion-exchange, normal-phase, reversed-phase and affinity chromatography. SEM images of bare (a,b) and treated (c,d) MSND particles are shown in Figure 4. 

The electroconducting boron-doped diamond phase has been used for electromodulated chromatography. So, the list of analytes could be quite broad. Considering the advantageous physicochemical properties, diamond-based stationary phases should be extremely useful for HPLC separations at high-pressures and high-temperatures.   

Is this research close to being suitable for commercialization?

Obviously, MSDN is not ready for commercialization as a chromatographic packing at the moment. The technology of the production of diamond-related materials, having developed surface area and uniform porous structure, needs further improvement. It should be noted that all of our chromatographic results have been obtained with carefully fractionated and purified materials, which are designed for polishing optics. Obviously, the producers of these abrasives have very different goals and are less worried about some of the parameters that are more important for chromatographers, such as the spherical shape of the particles, pore size and pore volume, and the chemical homogeneity of the surface. The preparation of MSND with optimized characteristics should provide better separation efficiency and well-defined selectivity.  

What areas do you intend to explore in the near future?

There is a growing interest in developing new types of diamond-based stationary phase. Actually, the chemical modification of separation selectivity is an obvious future direction because it opens up the possibility of obtaining chemically homogeneous surfaces and/or changing separation selectivity for some specific applications. The other option is to use nanodiamond particles as a material for coating other substrates, similarly to agglomerated ion-exchangers. This work is in progress in collaboration with research groups from within the Irish Separation Science Cluster (ISSC). 

 obtained a PhD (1982) and DSc (1999) in analytical chemistry from Lomonosov Moscow State University, Russia, where he performed most of his research. In December 2006 he joined the Australian Centre for Research on Separation Science (ACROSS) of University of Tasmania, Hobart, Australia as Professor in Separation Sciences. Professor Nesterenko has published over 220 scientific papers. He is also a member of Editorial boards of 

Encyclopedia of Analytical Chemistry/Academic Press

K.V. Purtov, A.P. Puzyr and V.S. Bondar, 

Pavel Nesterenko from ACROSS about his work on the potential of diamond-related substances as stationary phases

Diamonds are a Chromatographer's Best Friend?

The signal-to-noise ratio (S/N) in a liquid chromatography (LC) separation is usually defined as shown in Figure 1. The noise is measured between two lines bracketing the baseline and the signal is measured from the middle of the baseline to the top of the peak. S/N is merely the signal divided by the noise. There are some other ways to determine S/N, but that is not the point of the current discussion. My comments here will apply no matter what technique you use for measurement and calculation of S/N. 

Which is more important: signal or noise?

LCGC Europe-03-01-2010

Towards a Universal Detector for Small Molecule Applications: Direct-EI in LC–MS

Certificates for Chromatography Standards: Sense and Nonsense

Electromembrane Extraction: The use of Electrical Potential for Isolation of Charged Substances from Biological Matrices

Digital Update

Diamonds are a Chromatographer's Best Friend?

Enhancing Signal-to-Noise

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