36th International Symposium On Capillary Chromatography and the 9th GC×GC Symposium

International Symposium on Capillary Chromatography (ISCC)

 has established its reputation as a forum for microcolumn separation techniques. Since the first meeting in Hindelang in 1975, the most important developments in capillary gas chromatography, microcolumn liquid chromatography and electromigration techniques have been presented in this symposium series. 

The format and the atmosphere of the 36th meeting will be similar to the previous successful meetings, held in Hindeleng, Riva del Garda, Monterey, Baltimore, Gifu, Kobe, Wintergreen, Las Vegas, Dalia, Albuquerque, Portland and San Diego. Luigi Mondello from the University of Messina is the chairman of this year's event and Pat Sandra will be honorary chairman this year.  

, Italy, will accomodate the 36th meeting from 

. The four-day event will feature recent findings from leading academic and industrial experts in the form of lectures and posters. The conference offers sessions on capillary gas chromatography, microcolumn liquid chromatography, electromigration methods and microfabricated analytical systems, which are expected to cover lab-on-a-chip, column technology, coupled and multidimensional techniques, comprehensive techniques, hyphenated techniques, sampling and sample preparation, trace analysis and automation. 

Application sessions include environmental applications, energy/petrochemcal/industrial applications, biomedical/pharmaceutical applications and the analysis of natural products, food, flavours and fragrances. Workshop-seminars of instrument manufacturers and an extensive exhibition of instrumenation, accessories and supplies will run in parallel to the scientific programme. 

At the meeting, the 2012 Marcel Golay Award will be presented in recogntion of outstanding contributions in the field of separation science. Outstanding research work presented as oral or poster contributions by youngsters will be awarded with the Leslie Ettre Award for research on capillary gas chromatography applied to environmental or food analyses. 

To encourage scientific exchange and friendship building, the scientific programme will be spiced with the well-known "Riva Social Programme", which consists of a welcome reception, cocktail party, classical concert, cheese and wine evening, disco night and farewell cocktail. 

Considering the interest in comprehensive techniques, the 9th GC×GC Symposium will be organized during the same period to allow scientists to attend both meetings. The 9th GC×GC symposium will start on 27 May 2012 with a course presented by experts in the field covering the fundamental aspects of comprehensive techniques and a plenary session on 28 May 2012. 

For both meetings, abstracts for consideration as lecture or poster presentations can be submitted on-line at 

. All abstracts will be reviewed on the basis of scientific merit, novelty and practical application. Presenters at the meeting may also have their work published in a special volume of the 

Authors intending to submit papers for the symposium will be required to adhere to the following deadlines: A 300-word abstract must be received no later than 

. For abstract submission see the website: 

 Notification of acceptance will be mailed to the authors by 

Oud-Sint-Jan' Conference Centre, Bruges, Belgium

 Royal Flemish Chemical Society and Royal Society of Chemistry

Starling Hotel and Conference Centre, Geneva, Switzerland

Ornage County Convention Centre, Orlando, Florida, USA

New Munich Trade Fair Centre, Munich, Germany

Silesian School of Management, Katowice, Poland

 Institute of Environmental Engineering of Polish Academy of Science 

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No matter where liquid chromatography (LC) is used, it is never immune to problems.

One of the privileges of teaching liquid chromatography (LC) classes for a living is that I get to meet chromatographers from around the world during my travels. As I write this instalment, I'm just finishing six weeks of classes, where I have had a chance to discuss problems that attendees were having with a variety of LC applications. Although the topics in this column originate in Poland, Turkey and Argentina, they are no different than those that arise in Singapore, Galway, Charlottetown or Huntsville. Let's look at a couple of problems that students had related to both conventional LC and ultrahigh-pressure LC (UHPLC). These problems apply equally to both techniques. 

One of my students from Poland, who was just starting out using UHPLC, asked if she could inject 5 μL of sample onto her UHPLC column without any problems. Her conditions comprised a 75 mm × 2.1 mm column packed with 1.8-μm diameter particles operated at a flow rate of 0.8 mL/min. It is fairly easy to perform a few simple calculations and answer her question.  

A good rule of thumb (1) is that you can inject ≈ 15% of the volume of the first peak of interest without causing a more than ≈ 5% increase in peak broadening, as long as you use mobile phase for the injection solvent. We need to determine the volume of the first peak of interest to see how this applies in the present case. We will assume a retention factor, 

, of 1 for the first peak; you can make similar calculations for other retention factors.  

First, we need to estimate a column plate number (efficiency), 

 is the column length in millimetres and 

 is the particle diameter in micrometres. A well-packed column tested with an ideal solute will give larger values of 

, but equation 1 is a reasonable estimate for real samples. With our 

Next, it is necessary to estimate the column volume, 

 is the column diameter in millimetres. For the 75 mm × 2.1 mm column we get 0.165 mL. From this we can get the column dead time, 

, by dividing by the flow rate, 0.8 mL/ min: 0.21 min. (Note: If you are trying to repeat these calculations, carry along several extra decimal places of precision — I'm rounding the numbers for simplicity of presentation). 

With the knowledge of the column dead time, we can calculate the retention time, 

= 1 peak by rearranging the equation for 

The next to last step is to estimate the peak volume of the first peak. We estimated 

 with equation 1, but if we were measuring 

 from an isocratic chromatogram, we would use  

 is the baseline width of the peak measured between tangents drawn along the sides of the peak. Solving equation 4 for the peak width, we get  

Using our 15% rule of thumb from the beginning of our discussion, an injection of (0.15 × 11.8 μL) = 1.8 μL, or ≈ 2 μL would be allowed in the mobile phase. This is less than half the desired 5-μL injection, so it is expected that excess peak broadening would be observed with a 5-μL injection. Armed with this knowledge, I would perform a series of injections of 1, 2, 3, 4 and 5 μL and observe the effect on the peak width of the first peak of interest. I agree wholeheartedly with Izaak Kolthoff, the father of analytical chemistry, who is credited with the quote, "Theory guides, experiment decides." So, use the results of the injection-volume experiment for the final decision. 

The above example illustrates one of the challenges of UHPLC — reducing the sample volume sufficiently to avoid band broadening. Perhaps this could be accomplished if any preceding sample-preparation steps involved reconstitution of a dried extract. For example, if an evaporation-to-dryness step was followed by reconstitution in 50 μL of mobile phase, this volume could be reduced to 10 μL and now a 1-μL injection would contain the same mass of sample as 5 μL of the 50-μL volume. 

Because the band-broadening effect is worse for the first peak in the chromatogram, it may be possible to mitigate the problem by increasing the retention of the first peak. If you repeat the above calculations with several different 

-values for the first peak, you'll see that a 5-μL injection would be allowed with 

 = 4–5. This would likely be too long to wait (≈1 min) for the first peak for most impatient chromatographers — after all, you bought that UHPLC system so you could have short run times! 

A third option to solve this problem might be to use on-column concentration. When the injection solvent is more than 20% weaker than the mobile phase, it is possible to inject large volumes of sample because the weak solvent causes the sample to accumulate at the head of the column until the injection solvent washes through. For example, if the mobile phase were 65% acetonitrile and 35% buffer, injection in ≤45% acetonitrile would be expected to allow injection volumes much larger than the 2-μL injection calculated above. Thus, if the desired injection of 5 μL were in mobile phase, you could dilute the sample two-fold with buffer and inject 20 μL of the sample (now in ≈33% acetonitrile) and put the desired sample mass on the column with less chance of unwanted band broadening. 

As with the conclusion of the previous section, try one or more of these alternative solutions and see what works. 

A student from Buenos Aires was using LC for the analysis of a drug product. He was having problems with carryover, so he was experimenting with different autosampler wash solvents to see if he could mitigate the problem. With some wash solvents he noticed significant peak broadening for his sample, whereas others did not cause problems. Unfortunately, when he used the most effective wash solvent, 100% methanol containing 0.1% formic acid, he observed the worst peak shape. He wondered how he could select a wash solvent that would reduce carryover, yet maintain good peak shape. 

One of the most popular autosamplers in use today is the needle-in-loop design. This configuration typically uses a 100-μL sample loop that has the injection needle as a part of the loop. To inject, the needle is connected to the LC mobile-phase flow path with a high-pressure seal. The nice thing about this design is that, because the needle is in the flow path, 100% of the sample (up to the loop volume) is injected. However, the entire loop contents of 100 μL are injected every time, even with a small sample injection. For example, if a 1-μL injection is made, the loop contains 1 μL of sample plus 99 μL of solvent. Usually the loop is backflushed onto the column so that the sample goes onto the column first, followed by the remainder of the loop contents. If the loop is filled with mobile phase (or a weaker solvent), there is no problem; it is as if a 1-μL sample loop were used. However, if the solvent in the loop is stronger than the mobile phase, the injection plug can be distorted and cause band broadening. Obviously, it is desirable to avoid injecting an excess of strong solvent to avoid unwanted band broadening.  

So, how do we avoid injecting too much of a strong solvent? One way is to perform the wash cycle while the sample loop is in the flow path. With most LC systems, when the injection is made, the injection valve stays in the inject position during the run. If the autosampler wash cycle is performed while the injection valve is in the inject position, the remainder of the autosampler is washed with the wash solvent, but the sample loop is not.  

The sample loop is thoroughly flushed with mobile phase during the run, which is usually sufficient to clean it before the next injection — after all, the sample is soluble in the mobile phase, so the mobile phase should be good enough to flush the loop. If, however, the loop is washed in the load position, the wash solvent will remain in the loop when the next sample is aspirated. Thus, any remaining wash solvent will get injected with the sample. If this wash solvent is too strong, peak distortion is likely. Some autosamplers provide for the use of two wash solvents. If this is the case, the solution to the problem is simple. First, wash with the strong wash solvent in the load position to remove any residues from the sample loop, then wash with mobile phase (or a weaker solvent). Now when the sample is picked up, the remainder of the loop will be filled with a non-band-broadening solvent, so peak distortion should not occur. A final option would be to use a smaller-volume sample loop, if this is an option with your autosampler. Using a filled-loop injection with a 1-μL loop volume, for example, would allow injection of 1 μL of sample and because none of the wash solvent would remain in the loop, the choice of wash solvent composition would be of little concern. 

In Turkey, a student complained that he had purchased a reference standard that was claimed to be >99% pure in the certificate of analysis, but when he ran it on his LC system, he found impurity peaks that greatly exceeded the <1% claim. He showed me a chromatogram that he obtained with a large analyte peak and a smaller impurity peak that indeed was more than 1% of the reference standard. He also had a copy of the certificate of analysis that contained a chromatogram showing a single peak that looked similar to the one I've recreated in Figure 1. The mobile phase was 90% acetonitrile, 10% water on a C18 column for the certificate of analysis; unfortunately, I did not note the conditions for his analysis.  

Figure 1: Reconstructed chromatogram for a certificate of analysis. The arrow marks the column dead time. 

However, even with this limited amount of information, it is possible to make an educated guess as to the source of the problem. If you recall from previous instalments of this column, I've stated many times that the ideal isocratic chromatogram will have peaks in the 2 < 

 < 10 range, or if this is not possible, 1 < 

 < 20. This gives the analytes sufficient time to interact with the column to achieve "good" chromatography, yet keeps the run time from being excessive. Normally, we calculate the retention factor, 

 are the retention time and column dead time as noted in equation 3. I did not include a time axis in Figure 1, because I can't remember what it was in the certificate of analysis, but the units in equation 6 cancel, so we can use a ruler to calculate 

 from the chromatogram. The first disturbance in the baseline (arrow in Figure 1) marks 

 ≈ 0.3. This is much less than the desired minimum 

 << 1, there is little time for the analyte to interact with the column, and the chances of having unresolved peaks present is increased. This is especially important when using a chromatogram to certify peak purity. The small tail on the peak may be peak tailing that is normal with many peaks or it may be a subtle indication that an impurity is present. There is no excuse for producing a chromatogram like this for a certificate of analysis, except laziness, ignorance or impatience. It would be easy to increase 

 < 10 by changing to a weaker mobile phase, such as 70% acetonitrile, 30% water. At that point, a more convincing case could be made to show that the peak was indeed >99% pure. Although I don't remember the conditions of the user's chromatogram, I do remember that the analyte peak was significantly broader than that in the certificate of analysis, suggesting that the retention time was much larger. My answer to the question was that the certificate of analysis didn't convince me of the purity of the reference standard, so I would be more likely to trust the analyst's chromatogram in which 

It doesn't matter where we live; LC problems have no international boundaries. In fact, I've noticed that chromatographic terminology is often adopted directly into many different languages. In some ways, chromatography really is a universal language. It reminds me of a time when a visitor from one of the Asian countries visited my son's classroom when my son was about 12 years old. The visitor asked if the kids wanted to learn some of his language. All were excited about the prospect. The visitor carefully recited "computer", "Coca-Cola" and "McDonalds." Maybe he should have included "chromatography." 

, is vice president of LC Resources in Walnut Creek, California, USA. He is also a member of 

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

, 4A Bridgegate Pavilion, Chester Business Park, Wrexham Road, Chester, CH4 9QH, UK or e-mail the editor, Alasdair Matheson, at 

(1) L.R. Snyder, J.J. Kirkland and J.W. Dolan, 

Introduction to Modern Liquid Chromatography, 3rd Ed.

 (Wiley, Hoboken, New Jersey, 2010), 120. 

LC Troubleshooting

Liquid chromatography is never immune to unexpected problems.

LC Trouble Everywhere

LC–MS-based top-down proteomics involves the chromatographic separation and mass-spectrometric characterization of intact proteins without prior enzymatic cleavage into peptides. This article describes the preparation of monolithic reversed-phase (RP) columns with optimized porous structure and their performance separating intact proteins. Peak capacities of around 600 could be achieved within a 2 h gradient. The combination of high-resolution monolith chromatography with high-accuracy timeofflight mass spectrometry (TOF-MS) allowed intact proteins and protein isoforms that differ only in their oxidation state to be distinguished. The article also describes various MS fragmentation techniques for the identification and the more detailed characterization of intact proteins.

The concept of separation media based on monolithic materials can be traced back to the early 1950s, when Nobel Prize Laureates Martin and Synge discussed the benefits of these materials for different applications in chromatography (1). One of the first monolithic polymer gels for liquid chromatography (LC) was developed by Kubín et al. in 1967 (2). Highly swollen hydrogels, prepared in glass tubes from 2-hydroxyethyl methacrylate as the bulk monomer and ethylene dimethacrylate as the cross-linking monomer, were applied for the size-exclusion separations of water-soluble polymers. Rigid macroporous polymer-based monolithic columns were introduced in the early 1990s (3). This type of stationary phase is composed of a single monolithic unit with interconnected microglobules and macropores (flow-through pores), and is typically bonded to the column wall via a covalent bonding to enhance the robustness. 

Polymer monoliths are prepared from liquid precursors, that is, the bulk monomer and cross-linker dissolved in the porogen, allowing their 

preparation in virtually any format. The separation performance strongly depends on the porous structure of the monolithic material (4). It has been demonstrated that the composition of the polymerization mixture, including the type or ratio of the porogen solvents in the polymerization mixture (5), and the polymerization conditions, such as polymerization temperature and time (6), are key parameters that need to be controlled precisely. The surface chemistry and therefore selectivity can be tuned by incorporating 'functional' monomers in the polymer backbone (7). As a result of the absence of mesopores (stagnant zones inside polymer microglobules), the mass-transfer contribution to total band broadening is greatly reduced (8).  

The success of polymer monolithic columns for the reversed-phase gradient-elution separation of peptides in a LC–MS bottom-up proteomics approach has been demonstrated on several occasions (9–11). Karger et al. explored the use of 20 µm i.d. monolithic columns and established an HPLC–ESI–MS method yielding low-attomol detection sensitivity (12). Recently, we demonstrated a LC–MS–MS separation of a tryptic

 digest on a 1 m monolithic column yielding a peak capacity in excess of 1000 (13).  

LC–MS-based top-down proteomics is based on the chromatographic separation of intact proteins followed by their mass spectrometric elucidation. Single-stage MS provides information on the molar mass of intact proteins. The mass alone can give information about chemical or post-translational modifications of proteins, provided that the identity of the protein is already known. Tandem mass spectrometry MS

  can provide structural information and is essential for the identification of unknown proteins. In MS

  the selected precursor ions are fragmented in the gas phase using neutral gas molecules for collision-induced dissociation (CID) (14) and higher-energy collisional dissociation (HCD) (15); electrons for electron capture dissociation (ECD) (16); or radical anions for electron transfer dissociation (ETD) (17). In top-down proteomics high-resolution separation technology is of vital importance to address two bottlenecks: ion suppression and the spectral complexity. 

Here, the potential of capillary polymer monolithic columns is discussed for the separation of intact proteins, including protein isoforms. The effects of column properties (length and morphology) and gradient conditions on the separation performance were evaluated. The potential to achieve high-resolution protein separation is demonstrated with the liquid chromatography time-of-flight  mass spectrometry (LC–TOF-MS) analysis of a mixture containing 48 intact human proteins. In addition, the application of complementary fragmentation techniques (CID, HCD and ETD) is demonstrated for protein identification using a hybrid ion trap-orbitrap mass spectrometer. 

: Divinylbenzene (DVB, 65%) and styrene (S, >99%) were purchased from Merck (Hohenbrunn, Germany). Sodium hydroxide pellets were obtained from Merck (Overijse, Belgium). Acetonitrile (ACN, HPLC supragradient quality), formic acid (FA, ULC/MS quality), tetrahydrofuran (THF, > 99.9 %), aluminium oxide (Al

), toluene (99.8 %,), 3-(trimethoxysilyl)propyl methacrylate (98 %), methanol, 1-decanol (> 95.0 %, GC quality), trifluoroacetic acid (TFA, ULC/MS quality), carbonic anhydrase from bovine erythrocytes, ribonuclease A from bovine pancreas, lysozyme from gallus egg white, myoglobin from equine heart, trypsinogen from bovine pancreas and the "Universal Proteomics Standard 1" containing 48 human source or human sequence recombinant proteins (ABRF mixture) were purchased from Sigma-Aldrich (Bornem, Belgium). 

DVB and S monomers were purified by passing them over activated basic alumina. Water was purified in-house using a Millipore Simplicity system (Millipore, Bedford, MA, USA). Fused-silica capillary tubing (200 µm i.d. × 365 µm o.d.) was purchased from Aventes (Eerbeek, The Netherlands). 

HPLC experiments were performed using an UltiMate 3000 Proteomics MDLC system (Dionex Corporation, Germany) equipped with a membrane degasser, ×2 dual-gradient pump, a thermostatted flow manager and well-plate sampler applying a linear aqueous ACN gradient. For LC–TOF-MS analysis of intact proteins the HPLC system was coupled on-line with a maXis ultra-high-resolution qTOF mass spectrometer via a nano-electrospray interface (Bruker Daltonics, Bremen, Germany) equipped with a Proxeon steel emitter. Spectra were obtained in positive ion (PI) mode. LC–TOF-MS analysis of intact protein was performed on a 250 mm × 0.2 mm i.d. PepSwift monolithic capillary column purchased at Dionex Benelux. 

An Orbitrap Elite hybrid mass spectrometer (Thermo Fisher Scientific, Bremen, Germany) was used for MS–MS fragmentation experiments. Protein solutions were infused directly into the mass spectrometer. A solution of beta2microglobulin (0.5 µg/µL in 1:1 water/acetontrile containing 0.1% formic acid) was infused into the Orbitrap Elite mass spectrometer (Thermo Scientific) at 5 µL/min. The source voltage was set to 5 kV and source temperature to 275 °C. The AGC settings was 1E6 and 500 ms for MS and 5E4 and 250 ms for MS/MS. Five to 10 microscans were averaged for MS and MS–MS spectra. 

Scanning electron micrographs (SEMs) were obtained using a field emission scanning electron microscopy (FE-SEM) (Jeol JSM-7000F FE-SEM, Jeol Ltd., Tokyo, Japan) after sputtering a 20 µm coating on the monoliths with a Cressington 208 HR sputter coater. 

To demonstrate the effect of porogen composition of morphology, monolithic columns were prepared 

in 200 µm i.d. fused-silica capillaries from styrene and divinylbenzene precursors through a thermally initiated free-radical copolymerization reaction. Prior to the polymerization reaction, surface modification of the inner wall of fused-silica capillaries was performed with 3-(trimethoxysilyl)propyl methacrylate following an approach described by Svec (18). Polymerization mixtures comprising 40 w% monomers (20% S as bulk monomer and 20% DVB as cross-linker), 60 w% porogen (THF and decanol),and initiator (AIBN, 1 w% with respect to monomers) were degassed in an ultrasonic bath for 10 min. Surface-modified capillaries were filled with the polymerization mixture and then sealed with a septum. After polymerization at 70 °C overnight in a water bath, the columns were flushed with methanol and mobile phase.  

LC–TOF-MS analysis of intact proteins was performed on a 250 mm × 0.2 mm i.d. PepSwift monolithic capillary columns (Dionex Benelux, Amsterdam, The Netherlands). 

Control of monolithic structure and effect on gradient performance: 

Figure 1 shows scanning electron micrographs of the monolithic materials prepared. The close-up [Figure 1(b)] of the monolithic structure depicted in Figure 1(a) clearly shows the typical interconnected polymer microglobules and macropores in the polymeric structure. As a result of the absence of mesopores (stagnant zones in microglobules), polymer monolithic materials are conceptually very well-suited to perform large-molecule gradient separations because mass transfer is mainly driven by convection, rather than by diffusion. The monolith is covalently bonded to the fusedsilica capillary surface via a 3-(trimethoxysilyl)propyl methacrylate spacer to prevent channeling effects of analytes between the monolith and the wall. 

The porous structure of PS-DVB monoliths was systematically varied by changing the porogen ratio (THF/decanol) to maximize the peak capacity. With a decreasing amount of THF in the polymerization mixture, an increase in both the macropore and microglobule size can be observed (Figure 1). These findings can readily be explained by the solvating properties of the porogens: while THF is a thermodynamically "good" solvent for the polymer, decanol constitutes a "poor" solvent. Decreasing the amount of THF (thereby increasing the concentration of "poor" solvent) results in an earlier phase separation and results in the formation of a porous monolithic structure containing large macropores. Since the polymerization reaction is mainly continued within the nuclei, the size of the polymer microglobules increase. The resulting polymer monolith will accordingly exist of large polymer microglobules with large voids (macropores). THF is a "good" solvent for both monomers and polymers and increasing the THF content in the polymerization mixture means the phase separation is postponed. As a consequence, the monomer concentration in the nuclei is smaller, leading to the formation of a larger number of microglobules that are smaller in size [see the monolithic structure depicted in Figure 1(d)]. 

Columns of different length and morphology were evaluated in gradient LC mode by separating a standard mixture of proteins (ribonuclease A, lysozyme, trypsinogen, myoglobin and carbonic anhydrase) and varying the gradient duration. The peak capacity (

) was determined by averaging the 4σ peak width (

 the retention factor of the analyte at the moment of elution and 

 is the plate height. Figure 2 shows the effect of gradient time on peak capacity (a) and peak-production rate (peak capacity per unit time) (b) operating the columns at a flow rate of 2 µL/min. While longer gradient times and shallower gradients yield higher peak capacities, the peak capacity tends to reach a limit at longer gradient duration due to a linear increase in 

. However, with increasing gradient time, the peak-production rate dramatically decreases. Monoliths with smaller macropores and microglobules perform better in terms of peak capacity and in peak-production rate. Also, the optimum gradient time to achieve the maximum peakproduction rate shifts to slightly higher values [Figure 2(b)]. The longer monolithic column performs better in terms of peak capacity and peak-production rate when applying gradient times > 50 min, which is far from the optimal gradient duration required to reach the maximum peakproduction rate. However, this may be alleviated with the development of future monolithic nanomaterials that can be operated at ultra-high pressures. 

The potential of long monolithic columns for highly demanding separations is further demonstrated by a top-down LC–TOF-MS analysis of a complex mixture of intact proteins on a 250 mm monolithic column applying a 2 h gradient (Figure 3). The protein mixture contains 48 intact human proteins and the sample complexity is further increased by the presence of protein isoforms arising from co- and post-translational modifications, including oxidation, methylation and glycosylation (19, 20). The maximum peak capacity was around 600. The threedimensional representation of the LC–TOF-MS analysis is shown in Figure 3(b), depicting the retention time (

-axis). Figure 3(c) shows the charge envelope of a single protein with charge states ranging between 6+ and 10+ eluting between 46–48 minutes. After deconvolution of the mass spectrum the relative molecular weight (M

) was determined to be 8,381.4 Da, which corresponds to the theoretical molecular weight of interleukin-8 (M

 = 8,381 Da). Figure 3(d) shows a zoom spectrum of the isotopically resolved 8+ ion of this polypeptide (resolution ≈ 30,000 FWHM). 

Figure 4 clearly shows the high information content of the three-dimensional representation of proteins eluting between 88  min and 95 min. Whereas the TIC representation would only show co-eluting proteins, the three-dimensional representation allows for the deconvolution of co-eluting proteins, based on differences in 

 values. Protein identification, as shown in Table 1, was based on matching of experimental Mr values with theoretical masses obtained from the list of known proteins present in the 48 protein standard. The six charge envelopes that were baseline separated were consistent with their allocation to isoforms of peptidylprolyl 

 isomerase A. The mass difference of 16.0 Da could be indicative of the oxidation of methionine or cysteine residues. 

The three-dimensional representation in Figure 5(a) shows highly similar charge envelopes, indicative of the elution of protein isoforms, which are baseline separated. The average molecular weight of the most intense signal (signal 1) was determined to be 11,729 Da. The experimentally determined M

 values coincide well with the expected mass of beta-2-microglobulin (M

 = 11,731 Da). The mass difference of 2 Da can be explained by the formation of a disulfide bridge, which is a documented post-translational modification for this particular protein. The mass difference of 16 Da between peaks 1 and 2 could indicate protein oxidation. This post-translational modification makes the protein more polar and results in a decrease in retention time because of the reversed-phase (RP) character of the stationary phase. 

Application of complementary MS–MS fragmentation techniques: 

  experiments on the intact protein can add further confirmation to protein sequence and to the characterization of post-translational modification(s). MS–MS experiments were performed on beta-2-microglobulin (~12 kDa) using an Orbitrap Elite hybrid mass spectrometer which is capable of 240,000 resolution (at 

 = 400). Ion trap CID, HCD and ETD were performed on the +12 charge state of beta-2-micoglobulin and the HCD and ETD MS–MS spectra are shown in Figures 6(a) and (b), respectively.  

The multiply-charged ions in the ion trap CID and HCD spectra were assigned to b- and y-fragment ions, and sequence coverage with HCD fragmentation was found to be higher compared to ion trap CID. The ETD spectrum in Figure 6(b) revealed a homologous series of c- and z fragment ions. The sequence coverage of applying ion trap CID, HCD and ETD fragmentation is demonstrated in Figure 6(c). Applying complementary fragmentation techniques provided more reliable and detailed amino-acid sequence information. It is important to note that beta-2microglobulin contains a disulfide bridge and fragmentation was suppressed in the disulfide bonded loop (between amino acid 25 and 80). More structural information may be obtained after reducing the disulfide bridges before the LC–MS–MS experiment (21). 

The separation performance of capillary polymer monolithic columns that differ in porous structure and column length was evaluated and the gradient duration was optimized for the LC–MS analysis of intact proteins. The separation performance of the columns was assessed in terms of peak capacity and peak-production rate. Peak capacity can be interpreted as a direct measure for separation power, while peak-production rate can be used as a measure for effectiveness. Longer gradient times and shallower gradients yielded the highest peak capacities. However, with increasing gradient time, the peak-production rate decreased. 

The analysis of intact proteins using LC–TOF-MS allowed the tentative identification of proteins based upon their molecular weight. As demonstrated, this may be more confidently achieved by the application of MS/MS fragmentation techniques. The application of highresolution monolith chromatography in combination with complementary fragmentation techniques such as CID, ETD and HCD provides a unique platform to analyse proteins, including isoforms. Reduction of disulfide brides prior to LC–MS–MS analysis might be beneficial to obtain higher sequence coverage. 

This work was supported by a grant from the Research Foundation Flanders (G.0919.09) and is gratefully acknowledged. Bert Wouters acknowledges the financial support Institute for the Promotion of Innovation through Science and Technology in Flanders (IWT-Flanders). NEPAF is a CELS project funded by ONE NorthEast and partly financed by the European Regional Development Fund (ERDF). Franck van Veen (Dionex Benelux, Amsterdam, The Netherlands) is acknowledged for donation of the PepSwift monolithic columns. 

 obtained his Masters degree in Applied Engineering (Chemistry) in 2007 from the Artesis College University of Antwerp, and his Masters degree in Biomolecular Science in 2010 from the Vrije Universiteit Brussel in Brussel. He is a PhD student at the Department of Chemical Engineering at the Vrije Universiteit Brussel and focuses on the development of novel spatial multi-dimensional chromatography. 

 is a PhD student at the Department of Chemical Engineering at the Vrije Universiteit Brussel and focuses on the development and characterization of novel polymer monolithic structures for application in chromatography. He obtained a Masters degree in Bioengineering, medical biotechnology (Cellular & Genetic Biotechnology) with great distinction in 2009 from the Vrije Universiteit Brussel.  

 is Scientific Manager at NEPAF and is a visiting Research Professor at Northumbria University. After his post-doctoral research at the Fujita Health University in Toyoake, he was appointed Director of Mass Spectrometry in the Department of Clinical Pharmacology at the Royal College of Surgeons (RCSI), Dublin. He has performed extensive work in the application of NMR and mass spectrometry to solve the structure of lipoarabinomannan at both the University of St Andrews and Leeds University. Achim's key areas of expertise include analytical biochemistry, protein chemistry, liquid chromatography, mass spectrometry and bioinformatics. 

 is co-founder of LC Packings and is currently Managing Director of Dionex Benelux (Amsterdam, The Netherlands). His interests include column technology for nano- and bio-LC and LC–MS application development. 

 received her PhD degree in Chemistry (specializing in biological mass spectrometry) from the University of Manchester, United Kingdom. After postdoctoral research positions at the National Institutes of Health and California Institute of Technology she joined Thermo Fisher Scientific as a Proteomics Applications Specialist working on the Orbitrap mass analyser. 

 is the mass spectrometry Marketing Manager for Thermo Fisher Scientific in Europe. He has over 20 years of experience working with biological mass spectrometry. 

 is a professor in the Materials and Chemistry department of Vrije Universiteit Brussel and is responsible for the surface analysis facilities. He is also based part-time as a professor at TUDelft where he is cluster leader of the research on durability of materials within M2i.  

received his PhD degree in Chemistry (specializing in Analytical Chemistry) in 2005 from the University of Amsterdam, The Netherlands. After his postdoctoral research at the University of California in Berkeley, he joined Dionex and conducted research on packed and monolith column technology for ultra-high-pressure LC, two-dimensional LC and nano-LC. In 2009, the National Fund for Scientific Research (FWO, Belgium) awarded him an Odysseus grant. He currently holds a position as assistant professor at the Department of Chemical Engineering at the Vrije Universiteit Brussel. 

Describes the preparation of a reversed-phase monolithic column with an optimized porous structure to separate intact proteins using LC–MS.

The Potential of Polymeric Monolithic Capillary Columns for the LC–MS Analysis of Intact Proteins

This article describes a rapid heart-cutting liquid chromatography–gas chromatography (LC–GC) method for the analysis of mineral oil saturated hydrocarbons (MOSHs) found in a range of widely-consumed foods, including crisps, margarine, tinned tuna and vegetable oils.

The automated LC–GC experiments were performed using a system equipped with a syringe-type interface capable of both heart-cutting and comprehensive two-dimensional analysis. The first dimension separation was achieved on a silica column operated under isocratic conditions using hexane. The heart-cuts were then transferred to a programmed temperature vaporizer. After the large volume injection (LVI), the target analytes were rapidly separated (~ 9 min) using a micro-bore GC capillary column. The overall LC–GC run time enabled the analysis of around four samples in an hour. Various degrees of MOSH contamination — ranging from "not detected" to around 390 mg/kg — were discovered in the thirty samples that were subjected to analysis.

The contamination of foods with mineral oil saturated hydrocarbons (MOSHs) is an important issue. Mineral oil is derived from crude oil and mainly consists of MOSHs and mineral oil aromatic hydrocarbons (MOAHs). MOSHs are formed of straight and branched alkanes, as well as cyclic constituents such as naphthenes (1). Many papers based on this specific type of contamination have been reported over the last two decades. Grob et al. for example, reported various sources for this type of contamination, including mineral batching oil used for spinning jute, as well as lubricating oils and release agents used in the food industry (2,3). Printing inks that use mineral paraffins as solvents were found to have contamination levels between 10–100 mg/kg in foods contained in cardboard boxes (4). A case of mineral oil contamination in meats and eggs, caused by animal feed containing fat from waste collection sites, has also been reported (5). Another source of contamination is from the atmosphere. Neukom et al. investigated the presence of C

 MOSHs in particulate matter from polluted air and suggested that this was an important source of MOSH contamination in vegetable oils (6). 

In general, the presence of MOSHs in vegetable oils has been well-documented (6–9): Fiselier and Grob analysed sunflower oils using LC–GC analysis and discovered MOSH contamination levels between 2.7–32 mg/kg (7). It was suggested in this study that, apart from the atmosphere, contamination was also related to harvesting, storage, transport and processing. Two-hundred and twenty vegetable oil samples were analysed by Wagner et al., with levels of MOSH reaching 80 mg/kg (8). Recently, Tranchida et al. analysed several commercial vegetable oils and found some high levels of MOSH contamination, particularly for a pomace oil sample (9).  

The use of food grade (white) mineral oil that contains only saturated hydrocarbons and no aromatics is allowed in the food industry.  

The Joint FAO/WHO Expert Committee on Food Additives (JECFA) published a list of admissible daily intake (ADI) values for white mineral oils (10): for a high viscosity white oil the highest ADI value suggested was 20 mg/kg, while the lowest was 0.01 mg/kg for a low viscosity product. Recently, the European Food Safety Authority (ESFA) suggested an ADI value of 12 mg/kg bw (body weight)/day for high viscosity white mineral oils (11). 

At the moment there are no legal concentration limits for MOSHs in foods, except for one specification defined by the EU Commission (50 mg/kg) for Ukrainian sunflower oil, following a critical case of contamination (12). What is known is that MOSHs are probably the most abundant contaminants in the human body, with the intake starting from the first day of our lives (13). Furthermore, the exact toxicological effects of such a high and constant exposure to MOSHs is still not known (14). 

From an analytical perspective, heart-cutting LC–GC with a flame ionization detector (FID) is a suitable method for the determination of MOSHs in foods. Recently, Tranchida et al. developed a rapid, sensitive LC–GC method for the analysis of MOSHs in a selection of vegetable oils (9). The current research is based on the use of the fast LC–GC to analyse vegetable oils, as well as other commonly consumed foods, such as crisps, margarine and tinned tuna. Various degrees of contamination were found in the samples chosen. 

All the samples reported in Table 1 were purchased from supermarkets located in Messina, Italy, except for some of the extra virgin olive oils produced from a non-industrial source in the countryside near Messina (Table 1), kindly donated by staff from our laboratory. The vegetable oils were diluted to a final concentration of 25% v/v in hexane before LC–GC injection. The tinned tuna samples were filtered, and the vegetable oil collected and dried using Na

. The vegetable oil was filtered again and then diluted to a final concentration of 25% v/v in hexane. The crisp samples (500 mg) were extracted with hexane (1 mL) for 1 h, then dried under a gentle nitrogen stream and diluted to a final concentration of 25% v/v in hexane. The margarine samples (500 mg) were extracted three times with hexane (1 mL each), then dried under a gentle nitrogen stream and diluted to a final concentration of 25% v/v in hexane. 

 Table 1: Samples subjected to LCâGC analysis and their contamination values. 

 All two-dimensional LC–GC applications were performed on an LC×GC system (Shimadzu, Kyoto, Japan) consisting of a Shimadzu Prominence LC-20A system, equipped with a CBM-20A communication bus module, two LC-20AD dual-plunger parallel-flow pumps, a DGU20A online degasser, an SPD-20A UV detector, a CTO20A column oven and an SIL-20AC autosampler. Data were acquired by the LCsolution software (Shimadzu). 

 A 100 mm × 3 mm internal diameter (i.d.) × 5 µm 

 silica column (Supelcosil LC-Si, Supelco, Milan, Italy) was operated under isocratic conditions, using hexane as a mobile phase (0.35 mL/min). The injection volume was 20 µL. At the end of the heart cut, the column was backflushed with CH

. A Shimadzu AOC-5000 autoinjector equipped with a dedicated dual side-port syringe was used as a transfer device (a detailed description will be given in the Results and Discussion section).  

A Shimadzu GC2010 Plus, equipped with an Optic 3 injector (ATAS GC International, Eindhoven, The Netherlands). The Optic 3 injector was temperature-programmed as follows: from 75 °C (1 min) to 360 °C at 250 °C/min. The injection mode was split at a ratio of 200:1 for 1 min, then splitless for 1 min, then 50:1 for the remaining analysis time. Data were acquired by the GCsolution software (Shimadzu). 

 An SLB-5ms [silphenylene polymer, virtually equivalent in polarity to poly(5% diphenyl/95% methylsiloxane)] 15 m × 0.10 mm i.d. × 0.10 µm 

 column (Supelco) was heated from 50 °C (1 min) to 360 °C (4 min) at 70 °C/min. The hydrogen carrier gas was supplied at an initial pressure of 529 kPa (constant linear velocity was 100 cm/s). FID (360 °C) sampling frequency was 50 Hz.  

Multidimensional LC×GC software (Shimadzu, Duisburg, Germany) enabled the control of each instrument through the respective native software. The software controlled the transfer process, which will be described in detail in the Results and Discussion section.  

The multidimensional LC–GC system used in the present research can be used in either the heart-cutting or "comprehensive" mode. In the latter configuration, the entire initial sample is analysed in both dimensions. Chromatography band transfer is achieved through a modified 25-µL syringe. A schematic of the transfer device, in the cutting configuration, is illustrated in Figure 1. The lower part of the syringe barrel is linked, via two transfer lines, to the exit of the LC detector (line A) and to waste (line B). A Teflon plug is situated at the end of the syringe plunger, with the latter characterized by a lower outer diameter with respect to the i.d. of the barrel, enabling eluent flow inside the syringe. In the waste mode, the syringe plug is positioned below both lines and the effluent is directed to line B. In the cut position, the syringe plug is located between the two lines and the effluent flows through line A to the GC injector (the configuration represented in Figure 1). 

Figure 1: Schematic diagram of the syringe transfer device used for multidimensional LCâGC analysis in the cutting configuration. 

In the present work, the first-dimension effluent was directed to waste until 1.5 min. From 1.5–2.0 min the LC effluent (the MOSH-containing fraction) was directed to the GC injector. Dedicated LC×GC software controlled the transfer process: after 1.5 min, the LC pumps were deactivated and the syringe was directed to the injector port. In the latter position, the plunger was moved to the cut position, and the LC pumps were activated for a time corresponding to the transfer period, namely, 30 s (175 µL). At the end of the transfer process the pumps were stopped, the plunger injected the remaining volume of LC-effluent and the fast GC analysis started. After each stop-flow period the gradient programme can be resumed, using exactly the same solvent composition as before the interruption. There is also a software window for the automatic calculation of the transfer line delay. This option is useful to determine the delay time of the LC fraction, from the UV detector to the injector port as a result of the transfer line volume.  

The LC–GC method used, developed and validated in previous research is rapid and sensitive. The total analysis time is 14 min and the limit of quantification (LOQ) is 2 mg/kg (9). The first dimension is very useful because it frees the (possible) MOSH fraction from the lipids, which are retained on the silica phase. After transfer of the MOSH fraction, the primary column is backflushed with CH

. The scope of the present research was to apply the method not only to vegetable oils, but also to other foods which derive, or are prepared, using vegetable oil. Because MOSH contamination is also associated with industrial processing and transportation, a series of extra virgin olive oils, produced from a non-industrial source in the countryside near Messina, were evaluated.  

Prior to food analysis, a blank sample (hexane) was subjected to LC–GC analysis to assess the possible contamination of the mobile phase and/or of the instrumentation. As can be seen in Figure 2, if contamination was present it was below the limit of detection (0.6 mg/kg) of the method. The impurities eluting between 3–4 mins were of no concern from an analytical perspective because they did not co-elute with the MOSH fraction. 

Figure 2: GC chromatogram for a blank sample (hexane). 

In this study thirty food products were analysed consisting of four crisp samples, four margarine samples, five tinned tuna samples, and seventeen vegetable oil samples (which included fourteen extra virgin olive samples, one corn oil sample, one peanut oil sample and one sunflower sample).The results are listed in Table 1. All the values reported in the table are average results, derived from two consecutive LC–GC applications.  

For the potato crisp samples no information was given on the packaging on the type of vegetable oil used. A 500mg quantity was extracted for 1 h with 1 mL of hexane. The second extraction gave a negligible response. The crisp contamination levels were rather limited, with all but one of the samples under the 4 mg/kg level (for sample 01 no contamination was detected). However, for this type of food, the sample subjected to hexane extraction is obviously formed mainly of potato, containing only a minimum quantity of vegetable oil. For this reason, crisp sample 04, which was characterized by a concentration of MOSH ≈ 20 mg/Kg was most probably produced using a rather highly contaminated oil. Therefore, no estimation can be made on the degree of contamination of that vegetable oil. 

There was also no information on the type of vegetable oil used for the margarine samples. A 500-mg quantity was extracted for a 20 min period with 1 mL of hexane, three times consecutively. The fourth extraction gave a negligible response. Two of the margarine samples (01 and 02) were characterized by moderate contamination levels; whereas the other two samples presented a high degree of contamination (> 200 mg/kg), with one (03) nearing the 400 mg/kg mark. The amount of vegetable oil used in the production of the margarines was in the 60–70% range (reported on the container). Because such contamination levels are extremely high the following experiment was performed: 3 mL of hexane were dried under a nitrogen stream. The residue was dissolved in 1 mL of hexane and subjected to LC–GC analysis. Again, no response was observed. 

Regarding the vegetable oil used in the production of tinned tuna — in this case olive oil — the degree of variability was high: sample 01 was characterized by a low contamination level (8.9 mg/kg), samples 03 and 04 by moderate ones and samples 02 and 05 by a contamination slightly exceeding 100 mg/kg and 80 mg/kg respectively. Commercial olive oil is technically a mixture of refined and extra virgin olive oil. A fast GC–FID chromatogram for tuna sample 05 is shown in Figure 3. As can be seen, the chromatogram is characterized by a typical MOSH "hump" centred at the C

 n-alkane in this case and contained in the C

 range. Also highlighted in the figure are the predominant odd-numbered n-alkanes, which derive from the olive plant. 

Figure 3: GC chromatogram for the tinned tuna sample  05 with a MOSH concentration of 83.7 mg/kg. 

In this research five extra virgin olive oils (EVOOs), produced on family-run farms were also subjected to LC–GC analysis. For three of the EVOO samples the possible contamination was below the method's LOQ. The remaining two oils were characterized by low MOSH concentrations. It must be noted that the "family" oil group presented a 60% degree of negative response. It should be noted that no MOSHs were found in two out of nine commercial EVOO products; the remaining seven samples presented a low degree of contamination in the 2.5–10.5 mg/kg range, confirming the results obtained in previous research (9). It must also be added that in that study no EVOO samples were free from contamination in the four commercial products that were analysed. The production of EVOO essentially requires mechanical extraction only and the MOSH levels are, therefore, generally limited. The remaining three vegetable oils (corn oil, peanut oil and sunflower oil) contained moderate MOSH contamination. The production of seed oils is based on solvent extraction and a refining process and the possibility of MOSH contamination is, therefore, higher. 

The results obtained in the present study indicate that the level of MOSHs present in vegetable oils, and in foods containing such oils, is strongly related to the number of industrial phases that an oil or an oil-containing food is subjected to. The "family-sourced" oils were characterized by the lowest degree of contamination. As expected, vegetable oil-containing foods that are rarely produced using EVOO were nearly always contaminated. The only exception was a crisp sample (01) and this was probably because this type of food contains a low amount of vegetable oil, and not because the oil used was free from MOSHs. The highest concentration of MOSHs found was 388.4 mg/kg for the margarine sample (03). Such a degree of contamination is certainly an excessive one, a statement which is also valid for the second most contaminated sample (margarine 04 = 220.4 mg/kg). Although the production of margarine requires a certain amount of industrial processing, there is no way to pinpoint where such a high level of contamination comes from.  

The multidimensional LC–GC system is fairly new to this field and has been employed effectively and continuously during the last year. The instrumentation has also been used in the comprehensive two-dimensional mode. Currently, a rapid LC–GC method to analyse both MOSHs and MOAHs in foods is being developed. The presence of MOAHs in foods is certainly of greater concern from a toxicological perspective. 

 is Associate Professor in Food Chemistry at the School of Pharmacy in Messina, Italy. He has co-authored approximately 80 publications and presented work in over 130 oral/poster presentations in national and international meetings. His main research activities are focused on the analysis of volatiles and semivolatiles contained in complex samples, by using advanced gas chromatographic methodologies. In particular, he has acquired considerable experience in the use of fast and very fast gas chromatographic techniques. He is actively engaged in the development and application of classical multidimensional GC, comprehensive two-dimensional GC (GC×GC) and LC–GC (LC×GC) systems. 

 is currently a Ph.D. student in Food Chemistry and Safety at the University of Messina, Italy. His main research activities are focused on the development of heart-cutting and comprehensive multidimensional chromatography methods (GC–GC, LC–GC, GC×GC, LC×GC), combined with mass spectrometric detection, for food analysis.  

 is Full Professor of Food Chemistry at the University of Messina, Italy, and at the "Campus Biomedico" in Rome. Her research interests include the development of comprehensive chromatography, and high throughput separation methods applied to the study of Citrus products (essential oils and juices),compounds with a possible biological activity in natural samples (carotenoids, anthocyanins and coumarins), plant essential oils, the aromatic fraction of wine and other alcoholic beverages, and of triglycerides in food lipids. Prof Dugo is the author of approximately 130 scientific papers, and has been invited to be a speaker at several national and international congresses. 

 is Full Professor of Analytical Chemistry at the Dipartimento Farmaco-chimico of the University of Messina, Italy, and at the University "Campus Biomedico" in Rome. He is the author of approximately 200 scientific papers and 29 book chapters. In addition, he is co-editor of a book on multidimensional chromatography (Wiley), and editor of a book on comprehensive chromatography in combination with mass spectrometry (Wiley).  

His research interests include one-dimensional chromatography techniques, combined particularly with mass spectrometry, as well as multidimensional chromatography methods, such as LC–GC–MS, GC–GC– MS, GC×GC, LC×LC, LC×GC, and their use in the study of complex samples.  

(2) K. Grob, M. Lanfranchi, J. Egli and A. Artho, 

(3) K. Grob, A. Artho and M. Biedermann, J. Egli, 

Zeitschrift für Lebensmitteluntersuchung und -forschung,

(5) K. Grob, M. Vass, M. Biedermann and H.P. Neukom, 

(6) H. P. Neukom, K. Grob, M. Biedermann and A. Noti, 

(8) C. Wagner, H.P. Neukom, K. Grob, S. Moret, T. Populin, L.S. Conte, 

(9) P.Q. Tranchida, M. Zoccali, G. Purcaro, S. Moret, L. Conte, M. Beccaria, P. Dugo and L. Mondello, 

(10) Summary of Evaluations Performed by the Joint FAO/WHO Expert Committee on Food Additives, April 25 (2003). 

A fast heart-cutting liquid chromatography–gas chromatography (LC–GC) method for the analysis of mineral oil saturated hydrocarbons (MOSHs) in a range of widely consumed foods is described.

Rapid Multidimensional Liquid–Gas Chromatography for the Analysis of Saturated Hydrocarbon Contamination in Foods containing Vegetable Oil

In this month's instalment, we look at the results of a recent survey on high performance liquid chromatography (HPLC) columns, examining the trends in analytical and preparative column use and purchasing patterns since the last survey. Factors investigated in this study were mode and stationary phase usage, particle sizes and column dimensions, column budgets and the factors influencing buying decisions. Column lifetime and use of guard columns is also considered. Future column buying plans were explored.

 surveys its readers to obtain a current profile of users of high performance liquid chromatography (HPLC). The last survey on HPLC columns was conducted in 2009 (1). In the past, I have used results from these surveys to identify trends in column technology and HPLC in practice. In late 2010, a web-based survey was sent to subscribers whose primary chromatography technique was HPLC. A total number of 324 readers responded and this number was statistically sufficient to allow comparisons to previous survey data and to investigate trends.  

To ensure that the results of the current survey were compatible with those of previous surveys, I used the same methodology to report the results. Because many of the questions allowed respondents to give more that one answer, in some cases, I normalized response totals. Normalizing the results to a base of 100% makes it easier to compare the results of previous surveys with those of the present survey and to identify trends in the use of HPLC columns, modes and packings. Questions pertaining to mode usage, column lifetime, particle sizes usage, purchasing considerations and possible future needs were explored to understand selection criteria. 

To understand the current usage rate of instruments, a question was asked pertaining to number and type of instruments personally used per respondent per week. Questions in previous surveys were less specific and, therefore, laboratory managers occasionally responded with estimates for their entire laboratory, which tended to skew the results. Table 1 shows the average response for each category of instrument. The numbers reported should not be construed to mean that every respondent uses every type of instrument; they serve to provide an idea of the relative number of the various types of systems in general use. Later, I will look at the numbers of columns used by these instruments.  

Table 1:  Types of HPLC instruments used by survey respondents. 

According to the survey results, a user of conventional HPLC instruments is responsible for a weighted average of 2.6 units. Because each respondent may have been responsible for a single instrument or for multiple instruments, this weighted average was derived from looking at the total number of instruments of this type identified by all respondents and then dividing this number of the total number of respondents.  

For example, some respondents reported that they have only one conventional HPLC system while others reported that they have five instruments for which they are personally responsible. Based on the relative numbers of Table 1, for every one of these respondents that used a conventional LC system, only 1 in 16 would possess a capillary or nanoLC system; 1 in 4 might have an instrument dedicated to microbore columns; and 1 in 7 could have a preparative instrument in his or her laboratory. These numbers are slightly different from the 2009 survey (1), especially for conventional users, which was down from 3.6 per respondent (this was probably skewed by the laboratory manager contribution discussed above).  

Most liquid chromatographers use more than one chromatographic mode to solve specific separation problems. Because respondents were allowed to choose multiple modes, the total number of techniques used by respondents revealed that each used 3.2 modes on the average, in agreement with the last survey (1). This result doesn't mean that everybody is using all modes all the time, but that occasionally another mode may be used to solve a difficult separation problem. If one looks at the normalized data in Table 2, which compares the relative mode usage in 1997, 2007, 2009 and 2011, it should be no surprise that the results again show that reversed-phase chromatography was, by far, the most popular mode; according to the raw data, more than 93% of respondents use this mode and normal bonded phase usage was a distant second with 31%. These data have been corrected for those using ion pair chromatography (a subset of reversed-phase chromatography) and cyano columns for reversed-phase work. In earlier surveys, cyano columns were presumed to be mainly used in normal-phase separations, but this survey revealed that the predominant use of CN columns was in reversed-phase chromatography where this phase serves well as a short chain phase with unique selectivity.  

Table 2:  Analytical HPLC mode and stationary phase usage. 

Although the data in Table 2 show that reversed-phase chromatography has dropped on a relative basis, this drop is not due to its decrease in popularity (which remains extremely high), but rather from the increased use of other modes such as chiral and hydrophilic interaction chromatography (HILIC). HILIC, where respondents indicated a doubling in usage since 2007, is a separation technique for highly polar analytes that gets around some of the problems associated with reversed-phase chromatography, such as low retention or phase collapse (dewetting). HILIC uses a polar stationary phase such as bare silica gel, polar bonded phases (for example, diol) or specialty phases with dual functionality and requires a high percentage of a nonpolar mobile phase, usually acetonitrile, similar to the requirements for normal-phase chromatography.  

However, unlike normal-phase chromatography, which uses nonpolar solvents like hexane and methylene chloride and tries to exclude water from the mobile phase, HILIC requires some water in the mobile phase to maintain a stagnant enriched water layer on the packing surface into which analytes may selectively partition. In addition, water-miscible organic solvents are used. Under HILIC, polar analytes are well retained and elute in order of increasing hydrophilicity. Bare silica, specified for adsorption chromatography in the survey, is still a widely used HILIC phase, but the current survey did not explore its use in HILIC applications. 

Supercritical fluid chromatography (SFC), which employs columns similar to those used in HPLC, also has seen renewed interest, especially for preparative chiral separations. The use of chiral columns in SFC was not explored in the present survey, although a few respondents "wrote in" their use of HPLC columns in this increasingly popular chromatographic mode. 

The use of ion-exchange chromatography showed a slight upward swing in usage and is back to its historical levels. Included in this usage is ion chromatography, which is sometimes treated as a separate separation technique but in reality is ion-exchange chromatography because it employs the same separation principles and uses columns with ionic or ionizable functionalities. Ion-exchange chromatography is frequently used in proteomics where a large number of tryptic peptides are fractionated first by 2D LC with ion exchange followed by reversed-phase chromatography–mass spectrometry (MS) for separation and detection. It also finds use in amino acid separations and protein purification protocols. Although ion-pair chromatography also separates ionic and ionizable compounds, it is generally lumped with reversed-phase chromatography because it employs the same packing materials. Many chromatographers familiar with reversed-phase chromatography prefer to use ion-pair chromatography for ionic and ionizable compounds because the columns are more familiar to them and the principles of reversed-phase chromatography can be generally applied in method development. 

The normalized data in Table 3 further break down the reversed phases into individual organic moieties and clearly show that C18 (octadecylsilane) was most popular (the raw data showed that 92% of all users use this phase at some time) followed by C8 (octylsilane). The use of phenyl-bonded phase has showed steady usage as chromatographers exploit its unique selectivity compared to alkyl phases. The phenyl-hexyl and diphenyl phases that have seen some interest were not explored this time, but they will be included in future surveys. For the first time, I expanded the survey to include fluorinated phases that 14% of respondents mentioned that they have tried for separations where the conventional reversed-phase chromatography materials didn't do the job. In addition, graphitized carbon was added in this year's survey; only 4% of respondents indicated that they had tried this specialized material that is one of the more rugged reversed-phase packings. Again, just as in 2009, cyano phases used in aqueous solution showed quite strongly in the survey as a bonafide reversed-phase packing.  

Table 3: Stationary phases used in reversed-phase chromatography. 

Particle Sizes in Analytical and Preparative LC

Since the beginning of HPLC in the late 1970s, the trend has been to use columns packed with smaller spherical particles. Throughout most of the 1970s, 10-μm irregular particles were the norm because they were commercially available and slurry packing column efficiency was quite good. In the late 1970s, 5-μm spherical particles gave better performance because of improved mass transfer and better packing ability. Through occasional 

 column surveys, I started tracking particle size usage in the mid-1980s. Table 4 provides data of the use of various particle sizes from 1985 until 2011 (current survey). As one can see from the first survey, columns packed with 10-μm particles had already given way to 5-μm particles. These particles maintained their dominance through the 1990s and are still in widespread use today. There are many validated methods that have been developed on 5-μm particles, and users don't want to take the time to revalidate their method unless there is a strong driving force to do so. Nevertheless, as can be seen in Table 4, the use of even smaller particles (<2–4 μm) began in earnest in the mid-1990s and now these particles have caught up and surpassed the 5-μm particles. With the reduction in particle size, column efficiency improved further allowing a reduction in column length. Shorter columns are now in vogue. Not only does a shorter column provide faster separations but solvent use is reduced. Recently, the use of sub-2-μm particles has become fashionable in even shorter column lengths. In just a short time, the use of the sub-2-μm columns has doubled. Columns of 30–50 mm in length packed with these particles now provide plate counts formerly obtained on 15- and 25-cm columns packed with larger particles.  

Table 4: Analytical HPLC particle size trends 1985â2011*. 

In the current survey, we queried the use of 2–3 μm porous particles that have attracted the interest of some users. These columns are intermediate between the popular 3.0–3.5 μm columns and the newer sub-2-μm columns. They show lower pressure drops and efficiency than the sub-2-μm particles but better efficiency and higher pressure drop than the 3.0–3.5-μm particles. Since the last survey, the increasing use of the superficially porous particles (SPPs) (also referred to a solid core, core–shell and the like) has taken place. Even though the question for the survey was based on use of porous particles, I suspect that the respondents also took into account their increased use of SPP particles because the number reported in Table 4 was quite high for the relative usage of 2–2.9 μm particle columns. The SPP columns (with particle sizes of 2.6–2.7 μm) have quickly become a partial substitute for the sub-2-μm columns because they offer similar efficiency at half the pressure drop. Hence, users of conventional HPLC equipment can take advantage of ultrahigh-pressure liquid chromatography (UHPLC) separation capability. 

In comparing the use of preparative chromatography to analytical HPLC, 62% of those performing analytical separations do not use preparative chromatography. Not surprisingly, those that perform preparative separations tend to prefer larger particles as depicted in Table 5. Reasons for the preference of larger particles (10-μm and higher) are lower cost, lower pressure drop and loadability. For the latter consideration, the higher efficiency of smaller particles may be lost when overloaded conditions are employed. Even so, in the present survey, there has been an increase in respondents using smaller particles for preparative work (Table 5). For example, 18% of respondents use 5-μm particles for their preparative work, up from 9.7% in 2007.  

The results of a recent survey on stationary phases used in high performance liquid chromatography (HPLC) are revealed.

LCGC Europe-01-01-2012

Event News

LC Trouble Everywhere

The Potential of Polymeric Monolithic Capillary Columns for the LC–MS Analysis of Intact Proteins

Rapid Multidimensional Liquid–Gas Chromatography for the Analysis of Saturated Hydrocarbon Contamination in Foods containing Vegetable Oil

Current Trends in HPLC Column Usage