Incredible storage capacity at the touch of a button

The home user can now have 1 TB of storage at the touch of a button. 

Buffalo Technology has announced a latest addition to its portfolio of Network Attached Storage (NAS) products. The LinkStation Pro Duo [LS-W1.0TGL/R1] is designed for small office and home users (SoHo) wanting a cost-effective storage solution. The device offers a substantial 1 TB storage capacity, with RAID 0 and RAID 1 functionality options for additional security and resilience. The two 500 GB drives can also be accessed remotely via any web browser when users are out of the office. The large storage size and compact design of the LinkStation Pro Duo make it ideal as a main storage device. Back-ups can be made via a LAN connection to other Buffalo LinkStation devices and to external USB drives. For the SMB market, the LinkStation Pro Duo provides an additional storage or backup option, with easy integration into an existing network via Active Directory. Buffalo's web access feature allows users to instantly and securely access all digital files stored on the LinkStation Pro Duo, via an internet connection anywhere in the world. The free on-line service also allows remote workers across different departments in a networked office environment, or users in different geographic regions, to retrieve data from the device, quickly and easily. 

The LinkStation Pro Duo is compatible with Windows 2000, XP, Vista and supports Mac OS X and Apple Bonjour. 

Update your music collection with sounds from the past

Enjoy the best bands of the 70s, 80s and 90s with TAPE2PC from ION Audio. This revolutionary dual cassette deck allows you to convert tape cassette to MP3 on your computer, which you can then easily copy to CD, iPOD or other MP3 players. 

If left alone your tapes will deteriorate — eventually becoming unplayable. To avoid losing your music collection copy "tapes-to-PC" for safe, permanent storage. Simply plug TAPE2PC into your computers USB port (lead supplied), load the free software and begin converting tape to digital (no drivers needed). It's so simple to use it took under five minutes from opening the box to start copying tapes to the computer!? 

There is now an easy way to enjoy downloaded video on a television instead of your computer screen. The new Sansa TakeTV video player is a breakthrough device that gives you a smart, convenient and affordable way to take video from the PC to the TV, which is where it belongs. It's as simple as using a USB flash drive: just drag compatible video files to your TakeTV player, slip it into the TV cradle and press play on the included remote. No confusing Wi-Fi set-ups and no waiting to burn DVDs. 

   To share photos quickly and easily a wireless camera is a must. 

And, for those who do not want to invest in a new device then help is at hand. 

A company called Eye-Fi has developed a series of memory cards with built in wi-fi that automatically convert your camera into a wireless device. 

The 2GB cards automatically connect to a wi-fi network and can download photos to a folder on a PC or upload them to social networks or photo-sharing sites. 

The associated software takes care of log-ins and passwords and can even automatically resize photographs if the site requires it. 

For more information please visit http://

Articles

New Technologies

The home user can now have 1 TB of storage at the touch of a button.

The use of fragrances to generate pleasant and attractive smells in human bodies goes back thousands of years. These sample-types may be characterized by a considerable degree of complexity, as the number of natural and synthetic perfume formulation constituents in use is extremely high.

  The relationship between contact allergy and perfume ingredients has been widely discussed.

  Under a European regulation called the 

 the twenty-six most frequently recognized skin allergens must be labelled on the final cosmetic product if specific concentrations are reached: 10 mg/L and 100 mg/L in "leave-on" and "rinse-off" substances respectively. Twenty-four of the twenty-six skin sensitizors are volatile and can, therefore, be analysed by GC.  

Recently, a sample classification for the analysis of skin sensitizors was introduced: perfumes were included in class II, considering their medium to high complexity (commonly containing more than a hundred analytes). In these cases, a monodimensional gas chromatography (GC) application could be sufficient for satisfactory analyte determination. Whenever the complexity of a class II matrix exceeds the peak capacity of a single capillary column, then a multidimensional method must be used.

Comprehensive multidimensional gas chromatography (GC×GC) has been used for the analysis of complex perfumes.

  It was demonstrated that a single peak eluting from the primary column consisted of eight overlapping components.

  In such a situation, the reliable determination of a fragrance ingredient by using any of the most common gas chromatogrpahy mass spectrometry (GC–MS) approaches — such as full scan monitoring, multi-ion chromatogram (MIC), single ion monitoring (SIM) or peak deconvolution — is an arduous task. Classical MDGC has also been used for the analysis of allergens contained in a fragrance.

  The target analytes were freed from co-eluting compounds through two 1 min heart cuts, performed on the perfume. The extensive degree of overlapping was evident in the second dimension chromatogram. 

The research described in this article focuses on the use of an innovative valveless MDGC–MS system for the analysis of allergens in a complex perfume. The transfer device, which achieves two-dimensional (2D) analysis by pressure tuning at the column conjunction point, is simple to use and enables the transfer of multiple fractions during each application. Fourteen cuts were performed on the sample, with time windows determined using linear retention indices (LRIs). 

 A commercial perfume was purchased from a local store in Messina, Italy. Amyl cinnamaldehyde, anisyl alcohol, benzyl alcohol, benzyl cinnamate, methyl 2-octynoate, citral, cinnamaldehyde, benzyl benzoate, benzyl salicylate, cinnamyl alcohol, amyl cinnamyl alcohol, coumarin, eugenol, isoeugenol, farnesol (four isomers with the 

 isomers predominant), citronellol, geraniol, hydroxycitronellal, hexyl cinnamaldehyde, limonene, α-isomethylionone, lilial, linalool, lyral and and 1,4-dibromobenzene — the internal standard (IS) — were purchased from Sigma–Aldrich (Milan, Italy). The purity of each standard was verified and was accounted for prior to use, using GC–MS. A stock allergen solution (10000 mg/L) was prepared in ethanol and used for calibration purposes. Solutions containing all the 24 allergens were injected at different concentrations (from 1–10000 mg/L) considering their original purity.  

) was purchased from Supelco (Bellefonte, Philadelphia, USA).

Instrumentation and operational conditions:

 The Shimadzu MDGC system consisted of two GC–2010 gas chromatographs (defined as GC 1 and GC 2), an MS-QP2010 quadrupole mass spectrometer and an AOC-20i autosampler (Shimadzu Corporation, Kyoto, Japan). GC 1 presents a split/splitless injector and a flame ionization detector (FID), while GC 2 presents a split/splitless injector (not used for MDGC analysis), a FID (not used for for this application) and a rapid scanning quadrupole mass spectrometer. The MDGC transfer device, located in GC 1, is connected to an advanced pressure control (APC) unit which supplies carrier gas (He), at constant pressure.  

: An Equity-5MS (5% diphenyl + 95% polydimethylsiloxane) 30 m × 0.25 mm i.d. × 0.25 μm (Supelco) was used as column 1; an inlet head pressure of 100 kPa (He) was applied to the transfer device by means of an APC unit (P

); injection mode (280 °C): splitless (2 min); injection volume: 0.2 μL; carrier gas (He); Constant inlet head pressure (P

): 160 kPa; temperature programme: 40–260 °C at 1.5 °C/min; FID (280 °C); H

 flow: 50.0 mL/min; air flow: 400.0 mL/min; sampling rate: 80 msec. 

: An Omegawax (100 % polyethylene glycol) 30 m × 0.25 mm i.d. × 0.25 μm (Supelco) was used as column 2; temperature programme: 40 °C (26 min) to 260 °C (8 min) at 1.5 °C/min. All data were collected by the GC Solution software (Shimadzu) for the FID (GC 1) and by the GC–MS solution software (Shimadzu) for the MS (GC 2). The allergens were identified by using the FFNSC 1.2 mass spectral library database (Shimadzu).  

 Ion source: 200 °C; interface temp: 260 °C; interval scan: 40–400 

The heart of this innovative MDGC system consists of a simple transfer device for the rapid sequential re-injection of analyte "heart-cuts" from the first to the second dimension. The interface is thermally-stable, characterized by extremely reduced dead volumes and achieves analyte transfer through a Deans switching-type mechanism.

  Figure 1 reports two schemes of the transfer system device in the "stand-by" and "cut" position [Figure 1(a) and Figure 1(b) respectively]. In both configurations, the advanced pressure control (APC) supplies carrier gas at constant pressure (P

) to the external fused silica restrictor (defined as R

) and to the three-way solenoid valve (V). The latter is connected to two metallic branches, one with a fused silica restrictor (defined as R

 restrictor dimensions (length and internal diameter) generate a pressure drop (ΔP

) which is slightly lower than that relative to R

When the electrovalve is not activated [Figure 1(a)], the APC pressure is reduced on the side of the first dimension (P

), meanwhile it is able to reach the second dimension branch, passing through the electrovalve, without changes (P

). In such a pressure configuration, the analytes from the first dimension are directed to FID 1. The system is switched to cut configuration [Figure 1(b)], by simply activating the valve. The first dimension branch pressure is the same as described previously (P

, which, as discussed, is slightly lower than P

. In this instance, the first dimension flow is directed onto the secondary column. The FID 1 flame is maintained by a gas flow that derives from the APC unit.  

 Before a MDGC experiment it is of common to inject a target compound standard solution to define the heart-cut time windows. In the case of allergen solutions, it is well known that they are rather unstable and, hence, are characterized by a short life-time. A valid alternative was found: prior to the injection of a 100 mg/L allergen standard solution, a C

 alkane mixture was subjected to "stand-by" MDGC analysis. Experimental LRIs were derived for each skin sensitizor on the first dimension 5% diphenyl column; LRI cut windows were established by applying a ±10 LRI unit value, with respect to the experimental LRI value (Table 1). Once an LRI-cut window was established it was easy to derive a time window for each target compound. 

A total of fourteen cuts were defined because the retention time difference between several skin sensitizors was only slight. Through such an approach, the injection of an allergen solution before each multidimensional application (operational conditions may change in relation to the perfume complexity) would not be required: it would be only necessary to derive the LRIs through preliminary hydrocarbon analysis. It must be added that, as reported in the literature, LRI values calculated on a non-polar stationary phase are reproducible and may undergo only slight variations.

  Obviously, the allergen solutions would have to be injected for calibration curve construction, even though this would be done periodically and not on a daily basis. 

A chromatogram relative to the first dimension perfume chromatogram, after heart-cutting (the position of each cut is visualized), is shown in Figure 2. It must be emphasized that the degree of retention time shift during multiple heart-cutting was neglectable. This negative effect occurs when the first dimension gas flow in the stand-by and cut modes are different. In this case the pressure drop in the primary column is always constant: P

The second dimension total ion chromatogram (TIC) for the MDGC–MS result is shown in Figure 3. It can be observed that twelve target analytes were determined, nicely separated from other matrix interferences; many of these compounds underwent significant co-elution with other perfume volatiles in monodimensional applications. The general mass spectral purity was very good, with MS library similarities always over 90%. Analyte concentrations versus extracted ion responses were also calculated, through the construction of four-point calibration curves, using serial dilutions of the stock standard solution with 1,4-dibromobenzene as the IS; regression coefficients were always over 0.9995. The extracted ions used were those advised by International Fragrance Association (IFRA).

It must be added that only a single IS was employed because this eluted in the middle of the chromatogram (at approximately 72 min on the polar column). As can be observed from Table 1, the data derived is in good agreement with the labelled compounds. Four allergens (citronellol, geraniol, hydroxycitronellal, cinnamic alcohol), although reported on the label, were present in concentrations slightly lower than the limit. It is well known, though, that fragrance producers prefer to declare allergens in this type of situation. 

Table 1: Experimental LRI values, LRI windows, the 14 cut time windows and the concentrations determined for the allergens in the MDGCâMS experiment.   

The present research demonstrates the effectiveness and potential of a recently developed MDGC–MS system for the analysis of allergens in a complex perfume. The use of a twin-column system proved to be very useful in the elimination of matrix interferences. Furthermore, the addition of a third mass spectrometric dimension provided full structural elucidation for all compounds. Finally, the calculation of LRIs to define the cutting windows is a simple and effective procedure that can be applied generally.  

We would like to thank Shimadzu Corporation and Supelco for the continuous support.  

 is a full professor of analytical chemistry at the University of Messina, Italy and at the "Campus Biomedico" in Rome. His research interests include chromatographic techniques and the development of coupled techniques and their applications in the study of natural complex matrices. His current roles include being a member of the scientific committee of the International Symposium on Capillary Chromatography, chairman of the 35th International Symposium on Essential Oils, editor of

 is assistant professor in the Food Analysis Division at the School of Pharmacy of the University of Messina. His main interests regard the development of classical multidimensional and comprehensive chromatographic instrumentation in combination, particularly, with mass spectrometric detection.  

 is assistant professor in the Food Analysis Division at the School of Pharmacy of the University of Messina. His main interests regard the development and application of fast GC methods, as well as classical multidimensional and comprehensive chromatographic techniques.  

 is attending the third year of the PhD course in food chemistry and safety. His main interests regard multidimensional and comprehensive chromatographic techniques, such as LC×GC and MDGC, as well as fast GC–MS applications.  

 is an associate professor of food chemistry at the University of Messina, Italy. Her research interests include the study of the composition of citrus essential oils and the study of components with possible biological activity in natural matrices by chromatographic techniques. 

 is a full professor of food chemistry at the University of Messina, Italy. His main research interests cover the fields of food fats, citrus products and the development of innovative chromatographic techniques and methods for the study of complex matrices. He is a member of the Editorial Board of the 

 research and of the "Rivista italiana di Scienza dell'Alimentazione" (

) and chairman of the 35th International Symposium on Essential Oils. 

, (Royal Society of Chemistry, Cambridge, UK, 1999). 

2.  S.C. Rastogi, T. Menné and J. duus Johansen, 

7. Analytical procedure for the GC–MS quantification of potential fragrance allergens in fragrance compounds; version 1. 

 (International Fragrance Association: Brussels, Belgium, 2003) pp 1–10.

Coupling Matters

A total of fourteen cuts were defined because the retention time difference between several skin sensitizors was only slight.

Analysis of Allergens in Fragrances using Multiple Heart-cut Multidimensional Gas Chromatography–Mass Spectrometry

For the last six months, "LC Troubleshooting" has been concentrating on the process for the development of isocratic methods (those for which the mobile phase composition is constant throughout the run). This worked through the sequence of goal setting,

 selecting the right starting conditions,

 Hopefully you have gained additional insight into the liquid chromatography (LC) process, as well, so your methods will be less likely to fail and will be easier to troubleshoot.  

The first step in getting an acceptable separation is to get the retention times in the right general region.

  This is often is accomplished by changing the mobile phase organic content (%B) for a reversed-phase separation in 10% increments from 100% organic until the retention factors 

 are in the range of 1–20, or better yet, 2–10. Because peaks move in a regular fashion as the mobile phase strength is changed, we can generalize the effect of solvent strength as "The Rule of Three". This states that the 

-values change by approximately three-fold for each 10% change in organic. Because 

 changes three times per 10% change, a 20% change will result in approximately 3 × three-fold, or roughly 10-fold. Using such guidelines and a little common sense, we can usually find isocratic conditions that give us the 1 < 

 < 10 retention times in three to five runs. Wouldn't it be nice to be able to find these conditions in the very first run? Well, with the help of a gradient scouting run, you usually can. The use of a gradient scouting run to speed isocratic method development is the subject of this month's "LC Troubleshooting." 

A very efficient way to start an isocratic method development project is to make the first run as a scouting gradient run. With isocratic runs, you cannot often anticipate where peaks will come out in the first chromatogram, so you are forced to start with a solvent that is too strong (for example, 90% or 100% B). Then you work to weaker solvents in 10 or 20% steps using "The Rule of Three" for a guide until you achieve 2 < 

 < 10 for your sample. With gradient elution, you can predict in advance conditions that are likely to give the gradient equivalent of 2 < 

 < 10. This will occur for small molecules (molecular weights < 1000 Da) for full-range gradients that comprise ≈25 column volumes of solvent. For example, if we use the column recommended for starting with an LC–UV method,

  a 150 mm × 4.6 mm column packed with 5 μm particles, the column volume is approximately 0.01 × the column length (in millimetres), or ≈1.5 mL. This means that the gradient should be ≈37.5 column volumes; a 20 min gradient at 2 mL/min (40 mL volume) is close enough. For an unknown sample, we don't know the polarity range, so it is best to run a full-range gradient of 5–95% or 5–100% organic solvent (keep the percentage of organic solvent greater than 5% to avoid column dewetting). An example of such a gradient scouting run is shown in Figure 1 (the chromatogram is only shown until the last peak has been eluted). 

The first question after we run the scouting run should be, "Is a gradient or an isocratic run best for this sample?" An easy way to make this evaluation is to examine the fraction of the run time occupied by peaks. If the peaks take ≤25% of the run time, an isocratic run is the best choice. If ≥40% of the run is occupied by peaks, a gradient is advised. Between these limits, either an isocratic or gradient run can be possible. 

In the instance of Figure 1, the first peak is eluted at ≈10 min and the last peak at ≈12 min. So (12 – 10)/20 = 10%, which is less than 25%, so an isocratic run is recommended. There are several ways to select the isocratic conditions at this point, but one of the easiest is to use an Excel spreadsheet calculator that you can download for free from the LC Resources website (reference 7, go to the "Resources" page and download the file "scouting_gradients.xls").

The use of this calculator is shown in Figure 2. In the top section, fill in the run conditions for the scouting run. In this case, a 150 mm × 4.6 mm (15 cm × 0.46 cm) column was used. The equipment dwell volume was 1.5 mL and the flow-rate was 2.0 mL/min. The gradient was run from 5 to 100% organic in 20 min. At the bottom left, enter the retention times for the first (10.0 min) and last (12.0 min) peaks. The calculator then recommends the type of method for the next run (isocratic for this example) and conditions for the run (44% organic). I usually round this number to the nearest 5% for convenience. If you insist on running a gradient method, the conditions for the next run are shown (40–55% in 3 min), which adjusts the gradient to remove most of the wasted time before the first peak and after the last peak are eluted. 

The calculator requires that you know the dwell volume of your LC system. This is the volume from the point the solvents are mixed until they reach the head of the column. The calculation should be done for every LC system, and is straightforward using the directions in references 8 or 9, Section 4.3.1.2. High-pressure-mixing systems typically have dwell volumes in the 1.5–3.0 mL range, whereas low-pressure-mixing systems are a little larger, with typical volumes of 2.5–4.0 mL. LC systems designed for small-volume applications such as LC–mass spectrometry (MS) often have dwell volumes of 0.2–0.5 mL.

After the scouting gradient has been run and isocratic conditions have been determined to be appropriate, it is time to make the first isocratic run. The chromatogram for the isocratic conditions recommended from Figure 2 (rounded to 45% organic) is shown in Figure 3. The column dead time 

 is (1.5 mL/2.0 mL/min) ≈ 0.75 min, so 

) can be calculated from the retention times 

 of the first (1.8 min) and last (7.6 min); 1.4 < 

The run of Figure 3 can be satisfactory for many applications (resolution ≈2.0), but further fine-tuning might be desirable, as described in earlier instalments of this series. It is unlikely that the first isocratic run will be as good as in this example, but the retention should be in the right range to start fine-tuning the separation. It is highly unlikely that you would be as close to a 1 < 

 < 20 separation in two runs using the 100%, 90%, 80%... technique, so the gradient scouting run is a faster way to home in on the best isocratic conditions. 

The gradient scouting run, as used in the previous example, led us to an acceptable isocratic run in just two injections. Another strong advantage of the gradient scouting run is that it yields useful data if isocratic conditions are not possible. If the retention range for the sample is broad, such that the ratio of 

-values for the first and last peak exceeds 20, a gradient method usually is required. If you start method development with isocratic steps, you will realize after a few runs that the sample is too complex for isocratic separation, but the data you gathered isocratically will be of no use. You will have to start over with gradient runs. By using the gradient scouting run, you can evaluate the "isocratic or gradient" question based upon the initial run and proceed with the appropriate method development strategy with no wasted runs.  

We've spent enough time over the last few months discussing isocratic method development and I would like to get back to some more traditional troubleshooting problems, so we'll leave gradient method development for a future discussion. A good resource for gradient method development is reference 9, especially chapter 3. 

In this series of columns on method development for isocratic, reversed-phase separations, we've seen that a systematic approach will yield results in an efficient manner. The use of a gradient scouting run, as outlined here, can be a real time-saver at the beginning of the method development process. It can give us an idea of the complexity of the sample and using tools such as those shown in Figure 2, we can determine if an isocratic or gradient method will be required, as well as recommended conditions for the next isocratic (or gradient) run. 

 is vice-president of LC Resources, Walnut Creek, California, USA and a member of 

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

, Advanstar House, Park West, Sealand Road, Chester CH1 4RN, UK. 

Readers can also direct questions to the Chromatography Forum at 

9. L.R. Snyder and J.W. Dolan, High-Performance Gradient Elution (Wiley, New York, New York, USA 2007).

LC Troubleshooting

A very efficient way to start an isocratic method development project is to make the first run as a scouting gradient run.

The Perfect Method, Part 7: The Gradient Shortcut

The Chromatographic Society Spring 2008 Meeting and AGM Reading, UK

The meeting, being held 21–22 May, will review the latest developments in stationary phase technologies for liquid chromatography. It will also include lectures on emerging stationary phase chemistries, specialised functional phases and small particle size packings 

The event will be of interest to all practitioners of separation science, but in particular those using liquid chromatography for problem solving and method development. 

Day One highlights include Session One titled 

Current state-of–the art and the future

Overview of current technology and future

 with Dr Ron Majors (Agilent); Keynote Lecture Two: 

 with Dr Gert Desmet (Brussels Free University); Lecture One: 

 with Prof. Mel Euerby (Hichrom) and Lecture Two: 

Molecular mechanism of HPLC retention and column classification in view of QSRR

 with Prof. Roman Kaliszan (University of Gdansk, Poland).

Day Two highlights include Session Three titled 

 with Dr Roman Szucs (Pfizer); Lecture Four: 

 Stationary phase selectivity in preparative chiral SFC

 with Dr Mary Luz de la Puente (Lilly); Session Four titled 

 with Prof. Peter Myers (University of Liverpool); Lecture Seven: 

 with Dr Frederic Lynen (University of Ghent); Lecture Eight: 

 with Dr Richard Ansell (Leeds University) and Lecture Nine: 

From 1–4 April 2008, Munich will once again be the stage for Analytica 2008 — a leading trade fair offering marketable solutions in analytics, laboratory technology, biotechnology and the life sciences. 

Since 1968 Analytica has been held every two years and has become an established international meeting point for leaders in the laboratory industry. It is also recognised for being a communication and business event for the international community, offering the presentation of innovative products and services and of the reproduction of entire value-added chains in industrial and research laboratory applications.

Analytica 2008, which will be held at Fairground, Munich, will feature international events to help build business relationships on a global scale. These will include the Analytica Forum, which is a platform for the exchange of information and transfer of knowledge, and the European international research community summit.

For more information about Analytica 2008 contact Messe München GmbH, Messegelände, 81823 Munchen, Germany, tel: +44 89 949 11488, fax: +44 89 949 11489 or e-mail: 

32nd International Symposium on High Performance Liquid Phase Separations and Related Techniques Baltimore Marriott Waterfront Hotel, Baltimore, Maryland, USA Contact: Ms Janet Cunningham,  symposium/exhibit manager e-mail: 

HET Instrument 2008 — Analysis;  Biotechnology;  Laboratory Equipment Jaarbeurs Utrecht, Netherlands Organizer: FHI Federation of Technology Branches Contact: Ms Petra Bronsema tel: +31 33 465 75 07 fax: +31 33 461 66 38 e-mail: 

Forum LABO — Forum BIOTECH Porte de Versaille, Paris, France Organizer: Committee Interprofessional of Laboratory Suppliers (CIFL), 28, rue St Dominique, 75341  Paris, France Contact: Ms Christiane Turci tel:  +33 1 441898 62 fax:  +33 1 441898 63 web: http://

LABTECH Conference & Exhibition 2008 The Gulf International Convention and Exhibition Centre, PO Box 580, Kingdom of Bahrain, Manama, Bahrain Organizers: Saudi Aramco Co, ACS, SAICSC-ACS and Bahrain Society of Chemists Contact: Yaser Sonbul, Saudi Aramco Co. Box# 7010, N/A Ras Tanura, 31311, Saudi Arabia e-mail: 

10th European Meeting on Supercritical Fluids — Reactions, Materials and Natural Products Processing Congress Centre, Strasbourg, France Organizers: ISASF-ENSIC Contact: F Brionne, 1 Rue Grandeville, B.P. 45 I, Nancy, Cedex, F-54001, France tel: +33 3 83 17 50 03 fax: +33 3 83 35 08 11 e-mail: 

Event News

The meeting, being held 21–22 May, will review the latest developments in stationary phase technologies for liquid chromatography. It will also include lectures on emerging stationary phase chemistries, specialised functional phases and small particle size packings The event will be of interest to all practitioners of separation science, but in particular those using liquid chromatography for problem solving and method development.

Chromatographic Society Spring 2008 Meeting

Professor Gert Desmet, Transport Modelling & Analytical Separation Science, Vrije Universiteit Brussels, Brussels. 

: The Transport Modelling and Analytical Separation Science group is part of the Department of Chemical Engineering of the Faculty of Engineering Sciences. The group currently has eleven PhD students and one post-doc and is headed by Gert Desmet, who is also head of the department. The group focuses on the miniaturization of separation methods and on the investigation and the modelling of flow effects in commercial and innovative chromatographic systems. The activity of the group in the field of chromatography started in 1999 by practically demonstrating the giant leap in separation speed that can be achieved by using shear-driven flows in 100 nanometere-deep channels. 

Since then, the group has gained great expertise in the design, fabrication and testing of miniaturized or "on-chip" separation systems. In this area, the group is currently coordinating a large-scale project funded by the Flemish government to fabricate micro-machined chromatographic columns, in collaboration with seven partners. The group is also involved in several research projects comparing the performance limits of several novel commercial packing types (monoliths, sub-2 µm particles) and chromatographic operation modes (ultra-high pressures, high temperatures). For this work, the group cooperates extensively with groups of Kazuki Nakanishi (University Kyoto, Japan), Pat Sandra (Pfizer Analytical Research Center, Belgium), Peter Schoenmakers (University of Amsterdam, The Netherlands), Jean-Louis Rocca and Sabine Heinisch (University of Lyon, France), Jean-Luc Veuthey (University of Geneva, Switzerland) and many others.  

To support and guide this work, the group has also developed a substantial theoretical activity, partly based on the use of computational fluid dynamics. 

The main aim of this work is to obtain a better understanding of the relation between performance of high performance liquid chromatography (HPLC) supports and the packing structure and the flow and heat transfer characteristics of the columns. To conduct this theoretical work, the group uses a high-performance, 60-processor computer cluster.  

 Professor Eddy Esmans and Professor Filip Lemière, Nucleoside Research and Mass spectrometry Unit. University of Antwerp, Department of Chemistry, Antwerp,  

 This research unit has been involved in liquid chromatography mass spectrometry (LC–MS) research since 1978 when it purchased a direct liquid introduction (DLI) LC–MS system. 

 The group is currently involved in several projects, including the detection and analysis of DNA-adducts as initiators of tumour formation in real life samples; the identification of urinary nucleosides as early biomarkers for cancer (in collaboration with Professor Russell Newton and Professor Ed Dudley of Swansea University, UK); and the analysis of environmental samples for the occurrence of polyfluorinated molecules such as perfluorooctane sulphonic acid (PFOS) in collaboration with Professor Wim De Coen and Professor Ronny Blust from the University of Antwerp). 

 The team has also a large interest in plant proteomics in collaboration with Professor Harry Van Onckelen (University of Antwerp, Belgium) and Dr Erwin Witters (Flemish Institute for Technological Research & University of Antwerp, Belgium). 

The main analytical techniques used are LC–MS, specifically miniaturized electrospray ionization tandem mass spectrometry (ESI-MS–MS). In the case of plant proteomics an important amount of data is gathered by matrix assisted laser desorption/ionization time-of-flight–time-of-flight mass spectrometry (MALDI-TOF–TOF MS) after separating the proteins by two–dimensional gel electrophoresis (2D GE) or liquid chromatography (LC). ESI-MS–MS is used for the LC–MS–MS analysis of tryptic digests, if necessary. 

The group has been one of the pioneers of miniaturized LC–MS in these topics. As often is the case in the analysis of biochemical and biological samples a limited amount of sample is available and compounds of interest are present in very low quantities.  

Therefore LC–MS methodologies were developed using capillary – and nano-HPLC columns in reverse phase (RP) and hydrophilic interaction chromatography (HILIC) mode with an internal diameter of 300 µm and 75 µm respectively. These were coupled to nano-electrospray ionization (nESI) for the qualitative and quantitative analysis of the samples. Approximately 3700 times less sample was required for the 75 µm column compared with a 4.6 mm internal diameter (i.d.) column with no reduction in concentration sensitivity. In many cases the LC–MS analysis of the samples is performed in combination with column-switching techniques.  

 Professor Jos Hoogmartens, Professor Ann Van Schepdael and Professor Erwin Adams, Laboratory for Pharmaceutical Analysis, Catholic University of Leuven, Leuven. 

 The team's activities are targeted towards the application of chromatographic (gas, liquid) and electrophoretic techniques to pharmaceutical analysis. 

Liquid chromatography (LC) focuses on the development and validation of methods for the analysis of drug substances and drug products. Different detection methods are available: ultraviolet (UV), fluorescence (F), evaporative light scattering detection (ELSD), charged aerosol detection (CAD), pulsed electrochemical detection (PED) and mass spectrometry (MS). Several kinds of substances are studied, but stress falls on antibiotics and antiviral drugs. A number of methods developed have been incorporated in official compendia like the 

To help to select a suitable reversed-phase (RP) column, a characterization procedure has been developed using three simple chromatographic tests that allow the determination of four parameters for each column. Based on these, a ranking of the columns from most similar to dissimilar versus a freely to choose reference column can be obtained. More information can be found on 

pharm.kuleuven.be/pharmchem/Pages/7_CCS.html

Gas chromatography (GC) is applied to determine volatile substances in pharmaceuticals. Residual solvents are detected, but also volatile impurities and degradation products. To distinguish between them, mass spectrometry (MS) is necessary. However, quantitative application of MS is hampered by signal instability. Solutions to improve this and alternative detectors are currently being investigated.  

In the field of capillary electrophoresis (CE), the research is in two main areas: method development for the analysis and stability testing of drugs (with an emphasis on the impurity profiling of antibiotic substances) and the application of electrophoretically mediated microanalysis (EMMA). The EMMA aspect comprises the use of in-line systems that allow the interaction of different plugs of either enzyme and substrate or analyte and derivatization reagent, followed by the separation of the reaction products formed. This method has been applied to several types of enzymes and analytes, with the current focus on the cytochrome P450 enzymes because of their importance in drug behaviour in the human body. The laboratory is also establishing a capillary electrophoresis–mass spectrometry (CE–MS) system with a home-built interface. 

 Professor Bart Devreese and Professor Jozef Van Beeumen, Laboratory for Protein Biochemistry and Protein Engineering, Ghent University. 

 The unit for Mass Spectrometry and Proteomics within the Laboratory for Protein Biochemistry and Protein Engineering is involved in separation sciences in protein and proteomics research. The group pioneered the use of nano-liquid chromatography (nLC) combined with electrospray ionization mass spectrometry (ESI-MS) to identify two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) separated proteins, both from microbial and animal origin. Recent work involved the implementation of an interface between two dimensional strong cation exchange coupled to reversed-phase chromatography (2D-SCX×RP) capillary HPLC and matrix assisted laser desorption/ionization time-of-flight–time-of-flight mass spectrometry (MALDI-TOF–TOF MS) for the in-depth analysis of membrane proteins. 

There has also been a lot of research in novel protein identification strategies implementing chemical derivatization and tandem mass spectrometry (MS–MS). Current research efforts will focus on analysis of protein complexes, using miniaturized affinity separation techniques and on bottom-up proteomic analysis using chemical and metabolic stable isotope labeling. The group of Bart Devreese is a partner in a Flemish government-supported collaborative effort for developing microfluidic devices to be implemented in proteomic research involving pillar chromatography.  

This group also developed capillary electrophoresis–mass spectrometry (CE–MS) applications on hybrid quadrupole ion trap instruments for profound analysis of the carbohydrate composition and linkage analysis of glycoproteins. CIEF has been implemented to separate and characterize different glycoforms at the protein level. These methods were applied in the study of glycosidases and hemocyanins. 

The unit is imbedded in an environment within the Laboratory for Protein Biochemistry and Protein Engineering which also includes a structural biology, a protein and glycan engineering group that uses several preparative chromatography approaches for purifying proteins and glycoproteins. 

 Department of Analytical Chemistry and Pharmaceutical Technology, Pharmaceutical Institute, Vrije Universiteit Brussel led by Professor Johanna (An) Smeyers-Verbeke and Professor Yvan Vander Heyden for Analytical Chemistry, and by Professor Jacqueline Plaizier-Vercammen for Pharmaceutical Technology. 

 The Analytical Chemistry research group has separation science and applied chemometrics as the main research areas. The aim of the group is to evaluate the rational use of new techniques in pharmaceutical analysis, where techniques refer both to separation and data analysis techniques.  

In separation science, research is performed on chiral separations for specific cases, specified by industrial partners, and to define generic separation strategies in different techniques, such as capillary electrophoresis (CE), normal phase liquid chromatography (NPLC), reversed-phase liquid chromatography (RPLC), polar organic solvent chromatography (POSC), supercritical fluid chromatography (SFC) and capillary electrochromatography (CEC). In a second instance, the development of methodologies to obtain suitable fingerprints of herbal extracts by means of HPLC is investigated. Fingerprints are complex chromatograms, which allow the quality of an extract to be identified and evaluated. 

Drug impurity profiling by means of high performance liquid chromatography (HPLC) and the selection of dissimilar systems, that is, systems with different selectivities, are also studied. Monolithic columns and (sub)micron-sized particle-based columns are examined in CEC for their performance in the analysis of drug substances and their impurities, chiral separations and fingerprint development. Recently, a project was started to introduce separation techniques such as CE, in microbiology, and more specifically in virology.  

The data analysis aspects are mainly related to separation science data. They comprise, for example, the application of data pretreatment techniques, such as warping methods, variable reduction and visualization techniques, such as principal component analysis, pattern recognition methods and multivariate calibration techniques on fingerprint data. Predictive quantitative structure-retention relationship (QSRR) models are also constructed and allow predicting the chromatographic retention of solutes, based on their physicochemical or structural properties. Based on the same properties, QSAR models are built to predict the passage of drug molecules over cellular membranes, for example, the gastro-intestinal absorption or the blood–barrier passage. There is also a focus on method validation and on experimental designs. 

 Professor Luc Nagels, Organic Chromatographic Trace Analysis, Antwerp University, Antwerp. 

 The group is focused on applications of potentiometric sensors in high performance liquid chromatography (HPLC) and (more difficultly) in capillary electrophoresis (CE). Sensors which change surface potential when contacting analyte ions are an unexplored area of research in separation science. Other groups did pioneering work in the 1990s, but solving the puzzle of finding good sensor coatings for separation science has just started. One major problem until now was, surprisingly, the fact that electrochemists did not believe that potentiometric sensors reacted rapidly and sensitively to many industrial-, biotechnology- and pharmaceutically-oriented organic molecules. 

Even large and highly charged molecules, such as DNA and bioparticles, give surprisingly high responses. The study of the interaction of these highly charged molecules with surfaces is quite new. Electric surface potentials are part of nature, but are still badly understood. This is also the case for the interaction of small ionic organic compounds. It is to be expected that LC and other separation methods will bring potentiometry in another era, as they did with spectroscopic, for example ultraviolet (UV), and spectrometric, for example mass spectometry (MS), methods. 

The group firmly believes in the enormous potential of potentiometry for sensors. Separation methods provide the vehicle to explore them, and the sensors will be used as detection devices in separation methods. The group started a spin-off company, Octens, for dissolution applications in the pharmaceutical sector. 

 Professor Pat Sandra, Ghent University and the Research Institute for Chromatography, Ghent. 

 Research at the Ghent University is mainly in the framework of the Pfizer Analytical Research Centre (PARC), which was established in 2003 to drive innovation and productivity in pharmaceutical analytical sciences. PARC is a multidisciplinary collaboration with the aim of generating high-throughput analytical systems for pharmaceutical analysis and drug discovery, introducing novel approaches to pharmaceutical analysis such as green chromatography and making advances with innovative ideas and concepts for analytical work in the 21st century. These projects involve collaboration between experts in academia and industry. 

At present, eight postdoctoral and six PhD students work on the following research topics: the synthesis of thermoresponsive and polymeric stationary phases for liquid chromatography (LC); elevated temperature and high pressure liquid chromatography (HPLC); comprehensive liquid chromatography (LC×LC); supercritical fluid chromatography (SFC); multi-array microemulsion electrokinetic chromatography (MEEKC); the development of bioactivity post-column reactors; and the synthesis of receptors for endocrine disrupting chemicals (EDC) metabolomics and lipidomics. 

At the Research Institute for Chromatography (RIC), located in Kortrijk, eight professionals with a PhD degree perform research in gas chromatogrpahty (GC), liquid chromatography (LC) and supercritical fluid chromatography (SFC). These projects concentrate on method development, hardware development, training, consultancy and developing total analytical solutions. 

 Professor Joëlle Quetin-Leclercq, Professor Bernanrd Tilquin, Dr Marie-France Hérent, Dr Aubert Maquille and Dr Céline Rivière, Chemical and Physico-chemical analysis of Drugs and Pharmacognosy (CHAM) unit, Université catholique de Louvain, Faculty of Medicine, Department of Pharmacy, Brussels. 

Belgium Focus

Research Group: Professor Gert Desmet, Transport Modelling & Analytical Separation Science, Vrije Universiteit Brussels, Brussels.

Dow Deutschland Anlagengesellschaft mbH, Analytical Sciences, Rheinmünster, Germany, 

The Dow Chemical Company, Analytical Sciences, Midland, USA, 

Agilent Technologies Deutschland GmbH, Waldbronn, Germany. 

High-speed liquid chromatography is becoming important in many pharmaceutical and biochemical laboratories because of the demand to analyse complex samples quickly.

  The availability of small-particle columns (less than 2 µm particle size) has paved the way for LC separations with short analysis times, while maintaining — or even increasing — high separation efficiencies.  

There is a growing interest in this new generation of LC columns for a wide range of chemical applications, including small molecules, oligomers and polymers. The availability of high pressure liquid chromatography (HPLC) systems that operate at high pressures (> 400 bar) allows the use of long, small particle columns. Such columns are now available at lengths up to 150 mm. Thus, high resolution LC separations can be generated for difficult separations of complex mixtures. Recent experimental studies revealed that sub-2 µm particle columns show superior kinetic performance (peak capacity) compared with 3–3.5 µm particles, when operated at high-pressures and run times of less than 30 min.

Fast LC columns of different lengths were evaluated in terms of column performance. It should be noted that some of these were prototype/experimental columns. Some columns were coupled to assess the maximum possible plate numbers for high-efficiency separations. Experiments were performed with the 1200 Series Rapid Resolution LC system (Agilent Technologies, Waldbronn, Germany), which operates at pressures up to 600 bar. A fast scanning diode array detector (DAD) with a maximum scan rate of 80 Hz was used. 

Higher temperatures were recently applied to obtain high performance liquid phase separations with sets of coupled conventional columns.

  This concept was also applied to our investigations which include the separation of epoxy resins. Examples of fast LC for the determination of copolymer composition and functionality type distributions are described. Finally, ultra-fast separations of inhibitors were used to assess system performance. 

Figure 1 illustrates a typical high-resolution separation of SRM 869a (National Institute of Standards and Technology (NIST), Gaithersburg, Maryland, USA) — a shape selectivity and column test mixture,

  which is available from the National Institute of Standards and Technology (NIST). For this example, two small-particle columns were coupled for a total length of 150 mm. Excellent column efficiency was observed with plate numbers of N = 37000 for tetrabenzonaphthalene (TBN) or about 250000 plates/m. Figure 2 shows the performance of several individual and coupled columns. All columns were of 4.6 mm internal diameter (i.d.) with a 1.8 µm Zorbax SB-C18 silica (Agilent Technologies, Wilmington, Delaware, USA) as the packing material and some of these were prototypes. 

Theoretical and experimental plate numbers were evaluated for TBN at 80 °C and flow-rates near the optimum of the Van Deemter curve (1.5 mL/min). The efficiencies of these experimental columns varied. For example, the coupled 100 and 50 mm columns showed higher N values than a single 150 mm column. This was possibly because the shorter columns were packed more homogeneously or the prototype/research character of some of the longer columns. In general, it is more difficult to pack long columns with sub-2 µm particles. It is apparent that some efficiency was lost in column connections when constructing the very long columns. However, the availability of 50000–70000 plates for a given separation (400 mm total column length) may be very beneficial for highly complex mixtures, where high peak capacity is preferred over column selectivity and short analysis times.  

From a practical perspective it needs to be ensured that all fittings and connections from injector to detector (including column connections) are mounted carefully to allow for the lowest additional dead volume or extra-column band broadening. The influence of additional dead-volume will be more pronounced when going to smaller i.d. columns such as 3.0 mm or 2.1 mm i.d. The appropriate capillary connections are also important and should be below 150 µm i.d. to reduce extra-column band broadening. A thorough description of peak dispersion effects, including system dwell volume, can be found elsewhere.

  With an optimized set-up (125 µm i.d. capillaries, 2 µL flow cell, 80 µL solvent mixer) an extra-column band broadening of only 15 µL was determined using the procedure of Lauer and Rozing.

  This measurement did not include any additional dead-volume present in column-to-column connections. 

Several Zorbax Rapid Resolution (Agilent Technologies, USA) columns for high-resolution LC were used to separate complex samples. Epoxy resins are composed of various oligomers and different substitution patterns (Figure 3). They are therefore an ideal class of compounds for when high peak capacity is needed. An extended temperature range of the LC system with solvent-pre-heating and post-column cooling enables the use of longer columns without deteriorating the UV baseline performance. Total column lengths of 150 mm and 400 mm were used for this application. A temperature of 80 °C was sufficient to generate a backpressure of 552 bar with the 400 mm columns at about 40% water in acetonitrile. As can be seen from Figure 3 the peak resolution is excellent. It can be noticed though, that components that elute later are not fully resolved because of the presence of multiple isomeric constituents. 

The first three isomer peaks are fully baseline separated. Even minor peaks are observed inbetween the main isomers. For the p,p' peak of the n = 1 oligomer a shoulder is observed which indicates the presence of additional isomers. Such a separation has not been achieved before, and a total run time of about 12 min is still acceptable considering the wealth of information that is accessible. Separation is further improved because for the p,p' peak (n = 1) the isomer separation is more obvious in the lower chromatogram using the longer set of columns.  

Operation at high temperatures has advantages and limitations. The benefits are reduced viscosity of the mobile phase, which allows higher flow-rates to be applied, which reduces analysis times. Reduced viscosity is valuable, particularly for mobile phase gradients with a high percentage of water (> 50%) at starting conditions. However, a temperature increase shifts the minimum of the Van Deemter curve to higher linear flow-rates. Therefore to operate at optimal conditions the flow-rate must be increased.  

Temperature also has an effect on stationary phase selectivity and can be used to tailor a separation for optimal peak resolution.

  This means high-temperature LC can be beneficial or disadvantageous, depending on the components to be separated. Preheating of the mobile phase is required; otherwise peaks may broaden as a result of thermal mismatch. A possible drawback is the limited stability of silica-based packing materials at high temperatures (> 80 °C), however, it should be noted that manufacturers are investigating ways to increase stability in this regard. 

Polymer analysis by LC can be executed in three different modes: size-exclusion chromatography (SEC), LC at the critical point of adsorption [the liquid chromatography at critical conditions (LCCC)] and gradient LC (adsorption or precipitation– redissolution).

  The first methodology is governed by entropy (separation by hydrodynamic volume), adsorption LC is driven by enthalpic effects. LCCC is driven by a balance of enthalpic and entropic effects. Gradient LC of polymers enables separations according to differences in chemical composition distribution (CCD). For copolymer analysis this is a powerful tool to measure polymer composition and assess the distribution of the composition.  

LCGC Europe-03-01-2008

New Technologies

Analysis of Allergens in Fragrances using Multiple Heart-cut Multidimensional Gas Chromatography–Mass Spectrometry

The Perfect Method, Part 7: The Gradient Shortcut

Chromatographic Society Spring 2008 Meeting

Belgium Focus

Fast, Ultra-fast and High-resolution LC for Separation of Small Molecules, Oligomers and Polymers

The 2008 Update of US GMP — Why Not Include the Laboratory